[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Test item ‘Ester adipic acid and neodecanoic acid, 2-oxiranylmethyl ester, Batch No. LEZF 140027’ ( 2,3-epoxypropyl neodecanoate content of 0.5 %):

Ames Test (OECD 471): positive

in vitro gene mutation test (HPRT Test; OECD 476): negative

in vitro chromosomal mutation test (Micronucleus Assay, OECD 487): negative

Test item ‘Ester adipic acid and neodecanoic acid, 2-oxiranylmethyl ester, Batch No. GCZ-2 9065-2’ (2,3-epoxypropyl neodecanoate content of 0.05 %):

Ames Test (OECD 471): negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

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:

of July 2016

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

of August 1998

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room tempterature
- Stability under test conditions: given
- Solubility and stability of the test substance in the solvent/vehicle: stable in acetone for 4 and 24 hours
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: none

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM containing Hank's salts supplemented with 10 % FBS (except during 4 hour treatment), neomycin (5 µg/mL) and amphotericin B (1 %); for the selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine.
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from the liver of phenobarbital/beta-naphthoflavone induced rats.

Test concentrations with justification for top dose:

The dose range of the main experiment was set according to the stability and homogeneity data generated within the Envigo Study Number HH16TC. The individual concentrations were spaced by a factor of 2.0.
To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.

The maximum test item concentration of the pre-experiment (250 μg/mL) was based on the solubility properties of the test item in DMSO as determined in the solubility test. Since the stability in DMSO could not be analytically confirmed the solvent was changed to acetone. Based on the result of an additional solubility test with acetone, 1000 μg/mL was chosen as maximum concentration in the main experiment. The stability of the test item formulated in acetone was analytically determined and the data were provided prior to the start of the main experiment.

Main experiment (4 hours exposure) without and with S9-mix: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125.0, 250.0, 500.0, 1000.0 µg/mL
Phase separation was observed at 31.3 µg/mL and above without S9 and at 62.5 µ/mL and above with S9 mix. The cultures at 62.5 μg/mL and above without metabolic activation, and at 125.0 μg/mL and above with metabolic activation were not continued to avoid analysis of too many phase separating concentrations.
In the main experiment there was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item dissolved in acetone.

Vehicle / solvent:

- solvent(s) used: for the pre-experiment DMSO was used; for the main experiment acetone was used
- final concentration of solvent in the culture medium: 0.5 % (v/v)
- Justification for choice of solvent/vehicle: The solvent was chosen based on its compatibility with the test item, its solubility properties and its relative non-toxicity to the cell cultures.
All formulations were prepared freshly before treatment and used within two hours of preparation. The homogeneity and stability was confirmed for the test item in acetone formulations (within Envigo Study no. HH16TC).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Culture Medium:
For seeding and treatment of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, 10 % FBS (except during 4 hour treatment), neomycin (5 μg/mL) and amphotericin B (1 %). For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2.

Pre-Experiment:
A pre-test was performed in order to determine the toxicity of the test item. In addition the pH-value and the osmolarity were measured. The general culturing and experimental conditions in this pre-test were the same as described below for the mutagenicity experiment.

In this pre-test approximately 1.5 million cells were seeded in 25 cm² flasks 24 hours prior to treatment. After approximately 24 hours the test item was added and the treatment proceeded for 4 hours (duplicate cultures per concentration level). Immediately after treatment the test item was removed by rinsing with PBS. Subsequently, the cells were trypsinized and suspended in complete culture medium. After an appropriate dilution the cell density was determined with a cell counter. Toxicity of the test item is evident as a reduction of the cell density compared to a corresponding solvent control. A cell density of approximately 1.5 million cells in 25 cm² flasks is about the same as approximately 10 million cells seeded in 175 cm² bottles 24 hours prior to treatment with the main experiment.

The pre-experiment was performed in the presence and absence (4 h treatment) of metabolic activation. Test item concentrations between 2.0 μg/mL and 250.0 μg/mL were used. The highest concentration of the pre-experiment was based on the solubility properties of the test item in DMSO.

No relevant cytotoxic effects indicated by a relative cloning efficiency of 50 % or below were noted up to the maximum concentration with and without metabolic activation.

The test medium was checked for precipitation or phase separation at the beginning and at the end of treatment (4 hours) prior to removal to the test item. Phase separation occurred at 15.6 μg/mL and above without metabolic activation, and at 250.0 μg/mL with metabolic activation after 4 hours treatment.

Main Experiment:
Since the stability of the test item in acetone was determined analytically, acetone was used as solvent in the main experiment. The highest concentration of 1000.0 μg/mL used in the main experiment was based on the result of an additional solubility test performed with acetone.
In the pre- and main experiment there was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item in acetone.

Two to three days after sub-cultivation stock cultures were trypsinized at 37 °C for 5-10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10 % FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2 % in saline. Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7 to 1.2×10 E7 were seeded in plastic culture flasks. The cells were grown for 24 hours prior to treatment.

Treatment:
After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 μl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. 4 hours after treatment, this medium was replaced with complete medium following two washing steps with PBS.
Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2.0×106 cells per experimental point (concentration series plus controls) were subcultured in 175 cm² flasks containing 30 mL medium.
Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (cloning efficiency I) as measure of test item induced cytotoxicity. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2.
The colonies used to determine the cloning efficiency I were fixed and stained 6 to 8 days after treatment as described below.
Three or four days after first sub-cultivation approximately 2.0×106 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium.
Following the expression time of 7 days five 75 cm² cell culture flasks were seeded with about 4 to 5×105 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability (cloning efficiency II).
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
a) the mean values of the numbers of mutant colonies per 106 cells found in the solvent controls of both parallel cultures remain within the 95 % confidence interval of the laboratory historical control data range.
b) Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistical significant increase compared with the concurrent solvent control.
c) Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
d) An adequate number of cells and concentrations (at least four test item concentrations) are analysable even for the cultures treated at concentrations that cause 90 % cytotoxicity during treatment.
e) The criteria for the selection of the top concentration are fulfilled (see 3.5.2 Dose selection).

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95 % control limits).
A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (based 95 % control limits).
In cases when the response is neither clearly negative nor clearly positive as described above, or in order to judge the biological relevance of a result, the data should be evaluated by expert judgement or further investigations.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
A t-Test was not performed since all mean mutant frequencies were well within the 95 % confidence interval of our laboratory’s historical negative control data.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: no, but tested up to and including phase separating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

See Table 1 - Summary of results.
The cultures at 62.5 μg/mL and above without metabolic activation, and at 125.0 μg/mL and above with metabolic activation were not continued to avoid analysis of too many phase separating concentrations.
No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50 % was observed neither in the absence nor in the presence of metabolic activation.
No biologically relevant increase in mutation frequency as the mean of both parallel cultures was observed in the main experiment up to the maximum concentrations scored for gene mutations.
The mean mutant frequency obtained in the solvent controls was 18.2 without S9 mix and 20.8 with S9 mix. The values were well within the 95 % confidence interval of our laboratory’s historical negative control data and, thus, fulfilled the requirements of the current OECD Guideline 476. The range of the mean mutant frequencies of the groups treated with the test item was from 12.2 up to 27.7 mutants per 106 cells.
The linear regression analysis showed no significant dose dependend trend of the mutation frequency at any of the experimental groups.
EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Table 1: Summary of results (main experimen, 4 hour treatment)

	conc. µg/mL	PS	S9 mix	RACE (%)	mutant colonies/10 E6 cells
solvent control	-	-	-	100.0	18.2
positive control (EMS)	300.0	-	-	73.5	242.0
test item	2.0	-	-	83.3	27.7
test item	3.9	-	-	69.9	20.0
test item	7.8	-	-	82.5	20.8
test item	15.6	-	-	83.3	19.1
test item	31.3	PS	-	78.2	12.2
test item	62.5	PS	-	#	#
solvent control	-	-	+	100.0	20.8
positive control (DMBA)	2.3	-	+	99.6	132.9
test item	2.0	-	+	133.1	18.8
test item	3.9	-	+	114.1	19.1
test item	7.8	-	+	105.9	19.5
test item	15.6	-	+	101.5	17.5
test item	31.3	-	+	102.2	16.6
test item	62.5	PS	+	93.8	22.9
test item	125.0	PS	+	#	#

solvent control: 0.5 % acetone

PS: phase separation at the beninning and at the end of treatment

RACE: Relative Adjusted Cloning Efficiency I

# these and higher cultures were not continued to avoid analysis of too many phase separating concentrations

95 % confidence interval for all evaluable cultures without S9 mix: 2.7 -31.2

95 % confidence interval for all evaluable cultures with S9 mix: 2.9 -30.9

Conclusions:

negative

Executive summary:

The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster following OECD 476. The main experiment was performed with a treatment time of 4 hours with and without metabolic activation. Concentrations of 2.0 to 31.3 μg/mL with and 2.0 to 62.5 μg/mL without metabolic activation were evaluated in the main experiment due to phase separation at higher concentrations. No relevant cytotoxicity and no substantial and dose dependent increase of the mutation frequency was observed in the main experiment. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

of July 2016

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) No 2017/735: B49 “In vitro Mammalian Cell Micronucleus Test”

Version / remarks:

of February 2017

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room tempterature
- Stability under test conditions: given
- Solubility and stability of the test substance in the solvent/vehicle: stable in acetone for 4 and 24 hours
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: none

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Sex, age and number of blood donors if applicable: male donor (34 years old) for Experiment I, and from a female donor (33 years old) for Experiment II.
- Whether whole blood or separated lymphocytes were used if applicable: whole blood

Cytokinesis block (if used):

Cytochalasin B (4 μg/mL)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from the liver of Phenobarbital/beta-naphthoflavone induced rats.

Test concentrations with justification for top dose:

Exp. I, (4 hrs): 3.8, 6.6, 11.6, 20.3*, 35.5*, 62.2*, 109*, 190*, 333*, 1000* µg/mL (without and with S9 mix)
Exp. II, (20 hrs): 0.8, 1.6, 3.1, 6.3, 12.5, 25, 50, 100* µg/mL (solely without S9 mix)
* phase separation was observed at the end of treatment

The following concentrations were selected for reading:
Exp. I (without and with S9 mix): 6.6, 11.6 and 20.3 µg/mL
Exp. II: 25, 50, 100 µg/mL

The highest applied concentration in this study was based on the solubility properties of the test item and with respect to the current OECD Guideline 487.

Based on the solubility properties of the test item, 1000 μg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 3.8 to 1000 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, phase separation of the test item was observed at 20.3 μg/mL and above in the absence of S9 mix and at 62.2 μg/mL and above in the presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Using a reduced Cytokinesis-block proliferation index (CBPI) as an indicator for toxicity, no cytotoxic effects were observed in Experiment I after 4 hours treatment in the absence and presence of S9 mix. Based on the observed phase separation, 100 μg/mL were chosen as top treatment concentration for Experiment II.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone (final concentration: 0.5 %)
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
The homogeneity and stability was confirmed for the test item in acetone formulations (within Envigo Study no. HH16TC). All formulations were prepared freshly before treatment and used within two hours of preparation.

The osmolarity was determined by using an osmometer in Experiment I without metabolic activation. The pH was determined by using a pH meter in the solvent control and the maximum concentration without metabolic activation in Experiment I.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demeocolcine

Details on test system and experimental conditions:

Blood samples were drawn from healthy non-smoking donors not receiving medication. For this study, blood was collected from a male donor (34 years old) for Experiment I, and from a female donor (33 years old) for Experiment II. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately 2 – 2.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.

Culture conditions
Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Pre-Experiment:
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of reduction in the CBPI in comparison with the controls (% cytostasis) by counting 500 cells per culture. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay.
The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 40 hrs after start of the exposure.
This preliminary test was designated Experiment I, since the cultures fulfilled the acceptability criteria and appropriate concentrations could be selected for cytogenetic evaluation.

Pulse exposure (see also Table 1)
About 48 hrs after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 μL S9 mix per mL culture medium was added. After 4 hrs the cells were gently centrifugated, separated from the supernatant, washed and resuspended in complete culture medium for a 16-hour recovery period. After this period Cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

Continuous exposure (see also Table 1))
About 48 hrs after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with complete medium (with 10 % FBS) containing the test item. After 20 hours the cells were gently centrifugated, separated from the supernatant, washed and resuspended in complete culture medium. Cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

STAIN: Giemsa

NUMBER OF CELLS EVALUATED: At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is characterized by the percentages of reduction in the CBPI (Cytokinesis-block proliferation index) in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in all of the experimental conditions examined:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95 % control limit realized as 95 % confidence interval)
The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.
Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data (95 % control limit realized as 95 % confidence interval)
When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.

Statistics:

Statistical significance was confirmed by the Chi Square Test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

based on phase separation of the test item in culture medium

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

See Table 2 - Summary of results.
The highest treatment concentration in this study was 1000 μg/mL, limited by the solubility properties of the test item.
Phase separation of the test item in the culture medium was observed at the end of treatment in Experiment I in the absence of S9 mix at 20.3 μg/mL and above, in the presence of S9 mix at 62.2 μg/mL and above and in Experiment II at 100 μg/mL.
No relevant influence on osmolarity or pH was observed.
In Experiment I in the absence and presence of S9 mix after 4 hours treatment and in Experiment II in the absence of S9 mix after 20 hours treatment, no cytotoxicity was observed up to the highest evaluated concentrations, which showed phase separation.
In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed.
Demecolcine (50 ng/mL), MMC (0.8 μg/mL) and CPA (17.5 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

Table 2: summary of results

Exp.	Exposure period	Test item conc. (µg/mL)	proliferation index (CBPI)	Cytostasis (%)	Micronucleated cells (%)
1	4 h without S9 mix	solvent control acetone	1.93	-	0.95
		positive control MMC	1.87	6.1	9.45#
		6.6	1.90	3.0	0.65
		11.6	1.81	12.5	0.50
		20.3*	1.88	5.5	0.50
II	20 h without S9 mix	solvent control acetone	1.78	-	0.65
		positive control Demeocolcine	1.65	16.3	2.30#
		25.0	1.91	n.c.	0.80
		50.0	1.82	n.c.	0.55
		100*	1.66	14.8	0.85
I	4 h with S9 mix	solvent control acetone	1.89	-	0.65
		positive control CPA	1.60	32.6	4.20#
		20.3	1.84	5.7	0.50
		35.5	1.82	8.7	0.85
		62.2*	1.90	n.c.	0.55

* phase separation occurred at the end of treatment

# statistically significant increase

Conclusions:

negative

Executive summary:

The test item dissolved in acetone was assessed for its potential to induce micronuclei in human lymphocytes in vitro according to OECD TG 487. Phase separation of the test item in the culture medium was observed at the end of treatment in Experiment I in the absence of S9 mix at 20.3 μg/mL and above, in the presence of S9 mix at 62.2 μg/mL and above and in Experiment II at 100 μg/mL. No cytotoxicity was observed up to the highest evaluated concentrations, which showed phase separation. In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed. Appropriate positive controls showed distinct increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test substance is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to phase separating concentrations.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

of July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room tempterature
- Stability under test conditions: given
- Solubility and stability of the test substance in the solvent/vehicle: DMSO
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: none

Target gene:

mutant histidine gene

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix was made from the livers of phenobarbital/beta-naphthoflavone induced rats. The S9 mix comprised 10 % S9 fraction and the standard cofactors.

Test concentrations with justification for top dose:

The assay was performed in two independent experiments with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:
Experiment I: 33; 100; 333; 1000; 2500; and 5000 μg/plate with and without metabolic activation (plate incorporation)
Experiment II: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate with and without metabolic activation (pre-incubation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent has been chosen according to its solubility properties and its relative nontoxicity to the bacteria (Maron et al.; 1981). All formulations will be prepared freshly before treatment and used within two hours of preparation.
The test item was well soluble in DMSO. Due to methodological problems in analytics (DMSO gave interferences on the HPLC UV detector that masked the test item peak and could not be separated) no stability/homogeneity testing was initated in DMSO. However, since the test item showed a 100 % solubility and stability in acetone (see: Data generated in a separate GLP study (Envigo Ref No: HH16TC) conducted at Envigo Research Ltd.) it can be assumed that the test item is stable in nature and stability in DMSO is given.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylenediamine (4-NOPD) for TA 1537 and TA 98 without S9 mix; 2-amineoanthracene (2-AA) for all strains with S9-mix activity

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); pre-incubation

Experimental Performance
For each strain and dose level, including the controls, three plates were used.
Experiment I (Plate Incorporation)
The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
- 500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
- 100 μL Bacteria suspension (precultures),
- 2000 μL Overlay agar
Experiment II and IIa (Pre-Incubation)
The following materials were mixed in a test tube and incubated at 37 °C for 60 minutes.
- 100 μL Test solution at each dose level (solvent control, or positive control),
- 500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
- 100 μL Bacteria suspension (cf. 3.4.3 Precultures),
After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube.
The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.
In parallel to each test a sterile control of the test item was performed and documented in the raw data. Therefore, 100 μL (experiment I) or 50 μL (experiment II and IIa) of the stock solution, 500 μL S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.


NUMBER OF REPLICATIONS: all plates were prepared in triplicate

DETERMINATION OF BACTERIOTOXICITY
- Method: gross appraisal of background growth

Acceptability of the Assay
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
• regular background growth in the negative and solvent control;
• the spontaneous reversion rates in the negative and solvent control are in the range of our historical data;
• the positive control substances should produce an increase above the threshold of twofold (strains TA 98, TA 100, and WP2 uvrA) or threefold (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control;
• a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

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

Positive controls validity:

valid

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

Positive controls validity:

valid

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

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

See Table 1 - Summary of results.
Precipitation was observed at 5000 µg/plate in all preincubation cultures and in the plate incorporation test cultures without metabolic activation. In the plate incorporation test cultures with metabolic activation precipiation was observed at 2500 and 5000 µg/plate. The undissolved particles had no influence on the data recording.
The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.
No substantial increase in revertant colony numbers of the five tester strains was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix).
Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

Table 1: Summary of results

Metabolic activation	test group	dose level (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Exp. I without / with	solvent (DMSO)	-	10 / 12	14 / 12	32 / 35	109 / 144	46 / 58
	test item	3	9 /12	14 / 13	27 / 39	105 / 110	52 / 57
	test item	10	11 / 10	11 / 12	27 / 36	117 / 118	51 / 60
	test item	33	11 / 14	11 / 10	26 / 41	113 / 127	45 / 54
	test item	100	8 / 11	10 / 13	24 / 44	122 / 128	52 / 63
	test item	333	9 / 11	9 / 13	25 / 46	112 / 120	51 / 47
	test item	1000	9 / 12	6 / 9	22 / 40	127 / 138	42 / 60
	test item	2500	10 / 15P	7 / 12P	31 / 34P	104 / 110P	51 / 57P
	test item	5000	13P /15P	9P / 13P	23P / 35P	103P / 120P	47P / 57P
Exp. II without / with	solvent (DMSO)	-	12 / 11	9 / 11	27 / 47	114 / 114	37 / 54
	test item	33	10 / 12	9 / 10	33 / 37	106 / 111	47 / 48
	test item	100	14 / 13	13 / 14	34 / 44	131 / 109	40 / 44
	test item	333	14 / 11	9 / 13	30 / 37	113 / 112	50 / 53
	test item	1000	13 / 11	10 / 13	25 / 40	111 / 101	37 / 54
	test item	2500	12 / 15	13 / 10	25 / 41	114 / 111	39 / 50
	test item	5000	12P / 14P	12P / 7PM	30P / 37P	113P / 118P	39P / 34PM

P precipitate

M manual count

Executive summary:

The test item was evaluated in an Ames Test on Salmonella typhimurium strains TA 1535, TA 100, TA, 1537, TA 98, and E. coli strain WP2 uvrA, performed according to OECD TG 471. Doses of up to and including 5000 µg per plate did not produce bacteriotoxic effects. Substance precipitation that occurred at 2500 to 5000 µg per plate had no influence on bacterial growth and recording.

No increase in revertant colony numbers in the five tester strains was observed following treatment with the test substance.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item induced no gene mutations in the presence and absence of metabolic activation.

Therefore, the test substance is considered to be not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The registered UVCB substance ‘Ester adipic acid and neodecanoic acid, 2-oxiranylmethyl ester’ contains 2,3-epoxypropyl neodecanoate (EC-No. 247-979-2 / CAS-No. 26761-45-5) as minor constituent  in a concentration of < 1 %. According to the disseminated REACH registration dossier 2,3-epoxypropyl neodecanoate is an epoxide that is self classified for human health effects by the registrant as skin sensitizer (Skin Sens 1, H317) and mutagen (Muta 2, H341).  The generic concentration limit triggering classification for both effects according to EU Regulation 1272/2008 is ≥ 1 %. This concentration limit is not reached in ‘Ester adipic acid and neodecanoic acid, 2-oxiranylmethyl ester’.

Based on ECHA disseminated data, with regard to mutagenicity positive effects were recorded for 2,3-epoxypropyl neodecanoate in several Ames tests (OECD 471), specifically in Salmonella strain TA 1535 in cultures with metabolic activation. The results for TA 1535 were repeatedly negative when no metabolic activation system was added. For the strain TA 100 positive and negative results were recorded with 2,3-epoxypropyl neodecanoate alone. Thus, this result is not as reliable as this for TA 1535. No specific information is given in the ECHA disseminated data with regard to the concentrations yielding increased mutation frequencies, but treatments included up to 5000 µg/plate. In addition to the Ames test, 2,3-epoxypropyl neodecanoat is described by the registrant as inducer of mutagenic effects in liver, kidney and bone marrow, but not in sperms of the Muta Mouse in vivo (OECD 488) following oral exposure. In all other performed tests (e.g. in vitro mammalian cell chromosomal aberration tests, in vivo Alkaline Elution Assay and in vivo UDS test) 2,3-epoxypropyl neodecanoate showed no mutagenic activity. Thus, classification with Muta 2 (H341) seems appropriate.

The test item ‘Ester adipic acid and neodecanoic acid, 2-oxiranylmethyl ester, Batch No. LEZF 140027’, contains free 2,3-epoxypropyl neodecanoate in a concentration of 0.5 % (w/w; Currenta Report No. 2015/0032/22 of December 2018). This test item gave a positive result in the Ames test (OECD 471), however, in Salmonella strain TA 1535 with metabolic activation at high, precipitating concentrations only. The fact that mutagenic effects with LEZF 140027 in the Ames test were observed at precipitating concentrations indicates that not the main component in LEZF 140027, but a minor/highly soluble compound in the UVCB composition is responsible for the positive result. The data point to 2,3-epoxypropyl neodecanoate as cause for mutagenicity.

To verify this hypothesis a test item with a considerable lower 2,3-epoxypropyl neodecanoate  content of 0.05 % (w/w; Currenta Report No. 2018/0089/01 of November 2018) was investigated. This test item ‘Ester adipic acid and neodecanoic acid, 2-oxiranylmethyl ester, Batch No. GCZ-2 9065-2’ was tested in the Ames test (OECD 471). The outcome of the Ames test for the test item ‘Batch No. GCZ-2 9065-2’ was clearly negative. The mutation frequency was not increased by treatment with test item ‘Batch No. GCZ-2 9065-2’ in any of the Salmonella strains with and without metabolic activation, including TA 1535.

With the outcome of these comparative tests it is self-evident that 2,3-epoxypropyl neodecanoat is responsible for the mutagenicity of test item ‘Ester adipic acid and neodecanoic acid, 2-oxiranylmethyl ester, Batch No. GCZ-2 9065-2’ in the Ames Test. Reducing the 2,3-epoxypropyl neodecanoat  content from 0.5 % in ‘Ester adipic acid and neodecanoic acid, 2-oxiranylmethyl ester, Batch No. LEZF 140027’ to 0.05 % in ‘Ester adipic acid and neodecanoic acid, 2-oxiranylmethyl ester, Batch No. GCZ-2 9065-2’ changes the result from positive to negative.

The test item ‘Ester adipic acid and neodecanoic acid, 2-oxiranylmethyl ester, Batch No. LEZF 140027’, containing 2,3-epoxypropyl neodecanoat  in a concentration of 0.5 % (w/w; Currenta Report No. 2015/0032/22 of December 2018), was also tested in mammalian cells in vitro for the induction of gene mutations (HPRT Test in Chinese hamster ovary cells following OECD 476) and of micronuclei (in human lymphocytes following OECD 487). Both tests revealed negative results.

These results are also in line with the data shown in the ECHA disseminated tool for 2,3-epoxypropyl neodecanoat. Negative results are reported for 2,3-epoxypropyl neodecanoat in the chromosomal aberration test on Chinese hamster ovary cells and on rat liver cells in vitro, as well as in the alkaline elution and UDS test in vivo.

In conclusion, the results of mutagenicity tests performed with the two batches of the registered substance ‘Ester adipic acid and neodecanoic acid, 2-oxiranylmethyl ester’ indicate that the minor constituent 2,3-epoxypropyl neodecanoat is responsible for the positive outcome of the Ames Test. Since the concentration of 2,3-epoxypropyl neodecanoat in ‘Ester adipic acid and neodecanoic acid, 2-oxiranylmethyl ester’ is below the concentration limit of ≥ 1 % no classification for mutagenicity is warranted.

Justification for classification or non-classification

The results of mutagenicity tests performed with the two batches of the registered substance ‘Ester adipic acid and neodecanoic acid, 2-oxiranylmethyl ester’ indicate that the minor constituent 2,3-epoxypropyl neodecanoat is responsible for the positive outcome of the Ames Test. Since the concentration of 2,3-epoxypropyl neodecanoat in ‘Ester adipic acid and neodecanoic acid, 2-oxiranylmethyl ester’ is below the concentration limit of ≥ 1 % no classification for mutagenicity is warranted.