2-(3,5-dimethylhex-3-en-2-yloxy)-2-methylpropyl cyclopropanecarboxylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames test: not mutagenic in Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli WP2 uvrA with and without metabolic activation (OECD 471, GLP, K, rel. 1).

- in vitro cytogenicity / chromosome aberration study in mammalian cells : non-clastogenic without activation, weak clastogenicity with metabolic activation (OECD 473 , GLP, K, rel.1)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 25 to August 05, 2008.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other:

Remarks:

GLP study conducted according to OECD test Guideline No. 473 with the following deviations: tested too high with metabolic activation, excessive variability between duplicate cultures, no dose response and some individual cultures within HCD.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Adopted July 21, 1997.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Dated May 30, 2008.

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

German Statement of GLP compliance. Inspected on 02 September 2006 /Signed on 19 January 2007.

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Storage conditions: In the refrigerator at +2 to +8°C, protected from light

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 cell line supplied by Laboratory for Mutagenicity Testing, LMP,
Technical University Darmstadt, D-64287 Darmstadt, Germany were stored in liquid nitrogen in the cell bank of RCC Cytotest Cell Research GmbH allowing the repeated use of the same cell culture batch in experiments.
- Suitability of cells: Before freezing each batch was screened for mycoplasm contamination and checked for karyotype stability.
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37° C in 80 cm² plastic flasks (GREINER, D-72632 Frickenhausen). About 5 x 10e5 cells per flask were seeded into 15 mL of MEM (Minimal Essential Medium; SEROMED; D-12247 Berlin) supplemented with 10 % fetal calf serum (FCS; PAA Laboratories GmbH, D-35091 Cölbe). The cells were subcultured twice weekly. The cell cultures were incubated at 37° C in a humidified atmosphere with 1.5 %
carbon dioxide (98.5 % air).
- Modal number of chromosomes: The cells have a modal chromosome number of 22.
- Periodically checked for karyotype stability: YES; The cells have a stable karyotype.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: in-house preparation by RCC Cytotest Cell Research
- method of preparation of S9 mix: 8-12 weeks old male Wistar rats (from Harlan) were induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; D-22335 Hamburg) and β-Naphthoflavone p.o. (Aldrich, 82024Taufkirhcen) each on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCl solution (1:3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at -80° C. Small numbers of the ampoules were kept at -20°C for up to one week. The protein concentration was 33.6 mg/mL (Lot. no. 080508).
- concentration or volume of S9 mix and S9 in the final culture medium: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the following concentrations: 8 mM MgCl2; 33 mM KCl; 5 mM glucose-6-phosphate; 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During the experiment the S9 mix was stored in an ice bath.

Test concentrations with justification for top dose:

- Main Experiment (18-hour/4-hour without S9-mix): 10.8, 21.6, 43.3, 86.6, 173.1, 346.3, 692.5, 1385.0 (p) and 2770.0 (p) µg/mL
- Main Experiment, repeated due to strong cytotoxicity (18-hour/4-hour* without S9-mix): 10.8, 21.6***, 43.3***, 86.6***, 173.1, 346.3, 692.5, 1385.0 (p) and 2770.0 (p) µg/mL
- Main Experiment, repeated due to missing cytotoxicity (18-hour/4-hour** without S9-mix): 0.3, 0.7, 1.4, 2.7, 5.4, 10.8, 21.6, 43.3, 86.6 µg/mL.

- Main Experiment (18-hour/4-hour with S9-mix): 10.8, 21.6, 43.3, 86.6, 173.1, 346.3***, 692.5***, 1385.0*** (p) and 2770.0*** (p) µg/mL.

*: was repeated due to strong cytotoxicity
** was repeated due to missing cytotoxicity
*** Evaluated experimental points
P: phase separation

Detailed explanation for choice of top dose are explained on the Table 7.6.1/1: Dose selection.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol (E. Merck, 64293 Darmstadt, Germany, purity: 99.8 %, Lot no.: K38541383811 and K37361983723). The final concentration ofethanol in the culture medium was 0.5 % (v/v).
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

900 µg/mL (7.2 mM). Dissolved in nutrient medium.

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

1.4 µg/mL (5.0 µM). Dissolved in saline (0.9 % NaCl w/v)

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): The cells were seeded into Quadriperm dishes (Heraeus, 63450 Hanau, Germany) that contained microscopic slides (at least 2 chambers per dish and test group). In each chamber x 10000 - 60000 cells were seeded with regard to the preparation time
- Test substance added in medium (serum-free medium with S9-mix and MEM with 10 % FCS without S9-mix)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration: 4 hours with S9-mix and without S9-mix.
- Expression time (cells in growth medium): 14 hours with and without S9-mix.
- Fixation time (start of exposure up to fixation or harvest of cells): 18 hours

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Colcemid was added (0.2 Og/mL culture medium) to the cultures 15.5 hours after the start of the treatment. The cells on the slides were treated 2.5 hours later, in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37 °C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts, respectively). Per experiment two slides per group were prepared. After preparation the cells were stained with Giemsa (E. Merck, 64293 Darmstadt, Germany).
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): at least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. In each experimental group two parallel cultures were set up.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Evaluation of the cultures was performed using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates.
- Determination of polyploidy: yes
- Determination of endoreplication: yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: mitotic index (MI)
- Any supplementary information relevant to cytotoxicity: for evaluation of cytotoxicity indicated by reduced cell numbers two additional cultures per test item and solvent control group, not treated with colcemid, were set up in parallel. These cultures were stained after 18 hrs in order to determine microscopically the cell number within 10 defined fields per coded slide. The cell number of the treatment groups is given in percentage compared to the respective solvent control.

Rationale for test conditions:

This in vitro test was performed to assess the potential of test item to induce structural chromosome aberrations. Evaluation of cytogenetic damage induced in V79 cells (cell line from the lung of the Chinese Hamster) in the absence and the presence of metabolic activation was performed in one experiment at 18 hours preparation interval.

Evaluation criteria:

Acceptability of the assay:
The chromosomal aberration assay is considered acceptable if it meets the following criteria:
a) The number of aberrations found in the solvent controls falls within the range of historical laboratory control data range: 0.0 - 4.0 % aberrant cells, exclusive gaps.
b) The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratory’s historical control data:
EMS (400-900 µg/mL) : 7.0-51.0 %
CPA (1.0-2.0 µg/mL): 5.0-44.0 %

Evaluation of results:
A test item is classified as non-clastogenic if:
-the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
- no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as mutagenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
- and either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data (0.0 – 5.3% polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fisher’s exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria for the test item mentioned above are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant influence of the test item on the pH value was observed. In the main experiment pH solvent = 7.5 versus pH 2770 µg/mL = 7.4.
- Effects of osmolality: No relevant influence of the test item on the osmolality was observed. In main experiment solvent = 417 mOsm versus 2770 µg/mL = 360 mOsm.
- Precipitation: was observed after 4 hours treatment with 1385 Og/mL and above in the presence of S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: In the absence of S9 mix, no statistically significant or biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.5 - 2.5 % aberrant cells, excluding gaps) were close to the solvent control value (1.5 % aberrant cells,
excluding gaps) and within the range of the laboratory’s historical control data range (0.0 - 4.0 % aberrant cells excluding gaps). In contrast, in the presence of S9 mix statistically significant and biologically relevant increases in the number of cells carrying structural chromosome aberrations were observed at the three highest evaluated concentrations 692.5, 1385.0, and 2770 µg/mL . The aberration rates of the cells after treatment with the test item (5.5, 6.0, and 4.5 % aberrant cells excluding gaps, respectively) clearly exceeded the laboratory’s historical
control data (0.0 - 4.0 % aberrant cells excluding gaps).
ADDITIONAL INFORMATION ON CYTOTOXICITY: In the absence of S9 mix reduced cell numbers (23.1 % of control) and reduced mitotic indices (37.2 % of control) were observed after treatment with 86.6 µg/mL. In the presence of S9 mix neither reduced mitotic indices nor reduced cell numbers could be observed up to the highest required test item concentration

Conclusions:

Under the test conditions of this study, the test item is considered as negative for inducing chromosomal mutations in Chinese hamster cell line V79 under  non-activation conditions and weakly clastogenic (ambiguous response) under the activation conditions used in this assay.

Executive summary:

In an in vitro chromosome aberration test performed according to OECD guideline No 473 and in compliance with GLP, V79 cells (Chinese hamster cell line) were exposed to the test material diluted in Ethanol in one experiment.

According to the OECD Guideline only one experiment was performed, since the test item was considered to be mutagenic after 4 hours treatment. The chromosomes were prepared 18 hours after start of treatment with the test item. The exposure period was 4 hours with and without metabolic activation.

In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were scored for structural chromosome aberrations.

The highest applied concentration in the pre-test on toxicity (main study) (2770 μg/mL; approx. 10 mM) was chosen with regard to the molecular weight of the test item with respect to the current OECD Guideline 473.
Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation as following:

- Main Experiment (18-hour/4-hour without S9-mix): 10.8, 21.6, 43.3, 86.6, 173.1, 346.3, 692.5, 1385.0 (p) and 2770.0 (p) µg/mL
- Main Experiment, repeated due to strong cytotoxicity (18-hour/4-hour* without S9-mix): 10.8, 21.6***, 43.3***, 86.6***, 173.1, 346.3, 692.5, 1385.0 (p) and 2770.0 (p) µg/mL
- Main Experiment, repeated due to missing cytotoxicity (18-hour/4-hour** without S9-mix): 0.3, 0.7, 1.4, 2.7, 5.4, 10.8, 21.6, 43.3, 86.6 µg/mL.

- Main Experiment (18-hour/4-hour with S9-mix): 10.8, 21.6, 43.3, 86.6, 173.1, 346.3***, 692.5***, 1385.0*** (p) and 2770.0*** (p) µg/mL.

*: was repeated due to strong cytotoxicity
** was repeated due to missing cytotoxicity
*** Evaluated experimental points
P: phase separation

 

In the main experiment phase separation of the test item in culture medium was observed after 4 hours treatment with 1385 µg/mL and above in the presence of S9 mix. No relevant influence of the test item on the pH value or osmolarity was observed (solvent control 417 mOsm, pH 7.5 versus 360 mOsm and pH 7.4 at 2770 µg/mL).

 

None of the dose levels up to the cytotoxicity limit of 86.6 µg/mL in the absence of metabolic activation induced significant increases in the frequency of cells with aberrations in a single experiment. In the presence of metabolic activation the substance was not toxic at up to the maximum dose level of 2770 µg/mL equivalent to 10mM, but showed phase separation (precipitation) at the upper two dose levels. Four dose levels were evaluated for aberration frequency and the upper three dose levels exhbited frequencies of cells with aberrations that exceeded the threshold for a positive response. However, there was no dose-response relationship and marked inter-culture variation, rendering the results ambiguous or weakly clastogenic. The substance does not induce structural aberrations in the chromosomes of Chinese hamster cells line V79 under non-activation conditions, but was considered ambiguous or weakly clastogenic in the presence of activation. Both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells.The substance is therefore considered as negative for inducing chromosomal mutations in Chinese hamster cell line V79 under  non-activation conditions and weakly clastogenic (ambiguous response) under the activation conditions used in this assay.

This study is considered as acceptable and satisfies the requirement for chromosome aberration endpoint.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 July to 01 August, 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other:

Remarks:

Study performed according to OECD test guideline No. 471 and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines: Kanpoan No. 287 - Environment Protection Agency. Eisei No.127 - Ministry of Health&Welfare. Heisei 09/10/31 Kikyoku No.2 - Ministry of International Trade & Industry.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August, 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Directive 2000/32/EC, dated May 19, 2000.

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

German Statement of GLP compliance. Inspected on 02 September 2006 /Signed on 19 January 2007.

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Storage conditions: In the refrigerator at +2 to +8°C, protected from light

Target gene:

Histidine gene for Salmonella and Tryptophan gene for E.coli

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Not applicable.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: S9 fraction from Wistar Hanlbm rats treated with phenobarbital/beta-naphtoflavone
- method of preparation of S9 mix : the S9 is prepared from 8-12 weeks old male Wistar Hanlbm rats, weight approx. 220-320 g induced by applications of 80 mg/kg bw. Phenobarbital i.p. (Desitin; D-22335 Hamburg) and beta-naphtoflavone p.o. (Aldrich, D-89555 Steinheim) each on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with KCL solution (1+3) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampules at -80°C. Small numbers of the ampoules can be kept at -20°C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo(a)pyrene.
The protein concentration in the S9 preparation was 28.7 mg/mL (Lot no. R 160508) in the pre-experiment/experiment I and 31.4 mg/L (Lot no. R 290208) in experiment II.
- concentration or volume of S9 mix and S9 in the final culture medium: S9-fraction 10% v/v
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): no data

Test concentrations with justification for top dose:

Pre-experiment/Cytotoxicity test: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate in TA 1535, TA 1537, TA 98, TA100 and WP2 uvrA with and without S9 under the direct plate incorporation method.

Mutagenicity test:
Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2 uvrA with and without S9 under the direct plate incorporation method.
Experiment II: 33, 100, 333, 1000, 2500 and 5000 µg/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2 uvrA with and without S9 under the pre-incubation method.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol (MERCK, D-64293 Darmstadt; purity > 99%)

- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

- Justification for percentage of solvent in the final culture medium: no data.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene. 10 µg/plate in TA1535, TA 1537, TA 98, TA 100 and WP2 uvrA

Remarks:

With metabolic activation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other:

Remarks:

Without metabolic activation.

Details on test system and experimental conditions:

SOURCE OF TESTER STRAINS: Strains of salmonella typhimurium and E. Coli were obtained from Trinova Biochem GmbH (35394 GieBen, Germany).

METHOD OF APPLICATION: in agar (plate incorporation, pre-experiment and experiment I) and preincubation (Experiment II)

DURATION
- Preincubation period: 60 min at 37 °C
- Exposure duration: 37 °C for 48 h, in the dark.

NUMBER OF REPLICATIONS: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity evaluation of the test item was based on the decrease in the number of revertant colonies, or a clearing or diminution of the background lawn.

OTHER:
- A pre-experiment was performed with strains TA 98 , TA 100, TA 1535, TA 1537 and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions were the same as for the experiment I (plate incorporation test). The pre-experiment is reported as part of the main experiment I, since evaluable plates (>0 colonies) at five concentrations or more in all strains were used.

- After an incubation of about 48 hours at about 37 °C, the number of colonies per plate was counted.
- The number of revertant colonies per plate was counted and recorded by an automatic colony counter. Average plate counts was presented with the mean and the standard deviation for each set of triplicates per test item concentration and was used to calculate the ratio of colonies per exposed plate compared to the corresponding negative control.
- Precipitation of the test item was observed in the overaly agar in the test tubes and on the incubated agar plates from 2500 µg/plate up to 5000 µg/plate. The undissolved particles had no influence on the data recording.

Evaluation criteria:

- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
- A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
- An increase exceeding 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:

A statistical analysis of the data is not required.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed at 2500 - 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility:
Test item was found to be soluble when diluted 1/3 in ethanol.
- Precipitation: precipitation of the test item was observed in the overaly agar in the test tubes and on the incubated agar plates from 2500 µg/plate up to 5000 µg/plate. The undissolved particles had no influence on the data recording.

- Other confounding effects: None

CYTOTOXICITY TEST:
Toxic effects, evident as a reduction in the number of revertants, occurred in the test groups at 2500 and 5000 µg/plate (see Table 7.6.1/1)

MUTAGENICITY TEST:
- None of the concentrations assayed for the test item showed an increase in the R value either with or without S9 metabolic activation regardless of the procedure.
- No dose response for the test item was observed in any of the tested bacterial strains.

HISTORICAL CONTROL DATA
- Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data of the test facility.

Remarks on result:

other:

Remarks:

Ames test acceptance criteria: The bacterial reverse mutation test for the test item was considered valid as the following criteria were met: - regular background growth in the negative and solvent control. - The spontaneous reversion rates in the negative and solvent control are in the range of the historical data. - The positive control substances should produce a significant increase in mutant colony frequencies.

Table 7.6.1/1 Table of toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix
TA 1535	-	-	-	2500-5000
TA 1537	2500-5000	-	-	2500-5000
TA 98	-	-	-	-
TA 100	-	-	-	2500-5000
WP2 uvrA	-	-	-	-

-: no toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5).

Conclusions:

Under the test condition, test substance is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli WP2 uvrA were exposed to the test substance diluted in ethanol at the following concentrations both in the presence and absence of metabolic activation system (10% v/v S9).

Pre-Experiment/Experiment I: 0.3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate in TA 100, TA 98, TA 1535, TA 1537 and Escherichia coli WP2 uvrA with and without S9 under the direct plate incorporation method. 

Experiment II: 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate in TA 100, TA 98, TA 1535, TA 1537 and Escherichia coli WP2 uvrA with and without S9 under the pre-incubation method.

 

Vehicle and positive control groups were also included in mutagenicity tests.

 

Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data.

 Minor toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5) occurred in few strains at 2500 and 5000 µg/plate. None of the concentrations assayed for the test item showed an increase in the R value either with or without S9 metabolic activation regardless of the procedure. No dose response for the test item was observed in any of the tested bacterial strains.

 

Under the test condition, test substance is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

- In vivo micronucleus study: not clastogenic, did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse. (OECD 474, GLP, K, rel.1)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 17, 2008 To April 02, 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study according to OECD 474 without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Adopted July 21, 1997

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 2009-10-29 / Signed on 2009-03-30.

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Storage conditions: In the refrigerator at +2 to +8°C, protected from light

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse is an animal that has been used for many years as a suitable experimental animal in cytogenetic investigations. There are many data available from such investigatios, which my be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experimentations also may be useful for the design and the performance of the micronucleus test.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories Ltd.
- Age at study initiation: 8 to 12 weeks old
- Weight at study initiation: Mutagenicity Experiment: males mean value: 37.0 g (SD+/-2.6 g), females mean value 31.4 g (SD+/-3.0 g). Plasma Sampling: males mean value 39.2 g (SD+/- 2.6 g).
- Assigned to test groups randomly: yes, based on equalization of group mean body weights.
- Housing: Single (Cage type: Makrolon Type I, with wire mesh top).
- Diet: A pelleted standard diet (Harlan Laboratories) was provided ad libitum.
- Water: Animals were allowed free access to tap water (Gemeindewerke, 64380 Rossdorf, Germany)
- Acclimation period: no less than 5 days after receipt

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 25 - 85%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle.

IN-LIFE DATES: From: November 17, 2008 To April 02, 2009.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used:vegetable oil; Corn oil
- Justification for choice of solvent/vehicle: the vehicle was chosen to its relative non-toxicity for the animals.
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Lot/batch no. (if required): C8267 (Sigma)
- Purity: 100%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
All dose formulations were administered at a dose volume of 10 mL/kg by a single oral gavage. Oral gavage was performed using appropriate size disposable polypropylene syringes with gastric intubation tubes (needles).

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Single dose

Post exposure period:

- For low and mid-dose, positive control and vehicle: 24 hr
- For high dose: 24 and 48 hr

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle Group I (Corn Oil)

Dose / conc.:

187.5 mg/kg bw/day

Remarks:

Group II (Low dose)

Dose / conc.:

375 mg/kg bw/day

Remarks:

Group III (Mid dose)

Dose / conc.:

750 mg/kg bw/day

Remarks:

Group IV (High dose)

No. of animals per sex per dose:

- Pre-experiment: 2 animals/sex/dose (100, 500, 1000 and 750 mg/kg bw/day).
- Main Experiment: 6 animals/sex/dose (0, 187.5, and 375) and 12 animals/sex/dose at 750 mg/kg bw/day.

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control: cyclophosphamide (Sigma-Aldrich Vertriebs GmbH)
- Justification for choice of positive control(s): according to the OECD 474 guideline
- Route of administration: orally, once
- Doses / concentrations: 10 mL/kg bw
- Lot/batch no: C 0768
- Purity: > 98%

Tissues and cell types examined:

Bone marrow erythrocytes

Details of tissue and slide preparation:

Bone Marrow Collection and Slide Preparation: In the micronucleus test, at the scheduled bone marrow collection time, five mice per treatment were euthanized by CO2 asphyxiation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x G) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The mear was air dried and then stained with May-Grünwald (Merck, 64293 Darmstadt, Germany)/Giemsa (Merck, 64293 Darmstadt, Germany). Cover slips were mounted with EUKITT (Kindler, 79110 Freiburg, Germany). At least one slide was made from each bone marrow sample.
Scoring for Micronuclei: To control for bias, bone marrow slides were coded using a random number table by an individual not involved with the scoring process. Using oil immersion (1000X), the following cell populations and cellular components were evaluated and enumerated:
- Polychromatic erythrocytes (PCEs): Two-thousand PCEs per each animal were screened (scored) for the presence of micronuclei resulting in evaluation of a total of 10,000 PCEs per each treatment group.
- Micronuclei (M)
- The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes per each animal (PCEs/ECs ratio).

CRITERIA FOR DOSE SELECTION:
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items. The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours.
The administered volume was 10 mL/kg bw.
Three adequately spaced dose levels by a factor of 2 were administered, and samples were collected at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

OTHER:
Blood serum samples from satellite control and high dose animals were taken 1 and 4 hours after oral dosing and the plasma analyzed for the presence of the test item.

Evaluation criteria:

A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statisticals methods (nonparametric Mann-Whitney test will be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 4 animals (2 males and 2 females)/dose received orally a test substance formulated in corn oil at 100, 500, 1000 and 750 mg/kg bw.
- Solubility: no data
- Clinical signs of toxicity in test animals: No mortality was observed in any treatment group.
* The animals treated with 100 mg/kg bw did not express any toxic reactions.
* The animals treated with 500 mg/kg bw exhibited ruffled fur during the course of the study.
* The animals treated with 1000 mg/kg bw expressed toxic reactions, reduction of spontaneous activity, abdominal position and ruffled fur during the course of the study.
* The animals treated with 750 mg/kg bw exhibited ruffled fur during the course of the study.

Based upon the results of the dose range finding study, a dose of 750 mg/kg was estimated to be the maximum tolerated dose for male and female mice. Therefore, the doses of 187.5, 375 and 750 mg/kg were tested in the micronucleus test.


RESULTS OF DEFINITIVE STUDY
- Clinical Signs: no mortality was observed at all tested doses. The animals expressed clinical signs:
* The animals treated with the vehicle did not express any toxic reactions.
* The animals treated with 187.5 mg/kg bw exhibited ruffled fur during the course of the study.
* The animals treated with 375 mg/kg bw expressed ruffled fur during the course of the study.
* The animals treated with 750 mg/kg bw exhibited ruffled fur and reduction of spontaneous activity during the course of the study.

- Bone Marrow Evaluation: No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes in the 24 or 48 hr test substance groups relative to the respective vehicle control groups were observed.
- Ratio of PCE/NCE (for Micronucleus assay): the mean values of micronuclei observed after treatment with the test substance were near to the value of the vehicle control group.
- Appropriateness of dose levels and route: the bioanalysis showed that plasma samples of the animals treated with the high dose did not contain quantifiable amounts of the test item at both tested sampling intervals (1 and 4 h post treatment). The test item concentration in all plasma samples was below LLOQ. The ester compound of the test substance might have been hydrolyzed to the corresponding carboxylic acid by esterase enzymes, that are highly active in rodent plasma. Since no enzyme inhibitor was used during blood sampling, probably the hydrolysis of the test substance took place immediately. However, the bioavailability of the test substance could be confirmed, based on the clinical signs observed at all tested doses, which were taken to indicate that systemic absorption and exposure to the target tissue had been achieved.
- Statistical evaluation: statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test (see Table). In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with the test substance were near to the value of the vehicle control group.

Conclusions:

Under the test conditions of this study, the test substance did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Executive summary:

In an in vivo micronucleus test, performed according to OECD 474 and GLP, NMRI male and female mice were exposed to the test substance in corn oil by oral gavage. In the dose range-finding study, 4 mice (2 males and 2 females) were exposed to the test substance at 100, 500 or 1000 mg/kg bw. Based upon the clinical signs of toxicity observed in the dose range finding study, a dose of 750 mg/kg bw was estimated to be the maximum tolerated dose for male mice. Therefore, twelve mice (6 males and 6 females) per group were treated with the vehicle, positive control (cyclophosphamide monohydrate) or 187.5, 375, or 750 mg/kg bw of the test substance and were euthanized 24 hours after treatment. In addition, six mice per group were treated with the vehicle or 750 mg/kg bw of the test substance and were euthanized 48 hours after treatment. At the time of euthanasia, femoral bone marrow was collected and bone marrow smears (slides) were prepared and stained with Acridine orange stain (a nucleic acid specific stain). Bone marrow cells (polychromatic erythrocytes (PCEs); 2000 PCEs/animal) were examined microscopically for the presence of micronuclei (micronucleated PCEs). In addition, the ratio of polychromatic erythrocytes to total of 1000 erythrocytes (PCEs/ECs ratio) was determined as an indicator of bone marrow exposure to the test substance and subsequently as a measure of test substance cytotoxicity.. The animals dosed with the test substance at 750 mg/kg bw and three controls (only corn oil) were bled at 1, and 4, hours post-dose (controls only at 1 hour).

 

Plasma was analyzed as a confirmation of systemic exposure to the test substance. All measured concentrations of the test substance in the plasma samples of animals dosed with the test substance were below the lower limit of detection. No mortality was observed in any of the treatment groups, but clinical symptoms were observed in mice of the 187.5, 375, and 750 mg/kg bw dose groups. No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes in the bone marrow were observed in the test substance groups relative to the respective vehicle control groups suggesting that the test substance did not inhibit erythropoiesis. No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in the test substance groups relative to the respective vehicle control groups was observed at 24 or 48 hours after dose administration. The positive control induced a statistically significant increase in the incidence of micronucleated polychromatic erythrocytes relative to the vehicle control. The number of micronucleated PCEs in the vehicle control groups did not exceed the historical vehicle control range. Based upon this, all criteria for a valid test were met. Under the conditions of this test, a single oral administration of the test substance at doses up to and including 750 mg/kg bw (estimated maximum tolerated dose) did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow of NMRI mice. Therefore, the test substance was concluded to be negative in the in vivo micronucleus assay.

This study is considered as acceptable and satisfies the requirement for chromosome aberration endpoint.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Table 7.6/1: Summary of in vitro genotoxicity tests

 

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1 RCC	Ames Test (OECD 471) K, rel. 1, 2008)	Bacterial gene mutation	S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 bacteria	-S9 +S9	0.3 to 5000 µg/plate	-S9 : non mutagenic +S9 : non mutagenic
2 RCC	CA (OECD 473, K, Rel 1, 2009)	chromosomal aberration	V79 cells	-S9 +S9	21.6 to 86.6 346.3 to 2777.0 µg/mL	-S9: not clastogenic +S9: weakly clastogenic or ambiguous

 

 

Table 7.6/1: Summary of in vivo genotoxicity tests

 

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1 Harlan	Micronucleus assay (OECD 474, 2009, GLP, K, rel. 1)	Chromosomal aberration	Bone marrow erythrocytes cells	NA	187.5, 375 and 750 mg/kg	Not clastogenic

 

 

Gene mutation Assay (Test n°1) :

A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD guideline No. 471 with the substance(See Table 7.6/1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains under the test conditions, with any dose of the substance, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies.The substance is therefore considered as non-mutagenic according to the Ames test.

 

Chromosomal aberration (Test n°2)

The clastogenic potential of the substance was determined using an in vitro chromosome aberration test in CHO V79 cells (OECD 473), which measures the potential of a substance to increase the incidence the of structural chromosome aberrations in a cultured immortal cell line.

None of the dose levels up to the cytotoxicity limit of 86.6 µg/mL in the absence of metabolic activation induced significant increases in the frequency of cells with aberrations in a single experiment. In the presence of metabolic activation the substance was not toxic at up to the maximum dose level of 2770 µg/mL equivalent to 10mM, but showed phase separation (precipitation) at the upper two dose levels. Four dose levels were evaluated for aberration frequency and the upper three dose levels exhbited frequencies of cells with aberrations that exceeded the threshold for a positive response. However, there was no dose-response relationship and marked inter-culture variation, rendering the results ambiguous or weakly clastogenic. The substance does not induce structural aberrations in the chromosomes of V79 cells under non-activation conditions, but was considered ambiguous or weakly clastogenic in the presence of activation. Both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells.The substance is therefore considered as negative for inducing chromosomal mutations in V79 cells under non-activation conditions and weakly clastogenic (ambiguous response) under the activation conditions used in this assay.

 

Chromosomal aberration in vivo (Test n°1)

In an in vivo micronucleus test, performed according to OECD 474 and GLP, NMRI male and female mice were exposed to the test substance in corn oil by oral gavage. In the dose range-finding study, 4 mice (2 males and 2 females) were exposed to the test substance at 100, 500 or 1000 mg/kg bw. Based upon the clinical signs of toxicity observed in the dose range finding study, a dose of 750 mg/kg bw was estimated to be the maximum tolerated dose for male mice. Therefore, twelve mice (6 males and 6 females) per group were treated with the vehicle, positive control (cyclophosphamide monohydrate) or 187.5, 375, or 750 mg/kg bw of the test substance and were euthanized 24 hours after treatment. In addition, six mice per group were treated with the vehicle or 750 mg/kg bw of the test substance and were euthanized 48 hours after treatment. At the time of euthanasia, femoral bone marrow was collected and bone marrow smears (slides) were prepared and stained with Acridine orange stain (a nucleic acid specific stain). Bone marrow cells (polychromatic erythrocytes (PCEs); 2000 PCEs/animal) were examined microscopically for the presence of micronuclei (micronucleated PCEs). In addition, the ratio of polychromatic erythrocytes to total of 1000 erythrocytes (PCEs/ECs ratio) was determined as an indicator of bone marrow exposure to the test substance and subsequently as a measure of test substance cytotoxicity.. The animals dosed with the test substance at 750 mg/kg bw and three controls (only corn oil) were bled at 1, and 4, hours post-dose (controls only at 1 hour).

 

Plasma was analyzed as a confirmation of systemic exposure to the test substance. All measured concentrations of the test substance in the plasma samples of animals dosed with the test substance were below the lower limit of detection. No mortality was observed in any of the treatment groups, but clinical symptoms were observed in mice of the 187.5, 375, and 750 mg/kg bw dose groups. No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes in the bone marrow were observed in the test substance groups relative to the respective vehicle control groups suggesting that the test substance did not inhibit erythropoiesis. No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in the test substance groups relative to the respective vehicle control groups was observed at 24 or 48 hours after dose administration. The positive control induced a statistically significant increase in the incidence of micronucleated polychromatic erythrocytes relative to the vehicle control. The number of micronucleated PCEs in the vehicle control groups did not exceed the historical vehicle control range. Based upon this, all criteria for a valid test were met. Under the conditions of this test, a single oral administration of the test substance at doses up to and including 750 mg/kg bw (estimated maximum tolerated dose) did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow of NMRI mice. Therefore, the test substance was concluded to be negative in the in vivo micronucleus assay.

 

Overall Concusion

The substance is not mutagenic or clastogenic. The weak or ambiguous response seen in the clastogenicity assay in V79 cells is typical of high dose false positive responses in immortal cell lines. The absence of a dose response relationship, the weak and poorly reproducible frequency of cells with aberrations that only just exceeded the historical control limit , and the observation of 'phase separation' at the highest two dose levels indicates that the observations were spurious. Furthermore, a clear negative result in an in vivo mouse micronucleus study in which systemic exposure was confirmed, provides assurance that the in vitro response has no toxicological relevance.

 

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

 

Self classification:

Based on the available data, no additional classification is proposed regarding genetic toxicity according to the Annex I of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.