Merginol 901

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic potential of the substance was examined in two Ames tests (OECD 471, GLP), an HPRT test (OECD 476, GLP) and an in vitro micronucleus study (OECD 487, GLP). The test substance has been shown to be negative in these in vitro assays.

No in vivo genotoxicity data on the substance is available. Information is therefore derived from two in vivo micronucleus assays (OECD 474) with structurally related substances. Both substances are negative in this assay.

Taking all these assays into consideration, no indications exist for a mutagenic potential of the test substance.

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:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch identification: 0009554927
CAS Name: Fatty acids, C16-18 and C18-unsatd., epoxidized, Me esters, polymers
Date of production/supply: Mar 2013
Physical state, appearance: Liquid, yellowish

Target gene:

his, trp

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 from phenobarbital and beta-naphthoflavone induced rat liver

Test concentrations with justification for top dose:

33 - 5200 ug/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 4-nitro-o-phenylenediamine and 2-aminoanthracene (with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 37°C for 48 - 72 hours
NUMBER OF REPLICATIONS:
in triplicate

NUMBER OF CELLS EVALUATED:
all revertants / colonies counted

DETERMINATION OF CYTOTOXICITY
Toxicity detected by a
• decrease in the number of revertants
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
• reduction in the titer is recorded for all test groups both with and without S9 mix in all experiments

Evaluation criteria:

The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control data range under all experimental conditions in at least two experiments carried out independently of each other.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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

Untreated negative controls validity:

not applicable

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

Untreated negative controls validity:

not applicable

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

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

SOLUBILITY: Precipitation of the test substance was found from about 2 600 μg/plate onward with and without S9 mix.
TOXICITY: No bacteriotoxic effect was observed under all test conditions

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1999-06-30 until 1999-07-02

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

E.coli WP2 uvrA or S. typhimurium TA 102 strain not tested

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1983)

Deviations:

yes

Remarks:

E.coli WP2 uvrA or S. typhimurium TA 102 strain not tested

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1992)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Physical state: liquid, yellow-brown
- Analytical purity: 100 %
- Lot/batch No.: 00018009
- Expiration date of the lot/batch: 2000-01-08
- Storage condition of test material: at room temperature

Target gene:

His

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 mix of Phenobarbital/ß-naphtoflavone induced rats.

Test concentrations with justification for top dose:

8, 40, 200, 1000 and 5000 µg/plate

Vehicle / solvent:

ethanol diluted in nutrient medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See any other information on materials and methods incl. tables

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation (experiment I & II)
DURATION: 48 hours at 37°C
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY: Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

Evaluation criteria:

A combination of the following criteria was considered as a positive result:
- The plate background of non-converted bacteria did not show any growth reduction versus the respective negative controls.
- The spontaneaus mutation rates of each tester strain per plate were within the characteristic spontaneaus mutation range.
- As a rule, the positive controls showed mutation rates exceeding the control values of TA 100 at least by the factor 2.0 and those of the other tester strains at least by the factor 3.0 E03).
- At more than one dose tested, the test substance caused at least a 2.0-fold increase in comparison with the negative controls in the tester strain TA 100. For the other tester strains, an increase in the mutation rate of more than 3.0 above the corresponding negative controls was considered positive).

Statistics:

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

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA1538, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2012 - Jan 2013

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)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT gene

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver microsome preparations (S9 mix)

Test concentrations with justification for top dose:

The highest concentration used in the pre-test (5.0 μL/mL) was chosen with regard to the current OECD guideline. The undiluted test item was used at the maximum concentration, to generate the lower concentrations (between 39.1 μg/mL and 2500.0 μg/mL). The cytotoxicity was evaluated in the presence (4 h treatment) and absence of metabolic activation (4 and 24 hours treatment). Relevant cytotoxic effects were observed at 312.5 μg/mL and above with metabolic activation. After 4 and 24 hours treatment without metabolic activation the cell growth was completely
inhibited down to the lowest concentration.
Phase separation occurred at the lowest to the highest concentration with and without metabolic activation following 4 and 24 hours treatment.
Based on cytotoxicity and phase separation noted in the pre-experiment, the individual concentrations of the main experiments were selected.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): cell density at 1st subcultivation divided by the cell density at 1st subcultivation of the corresponding control) x100
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4h and 24h
- Harvest time after the end of treatment (sampling/recovery times): approx. 7 days after treatment

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days after treatment

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative survival (RS)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The stained colonies with more than 50 cells were counted.

Evaluation criteria:

The gene murtation assay is considered acceptable if it meets the following criteria:
- The number of mutant colonies per 10^6 cells found in the solvent controls falls within the laboratory historical control data.
- The positive control substances should produce a significant increase in mutant colony frequencies.
- The cloning efficiency II (absolute value) of the solvent controls should exceed 50%.
- The data of this study comply with the above mentioned criteria.

Statistics:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

exposure period	S9 mix	concentration in µg/ml
4 hours	-	0.63	1.3	2.5	5.0	10.0
4 hours	+	5.0	10.0	20.0	40.0	80.0
24 hours	-	2.5	5.0	10.0	20.0	40.0
4 hours	+	2.5	5.0	10.0	20.0	40.0

Conclusions:

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. Therefore, the substance is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The test item was assessed for its potential to induce gene mutations at the
HPRT locus using V79 cells of the Chinese hamster.
The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
Phase separation was observed in experiment I at 80.0 μg/mL with and at 20.0 μg/mL and above without metabolic activation. In experiment II turbidity occurred at 40.0 μg/mL with and without metabolic activation.
Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures occurred in the first experiment at 10.0 μg/mL without metabolic activation. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency I or relative cell density was covered without metabolic activation. The concentration range of the experimental parts with metabolic activation and 24 hour treatment without metabolic activation was limited by the solubility of the test item in culture medium.
No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration. The mutation frequency did not exceed the historical range of solvent controls. The induction factor of three times the corresponding solvent control was not reached or exceeded at any test point.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency. A single significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the first culture of experiment I without metabolic activation. However, the trend was judged as biologically irrelevant as the mutation frequency did not exceed the threshold described above.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 6.5 up to 26.8 mutants per 106 cells; the range of the groups treated with the test item was from 3.7 up to 43.1 mutants per 10^6 cells.

EMS (150 μg/mL) and DMBA (1.1 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

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.
Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan 2020 - May 2021

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:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt Kaiser-Friedrich-Straße 7, 55116 Mainz

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test item No.: 12/0028-3
- Batch identification: 0019913739
- Expiry date: 01 Nov 2021
- Purity: ~ 96%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance preparations.
- Storage stability: The stability of the test substance under storage conditions
is guaranteed until 01 Nov 2021 as indicated by the sponsor, and the sponsor holds this responsibility. The test facility is organizationally independent from the
BASF SE sponsor division.
- Storage condition of test material: Room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The substance was dissolved in DMSO. The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. To achieve a solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. The further concentrations were diluted from according to the planned doses. All test substance solutions were prepared immediately before administration.

FORM AS APPLIED IN THE TEST (if different from that of starting material):
- dissolved in the vehicle DMSO

OTHER SPECIFICS
- Physical state, appearance: liquid, viscous, yellowish, clear

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: blood samples from healthy non-smoking donors not receiving medication; fresh blood was collected from a single donor for each experiment
- Suitability of cells: The lymphocytes of each donor have previously shown to respond well to stimulation of proliferation with phytohemagglutinin (PHA) and to the used positive control substances.
- Sex, age and number of blood donors: Experiment I: one male donor (31 years old); Experiment II: one male donor (27 years old)
- Whether whole blood or separated lymphocytes were used if applicable: whole blood

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
• Culture medium: DMEM / Ham's F12 (1:1) (DMEM/F12) medium containing stable glutamine supplemented with 10% [v/v] fetal calf serum (FCS).
• Treatment medium (4 hours treatment without and with S9 mix): DMEM/F12 medium containing stable glutamine
• Treatment medium (20 hours treatment without S9 mix): DMEM/F12 medium containing stable glutamine and supplemented with 10% [v/v] FCS.

All media were supplemented with:
• 1% [v/v] penicillin/streptomycin (final concentration 100 μg/mL)
• 1% [v/v] HEPES buffer (1 M)
For the stimulation the medium was supplemented with:
• 0.5% [v/v] Phytohemagglutinin (PHA, stock solution 0.6 mg/mL, final conc.
3 μg/mL)
• 0.5% [v/v] sodium heparin (25000 IE)

For the Cytochalasin B (Cyt B) treatment the medium was supplemented with:
• 30 μL Cyt B (Cyt B, stock solution: 2 mg/mL in DMSO, final conc.: 6 μg/mL)

Cytokinesis block (if used):

The commonly applied cytokinesis block method using the actin polymerisation inhibitor cytochalasin B (Cyt B) was used to discriminate between proliferating and non-proliferating cells.
- 30 μL Cyt B (Cyt B, stock solution: 2 mg/mL in DMSO, final conc: 6 μg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: BASF SE; in an AAALAC approved laboratory in accordance with the German Animal Welfare Act and the effective European Council Directive
- method of preparation of S9 mix: At least 5 male Wistar rats [Crl:WI(Han)] received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally each on three consecutive days. 24 hours after the last administration, the rats were sacrificed, and the induced livers were weighed, washed and homogenized. After centrifugation of the homogenate at 9000 x g, portions of the supernatant (S9 fraction) were stored at -70°C to -80°C. The S9 mix was prepared freshly prior to each experiment (1 part of S9 fraction is mixed with 9 parts of S9 supplement (cofactors))

Test concentrations with justification for top dose:

Following the requirements of the current OECD Guideline 487 a test substance with defined composition should be tested up to a maximum concentration of 2 mg/mL, 2 μL/mL or 10 mM, whichever is the lowest. When the test substance is not of defined composition, e.g. substance of unknown or variable composition, complex reaction products or biological materials (socalled UVCBs), or environmental extracts, the top concentration should be higher to increase
the concentration of each of the components (e.g. 5 mg/mL). In case of toxicity, the top concentration should produce reduction of the proliferation index (CBPI) to 45 ± 5% (leading to a cytostasis increase of 55 ± 5%) of the concurrent vehicle control. For relatively insoluble test substances only one concentration should be tested showing turbidity or precipitation in culture medium at the end of exposure period.

In this study the concentrations are given as rounded values by using a dilution factor of 1.8, in general. At least three concentrations were evaluated to detect a possible dose-response relationship. At least 2 cultures were prepared per test group (referred to as A and B), and at least 1000 cells per culture were evaluated for the occurrence of micronucleated cells.

Vehicle / solvent:

- vehicle: DMSO
- Justification for choice of vehicle: Due to the limited solubility of the test substance in culture medium, DMSO was used as vehicle, which has been demonstrated to be suitable in the in vitro cytogenetic assay and for which historical control data are available.
- The final concentration of the vehicle DMSO in culture medium was 1% (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

The vehicle control cultures with and without S9 mix contained the vehicle selected for the test substance in the same volume and concentration as used in the test cultures.

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF TREATMENT/ EXPOSURE: :
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
Exp. I (With and without S9 mix); Exp. II (Without S9-mix)
- Stimulating time: 48h (Exp. I + Exp. II)
- Exposure duration: 4h (Exp. I); 20 h (Exp. II)
- Harvest time after the end of treatment (Recovery time): 16h (Exp. I)
- Harvest time: 20h (Exp. I + Exp. II)

FOR MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Cytochalasin B (30 μL Cytochalasin B (Cyt B, stock solution: 2 mg/mL in DMSO, final concentration: 6 μg/mL)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- cells were centrifugated (900 g, 5 min) and washed with HBSS
- cells were centrifuged (900 g, 5 min) and suspended in 0.0375 M KCl (37°C)
- incubation for 20 min at 37°C
- cells were fixed by adding of fixative (19 parts methanol and 1 part acetic acid)
- cells were centrifuged (900 g, 5 min, 4°C) and suspended in fresh fixative
- incubation for 20 min at 4°C
- fixation step will be repeated twice
- cells can be centrifugated directly (900 g, 5 min, 4°C), suspended in 1-2 mL fresh fixative and spread on slides
- slides were dipped in deionized water
- cells were pipetted on the slide and fixed by passing through a flame

- cells were stained with May-Grünwald (3 min) and 10% [v/v] Giemsa (in Titrisol, pH 7.2,10 min) and mounted

NUMBER OF CELLS EVALUATED:
- at least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group, were evaluated for the occurrence of micronuclei

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976).
- The diameter of the micronucleus was less than 1/3 of the main nucleus
- The micronucleus was not linked to the main nucleus and was located within the cytoplasm of the cell.
- Only binucleated cells were scored.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: determination of CBPI (Cytokinesis-block proliferation index) in 500 cells per culture (1000 cells per test group); A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis. CBPI = ((Mononucleate cells x 1) + (Binucleate cells x 2) + (Multinucleate cells x 3)) / Total number of cells

Evaluation criteria:

A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in the number of micronucleated cells was obtained.
• A dose-related increase in the number of cells containing micronuclei was observed.
• The number of micronucleated cells exceeded both the concurrent vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).

A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the number of cells containing micronuclei was observed under any experimental condition.
• The number of micronucleated cells in all treated test groups was close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit).

Statistics:

An appropriate statistical analysis was performed. The proportion of cells containing micronuclei was calculated for each test group. A comparison of the micronucleus rates of each test group with the concurrent vehicle control group was carried out for the hypothesis of equal proportions (i.e. one-sided Fisher's exact test, BASF SE). If the results of this test were statistically significant compared with the respective vehicle control (p ≤ 0.05), labels (s) were printed in the tables. In addition, a statistical trend test (SAS procedure REG) was performed to assess a possible dose-related increase of micronucleated cells. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report. The dependent variable was the number of micronucleated cells and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05. However, both, biological and statistical significance were considered together.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: pH values were not relevantly influenced by test substance treatment
- Data on osmolality: Osmolality was not relevantly influenced by test substance treatment
- Precipitation and time of the determination::
In this study, in the absence of S9 mix, test substance precipitation was observed macroscopically in culture medium at the end of treatment at 8.6 μg/mL and above in the 1st Experiment and macroscopically and microscopically at 20.00 μg/mL in the 2nd Experiment. In the presence of S9 mix precipitation was observed macroscopically and microscopically at 27.8 μg/mL and above in the 1st Experiment.

CYTOTOXICITY
- In this study, no reduced proliferative activity was observed either after 4 hours exposure interval in the absence and presence of S9 mix or after 20 hours continuous test substance treatment without S9 mix in the test groups scored for cytogenetic damage.

STUDY RESULTS
1st Experiment:
- without S9-Mix: values (0.3 – 0.6% micronucleated cells) were within the range of the 95% control limit of the historical negative control data range
- with S9-Mix: values (0.4% micronucleated cells) were within the range of the 95% control limit of the historical negative control data range
- No statistical significance compared to the vehicle control value

2nd Experiment:
- values (0.4 – 0.6% micronucleated cells) were within the range of the 95% control limit of the historical negative control data range
- No statistical significance compared to the vehicle control value

None of the data in the two experimens showed a positive dose response as assessed by a trend analysis.

- positive control substances MMC, Colchicine and CPA induced micronucleus frequencies in all independently performed experiments (compatible to the historical positive control data range)

HISTORICAL CONTROL DATA
- Positive historical control data: see tables 6 and 7
- Negative (solvent/vehicle) historical control data: see tables 8 and 9

Table 4: Summary table - experimental parts without S9 mix

Exp.	Exposure/ Recovery/ Preparation interval	Test groups [µg/mL]	S9 mix	Prec.*	Micro- nucleated cells** [%]	Cytotoxicity Proliferation index cytostasis [%]
1	4/16/20	Vehicle control (*1)	-	n.d.	0.6	0
		0.8	-	-	n.d.	-2.3
		1.5	-	-	n.d.	-4.7
		2.6	-	-	0.6	-3.1
		4.8	-	-	0.3	1.2
		8.6	-	+	0.4	-2.9
		15.4	-	+	n.p.	n.p.
		27.8	-	+	n.p.	n.p.
		50.0	-	+	n.p.	n.p.
		Positive control (*2)	-	n.d.	9.9 (S)	25.8
2	20/0/20	Vehicle control (*1)	-	n.d.	0.6	0
		0.33	-	-	n.d.	7.2
		0.59	-	-	n.d.	5.6
		1.06	-	-	n.d.	0.5
		1.91	-	-	n.d.	7.0
		3.43	-	-	n.d.	0.9
		6.17	-	-	0.4	3.9
		11.11	-	-	0.4	-0.6
		20.00	-	+	0.6	7.1
		Positive control (*3)	-	n.d.	6.7 (S)	21.8
		Positive control (*4)	-	n.d.	3.2 (S)	23.1

* Precipitation in culture medium at the end of exposure period (microscopically and macroscopically)
** Relative number of binucleated cells with micronuclei per 2000 cells scored per test group
(S) Frequency statistically significantly higher than corresponding control values
n.d. Not determined
n.p. Not continued due to strong test substance precipitation
(*1) DMSO 1% (v/v) (*2) MMC 0.31 μg/mL
(*3) MMC 0.04 μg/mL (*4) Col 0.05 μg/mL

Table 5: Summary table - experimental part with S9 mix

Exp.	Exposure/ Recovery/ Preparation interval	Test groups [µg/mL]	S9 mix	Prec.*	Micro- nucleated cells** [%]	Cytotoxicity Proliferation index cytostasis [%]
1	4/16/20	Vehicle control (*1)	+	n.d.	0.4	0
		0.8	+	-	n.d.	1.2
		1.5	+	-	n.d.	2.5
		2.6	+	-	n.d.	3.2
		4.8	+	-	n.d.	0.3
		8.6	+	-	0.2	4.3
		15.4	+	-	0.6	6.6
		27.8	+	+	0.6	7.4
		50.0	+	+	n.p.	n.p.
		Positive control (*5)	+	n.d.	2.9 (S)	13.7
		Positive control (*6)	+	n.d.	4.3 (S)	26.1

* Precipitation in culture medium at the end of exposure period (microscopically and macroscopically)
** Relative number of binucleated cells with micronuclei per 2000 cells scored per test group
(S) Frequency statistically significantly higher than corresponding control values
n.d. Not determined
n.p. Not continued due to strong test substance precipitation
(*1) DMSO 1% (v/v) (*5) CPA 2.50 μg/mL (*6) CPA 3.50 μg/mL

Table 6: HISTORICAL POSITIVE CONTROL DATA; WITHOUT S9 MIX, Mitomycin C and Colchicin; Period: April 2018 – November 2020

Micronucleated cells [%]
Exposure period	4 hrs	20 hrs	20 hrs
Substance and concentration	MMC 0.31 µg/mL	MMC 0.04 µg/mL	Col 0.05 µg/mL
Mean	9.1	4.1	4.0
Minimum	3.8	2.1	2.4
Maximum	15.1	7.1	7.2
Standard Deviation	2.99	0.93	1.05
No. of Experiments	56	48	45

Table 7: HISTORICAL POSITIVE CONTROL DATA; WITH S9 MIX, Cychlophosphamid; Period: April 2018 – November 2020

Micronucleated cells [%]
Exposure period	4 hrs
Substance and concentration	CPA 2.50 µg/mL
Mean	3.6
Minimum	1.9
Maximum	6.9
Standard Deviation	1.12
No. of Experiments	55

Table 8: HISTORICAL NEGATIVE CONTROL DATA; WITHOUT S9 MIX, ALL VEHICLES; Period: April 2018 – November 2020

Micronucleated cells [%]
Exposure period	4 hrs	20 hrs
Mean	0.5	0.5
Minimum	0.1	0.2
Maximum	1.0	1.2
Standard Deviation	0.18	0.17
95% Lower Control Limit	0.2	0.2
95% Upper Control Limit	0.9	0.9
No. of Experiments	59	54

Table 9: HISTORICAL NEGATIVE CONTROL DATA; WITH S9 MIX, ALL VEHICLES; Period: April 2018 – November 2020

Micronucleated cells [%]
Exposure period	4 hrs
Mean	0.6
Minimum	0.3
Maximum	1.3
Standard Deviation	0.24
95% Lower Control Limit	0.1
95% Upper Control Limit	1.1
No. of Experiments	59

Conclusions:

Under the experimental conditions chosen here, the conclusion is drawn that
the test substance has no potential to induce micronuclei (clastogenic and/or aneugenic activity) under in vitro conditions in primary human lymphocytes in the absence and the presence of metabolic activation.

Executive summary:

The test substance was assessed for its potential to induce micronuclei in primary human lymphocytes in vitro (clastogenic or aneugenic activity). Two independent experiments were carried out, with and without the addition of liver S9 mix from phenobarbital- and b-naphthoflavone induced rats (exogenous metabolic activation).
According to an initial range-finding solubility test for the determination of the experimental doses the following concentrations were tested. Test groups printed in bold type were evaluated for the occurrence of micronuclei:

1st Experiment

4 hours exposure, without S9 mix
0; 0.8; 1.5; 2.6; 4.8; 8.6; 15.4; 27.8; 50.0 μg/mL
4 hours exposure, with S9 mix
0; 0.8; 1.5; 2.6; 4.8; 8.6; 15.4; 27.8; 50.0 μg/mL

2nd Experiment
20 hours exposure, without S9 mix
0; 0.33; 1.59; 1.06; 1.91; 3.43; 6.17; 11.11; 20.00 μg/mL

A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group.
In this study, dimethyl sulfoxide (DMSO) was selected as vehicle. The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for primary human lymphocytes. The positive control substances, Mitomycin C (MMC), Colchicin and Cyclophosphamide (CPA), led to the expected increase in the number of cells containing micronuclei.
In this study, no cytotoxicity indicated by reduced proliferation index (CBPI) was observed up to the highest applied test substance concentration.
On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei  either without S9 mix or after adding a metabolizing system.
Thus, under the experimental conditions described, the test substance is considered to have no chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in primary human lymphocytes in the absence and the presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo genotoxicity data on the substance is available. Information is therefore derived from two in vivo micronucleus assays (OECD 474) with structurally related substances. Both substances are negative in this assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1985-07-08 until 1985-08-01

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: micronucleus assay

Specific details on test material used for the study:

- Name of test material: LA 990
- Substance type: organic
- Physical state: viscous liquid, beige-coloured
- Lot/batch No.: 37-4-222
- Stability under test conditions: stable under test conditions
- Storage condition of test material: at room temperature

Species:

mouse

Strain:

other: CWF 1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, 4799 Borchen, FRG
- Age at study initiation: appox. 8 weeks
- Weight at study initiation: 17 -30 g
- Housing: Male mice individually in macrolon cages type I; female mice up to 4 animals per cage in macrolon cages type II.
- Diet: Altromin No. 1324, 10 mm pellet diameter, supplied from Altrogge Spezialfutter.
- Water: Drinking water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21°C
- Humidity: 30 - 70 %
- Photoperiod: 12 hours dark/ light cycle

Route of administration:

oral: gavage

Vehicle:

- Vehicle: distilled water with carboxymethyl cellulose 2% and cremophore 0.5 %
- Justification for choice of vehicle: preparation of a suspension for adequate administration
- Amount of vehicle: 20 mL/kg

Frequency of treatment:

once

Post exposure period:

10000 mg/kg bw: 24, 48, and 72 h
5000 mg/kg bw: 24 h
1000 mg/kg bw: 24 h

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

Dose / conc.:

10 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

7 animals per sex per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamid 10 mg/kg bw intraperitoneal (seven male and female animals)

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
24, 48 and 72 h after the administration the animals were sacrificed with an overdose of carbonmonoxide. Bone marrow was extracted from the femur and prepared adequately. Three slides were prepared for each animal. The slides were air-dried at least overnight and then stained with Giemsa according to modification of Gollapudi and Kamra. The slides were fixed in 100 % methanol for 5 min., then rinsed twice in distilled water and thereafter stained in Giemsa solution. After air -drying the back side was cleaned, necessary with ethanol and then dipped for 3 min. in xylol.

METHOD OF ANALYSIS:
From the three slides prepared per animal, one slide was chosen and randomly scored. The slides of five male and five female animals per treatment group were scored microscopically at a magnification of 1000. The number of micronucleated cells was counted in 1000 polychromatic erythrocytes/ per animal. The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Averages and standard deviations were calculated after decoding the complete scoring results.

Evaluation criteria:

- A micronucleus test is considered acceptable if it meets the following criteria: The positive control substance induced a statistical significant increase in the frequency of micronucleated polychromatic erythrocytes - the incidence of micronuclei should reasonably fall within the historical control data range of the laboratory.
- A test item is considered positive in the micronucleus test if it induced a biologically as well as statistically significant increase (p < 0.05) in the frequency of micronuclei at any dose or at any sampling either in the male or in the female groups.
- A test substance is considered negative in the micronucleus test if none of the tested doses or sampling times showed a statistically significant (p < 0.05) increase in the incidence of micronuclei, neither in male nor in female groups.

Statistics:

Statistical analysis of data was performed by calculating the statistical significance versus negative controls with the aid of the tables of Kastenbaum and Bowman. The preceding criteria are not absolute and other factors may influence the final evaluation decision.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
At the highest test item concentration of 10000 mg/kg bw, no effect on the numerical ratio between polychromatic and normchromatic erythrocytes was observed. Furthermore, the test item did not cause a significantly elevated number of micronucleated polychromatic erythrocytes when compared to the concurrent negative control. Thus the lower test item concentrations of 5000 and 1000 mg/kg bw were not microscopically evaluated. Besides the death of one animal in the 5000 mg/kg bw does group, not toxic effects were observed. The death of this animal could not be related to the test item exposure.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1989/10/17- 1990/01/30

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(draft)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: micronucleus assay

Specific details on test material used for the study:

- Name of test material: Stabiol VP 1711
- Substance type: Fatty acid C18 and C18-unsaturated epoxidized, ester with ethylene glycol
- Physical state: white powder
- Lot/batch No.: Nr. 41, 041/8/055
- Stability under test conditions: not given
- Storage condition of test material: at room temperature, at 20 °C the test item only hydrolyses 1 % /day

Species:

mouse

Strain:

other: CFW 1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, 4799 Borchen, Germany
- Age at study initiation: Aged 7 to 8 weeks
- Weight at study initiation: Between 20.7 g and 32.8 g for the males; between 19.5 g and 24.8 g for the females.
- Housing: Male mice were housed individually macrolon cages type I; female mice were housed in groups up to three in macrolon cages type II.
- Diet: Altromin No. 1314 , 10 mm pellet diameter, supplied from Altrogge Spezialfutter.
- Water: Drinking water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21°C
- Humidity: 30 - 70 %
- Photoperiod: 12 hours dark/ light cycle

Route of administration:

oral: gavage

Vehicle:

- Vehicle: arachis oil
- Justification for choice of solvent/vehicle: solubility of the test item and not toxic to rats.
- Concentration of test material in vehicle: 3000, 4000, 5000 mg/kg bw for the dose range finding study, 5000 mg/kg bw for the main trial.
- Amount of vehicle: 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Solutions were prepared on the day of administration. The stability of CP at room temperature is good. In aqueous solution at 20°C only 1 % of CP is hydrolyzed per day.

Duration of treatment / exposure:

24, 48 or 72 hours

Frequency of treatment:

once

Post exposure period:

none

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

dose range finder: 3 groups of 2 males and 2 females
main trial: 5 groups of 6 males and 6 females

Control animals:

yes, concurrent vehicle

Positive control(s):

- Justification for choice of positive control: Cyclophosphamide was selected due to its genotoxic potential after metabolic activation and is recommended as positive control in the guideline.
- Route of administration: Intraperitoneal administration
- Doses/ concentrations: 10 mg/kg bw in bidistilled water

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: based on a dose range finding study

TREATMENT AND SAMPLING TIMES: The animals of all treatment groups received a single dose at a volume of 10 mL/kg bw

DETAILS OF SLIDE PREPARATION:
Bone marrow smears from both femurs of each animal were prepared. The slides were air-dried at least overnight and then stained with Giemsa according to modification of Gollapudi and Kamra. The slides were fixed in 100 % methanol for 5 min., then rinsed twice in bidistilled water and thereafter stained in Giemsa solution. After air -drying the back side was cleaned, necessary with ethanol and then dipped for 3 min. in xylol.

METHOD OF ANALYSIS:
From the three slides prepared per animal, one slide was chosen and randomly scored. The slides of five male and five female animals per treatment group were scored microscopically at a magnification of 1000. The number of micronucleated cells was counted in 1000 polychromatic erythrocytes/ per animal. The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Averages and standard deviations were calculated after decoding the complete scoring results.

Evaluation criteria:

- A micronucleus test is considered acceptable if it meets the following criteria: the positive control substance induced a statistical significant increase in the frequency of micronucleated polychromatic erythrocytes- the incidence of micronuclei should reasonably fall within the historical control data range of the laboratory.
- A test item is considered positive in the micronucleus test if it induced a biologically as well as statistically significant increase (p < 0.05) in the frequency of micronuclei at any dose or at any sampling either in the male or in the female groups.
- A test substance is considered negative in the micronucleus test if none of the tested doses or sampling times showed a statistically significant (p < 0.05) increase in the incidence of micronuclei, neither in male nor in female groups.

Statistics:

Statistical analysis of data was performed by calculating the statistical significance versus negative controls with the aid of the tables of Kastenbaum and Bowman. The preceding criteria are not absolute and other factors may influence the final evaluation decision.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: the dose range finding study determined 5000 mg/kg bw as final concentration for the main trial.
- Clinical signs of toxicity in test animals: no further signs of toxicity were observed.
- Rationale for exposure: based on the non toxic effect in the dose range finding study.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: no
- Ratio of PCE/NCE: mean of animal 1 - 5 at a dose of 5000 mg/kg bw = 1.24
- Statistical evaluation: The investigated test item did not induce a statistically significant (time-dependent) increase in the number of micronucleated polychromatic erythrocytes in the bone marrow of male or female mice.

Additional information

The mutagenic potential of the substance was examined in two Ames tests (OECD 471), an HPRT test (OECD 476) and an in vitro MNT study (OECD 487). None of the tests gave any indication for a mutagenic potential.

No in vivo genotoxicity data on the substance is available. Information is therefore derived from two in vivo micronucleus assays (OECD 474) with structurally related substances. Both substances are negative in this assay.

The studies are summarized below.

Ames test:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. Tester strains TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA were incubated at concentrations of 33 μg - 5 200 μg/plate (SPT) 33 μg - 5 200 μg/plate (PIT). A Standard plate test (SPT) as well as a preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats) was performed. Precipitation of the test substance was found from about 2 600 μg/plate onward with and without S9 mix. No bacteriotoxic effect was observed under all test conditions. A biologically relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation (BASF 2014).

Ames test:

The test item was tested for its mutagenic potential in two independent experiments according to the OECD guideline 471 at a concentration range of 8.0 - 5000 µg/plate. The following Salmonella typhimurium strains were used: TA 1535, TA 1537, TA 1538, TA 98 and TA 100. The test substance was tested in the absence and presence of S9 -mix which was obtained from phenobarbital/ ß-naphtoflavone treated rats. In both test series the plates were incubated for 48 h at 37°C. The findings show that the test item did not induce any reverse mutations in the absence or presence of S9 mix. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, that the test article did not induce point mutations by base-pair changes or frame shifts in the genome of the strains used. Therefore, the test item is considered to be not mutagenic in this Salmonella typhimurium reverse mutation assay (Henkel 1999).

HPRT test:

 

The test item was assessed for its potential to induce gene mutations at the
HPRT locus using V79 cells of the Chinese hamster.
The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
Phase separation was observed in experiment I at 80.0 μg/mL with and at 20.0 μg/mL and above without metabolic activation. In experiment II turbidity occurred at 40.0 μg/mL with and without metabolic activation.
Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures occurred in the first experiment at 10.0 μg/mL without metabolic activation. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency I or relative cell density was covered without metabolic activation. The concentration range of the experimental parts with metabolic activation and 24 hour treatment without metabolic activation was limited by the solubility of the test item in culture medium.
No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration. The mutation frequency did not exceed the historical range of solvent controls. The induction factor of three times the corresponding solvent control was not reached or exceeded at any test point.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency. A single significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the first culture of experiment I without metabolic activation. However, the trend was judged as biologically irrelevant as the mutation frequency did not exceed the threshold described above.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 6.5 up to 26.8 mutants per 106 cells; the range of the groups treated with the test item was from 3.7 up to 43.1 mutants per 10^6 cells.

EMS (150 μg/mL) and DMBA (1.1 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

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.
Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

In vitro micronucleus study (MNT):

The test substance was assessed for its potential to induce micronuclei in primary human lymphocytes in vitro (clastogenic or aneugenic activity). Two independent experiments were carried out, with and without the addition of liver S9 mix from phenobarbital- and b-naphthoflavone induced rats (exogenous metabolic activation). According to an initial range-finding solubility test for the determination of the experimental doses the following concentrations were tested. Test groups printed in bold type were evaluated for the occurrence of micronuclei:

1st Experiment

4 hours exposure, without S9 mix
0; 0.8; 1.5; 2.6; 4.8; 8.6; 15.4; 27.8; 50.0 μg/mL
4 hours exposure, with S9 mix
0; 0.8; 1.5; 2.6; 4.8; 8.6; 15.4; 27.8; 50.0 μg/mL

2nd Experiment
20 hours exposure, without S9 mix
0; 0.33; 1.59; 1.06; 1.91; 3.43; 6.17; 11.11; 20.00 μg/mL

A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group. In this study, dimethyl sulfoxide (DMSO) was selected as vehicle. The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for primary human lymphocytes. The positive control substances, Mitomycin C (MMC), Colchicin and Cyclophosphamide (CPA), led to the expected increase in the number of cells containing micronuclei. In this study, no cytotoxicity indicated by reduced proliferation index (CBPI) was observed up to the highest applied test substance concentration. On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei  either without S9 mix or after adding a metabolizing system. Thus, under the experimental conditions described, the test substance is considered to have no chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in primary human lymphocytes in the absence and the presence of metabolic activation.

Micronucleus test in vivo (CAS 151661-88-0):

The substance was tested in vivo for its DNA damaging potential in a micronucleus test. The study was performed in albino mice of the strain CFW1 following the draft OECD 474 (initial assessment). Six male and six female mice were used per experimental group. The test item was administered by oral gavage, using arachis oil as vehicle. Based on the results of the dose range finding study a dosage of 5000 mg/kg bw was applied in the main trial. The test item was administered once, and six male and six female animals per group were sacrificed at time intervals of 24, 48 and 72 hours after the administration. Bone marrow smears from both femurs of each animal were prepared and the bone marrow preparations from the first five animals of each group were examined for micronuclei in 1000 polychromatic erythrocytes of each animal. A vehicle control and MMS as positive control were included in this study. No statistically significant enhanced mean values of micro-nucleated cells in polychromatic erythrocytes were observed at all examined time intervals compared to negative control values. Toxic effects as indicated by an enhanced mortality rate or by a reduction in the ratio of polychromatic to normochromatic erythrocytes (PCE/ NCE) were not noticed. Under the experimental conditions selected the test item did not induce chromosomal mutations in the bone marrow of mice. The test item is considered to have no DNA damaging potential in mice in vivo (Henkel 1990).

  

Micronucleus test in vivo (Oleic methyl ester, epoxidized, reaction products with glycerol):

The structural analogue substance (read across) was tested in the micronucleus test for the incidence of chromosomal aberration in male and female mice according to OECD guideline 474. Seven animals /sex were treated with 10000 mg/kg bw which was suspended in water with the aid of CMC (2 %) and cremohore (0.5 %) and was administered by oral gavage. Bone marrow smears from both femurs were prepared from each animal 24, 48 and 72 hours after the administration of the product and evaluated for micronuclei. In addition, one treatment group obtained an extra dosages of 5000 mg/kg bw and one group of 1000 mg/kg bw 24 h after the first application by oral gavage. The group treated with 10000 mg/kg bw showed a micronucleated cell count which was comparable to the current controls. Therefore the additional dosages of 5000 and 1000 mg/kg bw were not evaluated. It was concluded from the obtained results that there was no evidence of mutagenic potential of the test item, when administered orally in this in vivo micronucleus test (Henkel 1985).

Justification for classification or non-classification

The substance was not mutagenic in the Ames test, in the HPRT assay in V79 cells and in the in vitro micronucleus assay. Furthermore, structural analogues were not clastogenic in vivo in the mouse micronucleus test. Therefore, based on the available data, classification for genetic toxicity is not warranted in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.