Reaction mass of 1-methyl-3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde and 1-methyl-4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay, performed according to OECD guidelines and GLP principles.

The mutagenic activity of Precyclemone B was tested in the mouse lymphoma assay conducted according to OECD 476 guideline and GLP principles. Based on the absence of significant increase in the number of small and large colonies, it is concluded that Precyclemone B is not mutagenic in the mouse lymphoma L5178Y test system.

The ability of Precyclemone B to induce micronuclei in cultured human peripheral lymphocytes was investigated in an in vitro micronucleus assay conducted according to OECD 487 guideline and GLP principles. Based on the absence of significant statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, it is concluded that Precyclemone B is not clastogenic or aneugenic in human lymphocytes.

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:

04 May, 1999 - 21 May, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

yes

Remarks:

Not always dosed high enough. As experiment 1 or 2 shows toxicity in all strains, this deviation does not effect the outcome of the study.

Qualifier:

according to guideline

Guideline:

other: Methods for the determination of physio-chemical properties, toxicity and ecotoxicity: Annex V to Directive 79/831/EEC. B14. Salmonella typhimurium - Reverse mutation assay (1992).

Deviations:

yes

Remarks:

Not always dosed high enough. As experiment 1 or 2 shows toxicity in all strains, this deviation does not effect the outcome of the study.

Principles of method if other than guideline:

The proposals of the ICH working group for genotoxicity testing

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Precyclemone B
- Appearance: colorless liquid
- Analytical purity: 99.3 % (GLC, sum of two peaks)
- Lot/batch No.: 9000320077
- Expiration date of the lot/batch: 25-03-2000
- Storage condition of test material: refrigerator

Target gene:

- S. typhimurium: Histidine gene

Species / strain / cell type:

S. typhimurium, other: TA1535, TA97, TA98, TA100 and TA102

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Preliminary test (without and with S9) TA100: 50, 158, 500, 1580 and 5000 µg/plate

Experiment 1: TA1535, TA97, TA98, TA100 and TA102:
Without and with S9-mix: 2, 6.32, 20, 63.2 and 200 µg/plate

Experiment 2: TA1535, TA97, TA98, TA100 and TA102:
Without and with S9-mix: 1, 3.16, 10, 31.6 and 100 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test compound was miscible in DMSO and it is to be expected that no gross degradation is occuring when dissolved in DMSO for the test duration (≤ 6 hours).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 (TA100 and TA1535 1.0 µg/plate)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9 (TA98 0.5 µg/plate)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: ICR-191 1 µg/plate in DMSO for TA97

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 (TA102 0.4 µg/plate)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation, exp. 1) and preincubation (exp. 2)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain, duplicates for postive controls. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A positive result is defined as a reproducible, dose-related increase in the number of his+ revertants. The increase should reach at least a doubling of the number of spontaneous revertants for TA1535 and TA98. For TA97, TA100 and TA102 a 1.5-fold increase over control values might be indicative of a mutagenic effect provided the negative control values fall within the historical control data. Other investigators have set higher limits for a mutagenic response (factor 3 and 2 for the respective groups of strains). These rules of thumb have a questionable scientific foundation (Claxton et al. 1987) and biological relevance should always be taken into account. A negative result is defined as the absence of a reproducible increase in the number of his+ revertant colonies.

Species / strain:

S. typhimurium, other: TA1535, TA97, TA98, TA100 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: small droplets were visible at dose levels of 1580 µg/plate and above.

RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA100, toxicity was observed at dose levels of 50 μg/plate and above in the absence of S9-mix. In the presence of S9-mix, toxicity was observed at dose levels of 158 μg/plate and above.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The mutant frequencies of the controls were in the range of the historical control values and the data published in the literature (Maron and Ames, 1983; Levin et al., 1982).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1:
TA1535: without S9: 63.2 µg/plate and above and with S9: no toxicity
TA97: no toxicity
TA98: without and with S9: 200 µg/plate
TA100: without and with S9: 63.2 µg/plate and above
TA102: without and with S9: 63.2 µg/plate and above
Experiment 2:
TA1535: without S9: 10 µg/plate and above and with S9: no toxicity
TA97: without S9: 100 µg/plate and above and with S9: no toxicity
TA98: without S9: 10 µg/plate and above and with S9: no toxicity
TA100: without S9: 10 µg/plate and above and with S9: 100 µg/plate
TA102: without S9: 10 µg/plate and above and with S9: 100 µg/plate

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay, performed according to OECD guidelines and GLP principles.

Executive summary:

The genetic toxicity of the substance was assessed using Salmonella typhimurium TA97, TA98, TA100, TA102 and TA1535 strains, in accordance with OECD guidelines (OECD 471, 1997) and GLP principles. The first experiment was a standard assay, concentrations up to 200 µg/plate, and the second experiment was a preincubation assay with concentrations up to 100 µg/plate. This was based on a dose range test with TA100 showing toxicity at dose levels of 50 μg/plate and above in the absence of S9-mix. In the presence of S9-mix, toxicity was observed at dose levels of 158 μg/plate and above.

In the main test, toxicity was observed in several strains depending on strain and metabolic activation conditions.

All bacterial strains showed negative responses over the entire dose range, i.e. no biologically significant dose-related increase in the number of revertants in two independently repeated experiments with and without metabolic activation up to the highest concentration. Based on the results it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 September, 2014 - 27 October, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1997)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

(2008)

Deviations:

no

Principles of method if other than guideline:

The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Precyclemone B
- Chemical name (Major Isomer): 1-Methyl-4-(4-methylpent-3-en1-yl)cyclohex-3-ene-1-carbaldehyde
- Chemical name (IUPAC): Reaction mass of 1-methyl-3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde and 1-methyl-4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde
- EC Number: 915-712-5: 257-941-7: 257-942-2
- Description: Clear colourless liquid
- Analytical purity: 99.4% (sum of the two peaks)
- Batch No.: SC00010316
- Expiration date of the batch: 24 July 2015
- Storage condition of test material: In refrigerator (2-8°C) protected from light
- Test substance handling: Use amber-coloured glassware or wrap container in aluminium-foil.

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
- RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw)

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3 hours treatment: 17, 52, 100, 164 and 512 µg/mL
Without S9-mix, 24 hours treatment: 17, 52, 100, 164 and 512 µg/mL
Experiment 1:
Without and with S9-mix, 3 hours treatment: 0.1, 0.5, 1, 5, 10, 20, 25, 30, 35, 40, 45 and 50 μg/mL
The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix, 3 hours treatment: 0.1, 0.5, 1, 5, 10, 20 and 25 µg/mL
With S9-mix, 3 hours treatment: 1, 5, 10, 20, 30, 35, 40 and 45 µg/mL
Experiment 2:
Without S9-mix, 24 hours treatment: 0.05, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40 and 45 µg/mL
The dose levels selected to measure mutation frequencies at the TK-locus were: 0.1, 0.5, 1, 5, 10, 15, 20 and 25 µg/mL

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: Test compound was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines. Amber-coloured vials or tubes wrapped in aluminium-foil were used when preparing the test solutions. Precyclemone B concentrations were used within 2 hours after preparation.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 Migrated to IUCLID6: 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 Migrated to IUCLID6: 7.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10E6 survivors and ≤ 170 per 10E6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 per 10E6 survivors, and for CP not below 700 per 10E6 survivors.

DATA EVALUATION
In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test substance is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Statistics:

The global evaluation factor (GEF) has been defined by the IWTGP as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
-Precipitation: Precipitation in the exposure medium was observed at dose levels of 164 µg/mL and above

RANGE-FINDING/SCREENING STUDIES:
-At the 3 hours treatment time, both in the absence and presence of S9-mix, hardly any toxicity in the relative suspension growth was observed at the test substance concentration of 17 μg/mL. No cell survival was observed at test substance concentrations of 52 μg/mL and above. At the 24 hours treatment time, in the absence of S9-mix, the relative suspension growth was 68% at the test substance concentration of 17 μg/mL compared to the relative suspension growth of the solvent control, No cell survival was observed at test substance concentrations of 52 μg/mL and above.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.
Mutation frequencies in cultures treated with positive control chemicals were increased 13- and 9-fold for MMS in the absence of S9-mix, and 13-fold for CP in the presence of S9-mix. In addition the observed mutation frequencies of the positive control substances were within the acceptability criteria of this assay. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1:
In the absence of S9-mix, the dose levels of 30 to 50 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. The relative total growth of the highest test substance concentration was reduced by 74% compared to the total growth of the solvent controls. In the presence of S9-mix, the dose levels of 0.1 to 30 μg/mL showed no cytotoxicity. Therefore, the dose levels of 0.1, 0.5 and 25 μg/mL were not regarded relevant for mutation frequency measurement. The dose level of 50 μg/mL was not used for mutation frequency measurement, since this dose level was too toxic for further testing. The relative total growth of the highest test substance concentration was reduced by 93% compared to the total growth of the solvent controls.
Experiment 2:
The dose levels of 0.05 to 10 μg/mL showed no cytotoxicity. Therefore, the dose level of 0.05 μg/mL was not regarded relevant for mutation frequency measurement. The dose levels of 30 μg/mL and above were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. The relative total growth of the highest test substance was reduced by 88% compared to the total growth of the solvent controls.

Remarks on result:

other: strain/cell type: Test system L5178Y/TK+/-3.7.2C

Remarks:

Migrated from field 'Test system'.

Conclusions:

The mutagenic activity of Precyclemone B was tested in the mouse lymphoma assay conducted according to OECD 476 guideline and GLP principles. Based on the absence of significant increase in the number of small and large colonies, it is concluded that Precyclemone B is not mutagenic in the mouse lymphoma L5178Y test system.

Executive summary:

The mutagenic activity of Precyclemone B was tested in the mouse lymphoma assay conducted according to OECD 476 guideline and GLP principles.

In the first experiment, Precyclemone B was tested up to and including concentrations of 25 and 45 μg/mL in the absence and presence S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was reduced to 26% and 7% in the absence and presence of S9-mix, respectively. In the second experiment, Precyclemone B was tested up to and including concentrations of 25 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 12%.

In the absence of S9-mix, Precyclemone B did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time. In the presence of S9-mix, Precyclemone B did not induce a significant increase in the mutation frequency.

It is concluded that Precyclemone B is not mutagenic in the mouse lymphoma L5178Y test system.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 March, 2014 - 10 July, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test, 2010)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.49 “InVitro Mammalian Cell Micronucleus Test" (2012).

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

- Name of test material (as cited in study report): Precyclemone B
- Chemical name (IUPAC): Reaction mass of 1-methyl-3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde and 1-methyl-4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde
- CAS Number: 52475-86-2 : 52474-60-9
- EC Number 915-712-5 : 257-941-7 : 257-942-2
- Description: Clear colourless liquid
- Analytical purity: 99.4% (sum of two peaks)
- Batch No.: SC00010316
- Expiration date of the batch: 24 July 2015
- Storage condition of test material: In refrigerator (2-8°C) protected from light
- Test substance handling: Use amber-coloured glassware or wrap container in aluminium-foil.

Species / strain / cell type:

lymphocytes: human peripheral blood, male < 35 years

Details on mammalian cell type (if applicable):

Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.

- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.

- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin was added.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight)

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3hr exposure; 27 hr fixation: 3, 10, 33, 100 and 333 µg/mL
Without S9-mix, 24 exposure; 24 hr fixation: 3, 10, 33, 100 and 333 µg/mL
First cytogenetic test:
Without S9-mix, 3hr exposure; 27 hr fixation: 30, 50, 70, 90, 100, 150 and 200 µg/mL
With S9-mix, 3hr exposure; 27 hr fixation: 30, 50, 70, 100, 125, 150, 175, 200, 225 and 250 µg/mL
The dose levels selected for scoring of micronuclei:
Without S9-mix, 3hr exposure; 27 hr fixation: 30, 70 and 100 µg/mL
With S9-mix, 3hr exposure; 27 hr fixation: 30, 70, 125 and 150 µg/mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 10, 30, 40, 50, 60, 70, 80 and 100 µg/mL
The dose levels selected for scoring of micronuclei:
Without S9-mix, 24 hr exposure; 24 hr fixation: 10, 30 and 50 µg/mL

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: Test compound was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: mitomycin C 0.25 µg/mL

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: colchicine: 0.1 µg/mL

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 Migrated to IUCLID6: 15 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 46 ± 2 hr
- Exposure duration:
Short-term treatment
Without and with S9-mix: 3 hr treatment, 24 hr recovery/harvest time
Continuous treatment
Without S9-mix: 24 hr treatment/harvest time

ARREST OF CELL DIVISION: 5 µg/mL Cytochalasine B
STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: at least 1000-2000/culture (mono- and binucleated cells)

DETERMINATION OF CYTOTOXICITY
- The cytostasis/cytotoxicity was determined using the cytokinesis-block proliferation index (CPBI index)

Evaluation criteria:

An in vitro micronucleus test was considered acceptable if it meets the following criteria:
a) The positive control substance colchicine produced at least a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number mononucleated cells with micronuclei and the positive control substances MMC-C and CP produced at least a statistically significant increase in the number of binucleated cells with micronuclei.
b) A homogeneous response between the duplicate cultures is observed.

A test substance was considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if:
a) It induces a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono or binucleated cells with micronuclei.
b) A statistically significant and biologically relevant increase is observed in the number of mono or binucleated cells with micronuclei in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono and binucleated cells with micronuclei.
b) The number of mono and binucleated cells with micronuclei was within the laboratory historical control data range.

Statistics:

The incidence of micronucleated cells (cells with one or more micronuclei) for each exposure group was compared to that of the solvent control using Chi-square statistics.

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 150 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 100 µg/mL and above in the absence of S9, 3 hr treatment/24 hr fixation; at dose levels of 50 µg/mL and above in the absence of S9 for the continuous treatment of 24 hr and at dose levels of 125 µg/mL and above in the presence of S9, 3 hours treatment, 24 hours fixation.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Appropriate toxicity was reached at the dose levels selected for scoring.

Conclusions:

The ability of Precyclemone B to induce micronuclei in cultured human peripheral lymphocytes was investigated in an in vitro micronucleus assay conducted according to OECD 487 guideline and GLP principles. Based on the absence of significant statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, it is concluded that Precyclemone B is not clastogenic or aneugenic in human lymphocytes.

Executive summary:

The ability of Precyclemone B to induce micronuclei in cultured human peripheral lymphocytes was investigated in an in vitro micronucleus assay conducted according to OECD 487 guideline and GLP principles.

In the first experiment, Precyclemone B was tested up to and including concentrations of 100 and 150 μg/mL in the absence and presence S9-mix, respectively. The incubation time was 3 hours. Appropriate toxicity was reached at these dose levels.

In the second experiment, Precyclemone B was tested up to and including 50 μg/mL for a 24 hours exposure time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

Reliable negative and positive controls were included showing that the test conditions were adequate.

Precyclemone B did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two independently repeated experiments.

It is concluded that Precyclemone B is not clastogenic or aneugenic in human lymphocytes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)