Cyclopropanemethanol, 1-methyl-2-[(1,2,2-trimethylbicyclo[3.1.0]hex-3-yl)methyl]-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Bacterial Mutation Test (OECD 471,1983,1994; EU Method B.14, 1992, GLP, K rel.1): not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98, TA100 and TA102

- Chromosome aberration test (OECD 473, 1997, EU Method B.10, 1992, GLP, K rel. 1): not clastogenic in either the absence or presence of metabolic activation.

- Mammalian Cell Gene Mutation Assay (OECD 476, GLP, K rel. 1): not mutagenic to CHO cells with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 13 October 1999 to 24 February 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Substance identity: Javanol
Appereance: Liquid

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 (Preparation by RCC Cytotest Cell Research):

The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats, strain Wistar Hanlbm (BRL, CH-4414 Fuillinsdorf; weight approx. 220 - 320 g) which received daily applications of 80 mg/kg b.w. phenobarbital i.p. dissolved in deionised water (Desitin; D-22335 Hamburg) and B-naphthoflavone orally dissolved in corn oil (Aldrich, D- 89555 Steinheim) on three subsequent days. The livers were prepared 24 hours after the last treatment.

After decapitation of the anaesthetised animals, the livers of the animals were removed, washed in 150 mM KCI and homogenized. The homogenate, diluted 1+3 with KCl was centrifuged at 9000 g for 10 minutes (4° C). Aliquotes of the supernatant containing the microsomal fraction 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 content was determined using the analysis kit of Bio-Rad Laboratories, D-80939 Miinchen: Bio-Rad protein assay, Catalogue No. 5000006.

The protein concentration in the S9 preparation is usually between 20 and 45 mg/ml. The protein concentration was 30.3 mg/ml (Lot. No.: 240999) in the pre-test and experiment I 23.9 mg/ml (Lot. No.: 121199) in experiment II.


S9 Mix:

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:
8mM MgCl,
33 mM KCl
5 mM_glucose-6-phosphate
4mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al. (1).

Test concentrations with justification for top dose:

Range-Finder/Pre-test:
The highest concentration used in the pre-test was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests. With respect to the molecular weight of Javanol, 2230 microgram/mL was applied as the top concentration for treatment of the cultures in the pre-test. Test item concentrations between 17.4 and 2230 microg/ml (with and without S9-mix) were chosen for the evaluation of cytotoxicity.

Using reduced cell numbers as an indicator for toxicity in the pre-test, strong toxic effects were observed after 4 and 24h treatment with 34.9 microgram/mL and above in the absence of S9 mix and with 69.7 microg/ml and above in the presence of S9 mix. Considering the toxicity data of the pre-test, 50 microgram/mL (without S9 mix) and 75 microgram/mL(with S9 mix) were chosen as top concentrations for 4h treatment in experiment 1. For the 24h continuous treatment, 40 microg/ml were chosen as top concentration in the absence of S9 mix.

Dose selection of experiment 2 was influenced by the results of experiment 1. In the presence of S9 mix in experiment 1 reduced cell numbers were observed after 4h treatment with 45 microgram/mL. Therefore, 60 microg/ml were chosen as top treatment concentration in experiment 2. In the absence of S9 mix 18h treatment with 10 microg/ml induced strong toxicity indicated by reduced miotic indices. Therefore, 15 microgram/mL were chosen as top treatment concentration for 28h continuous exposure. The applied concentrations in the cytogenetic experiments are presented in table 2 page 15.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Treatment

Exposure period 4 hours
The culture medium of exponentially growing cell cultures was replaced with serum-free medium (for treatment with S9 mix) or complete medium (for treatment without S9 mix) with 10 % FCS (v/v), containing the test item. For the treatment with metabolic activation 50 ul/ml S9 mix per ml culture medium were added. Concurrent negative, solvent and positive controls were performed. After 4 h the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time.
The "Saline G" solution was composed as follows (per litre):
NaCl 8000 mg KCl 400 mg Glucose 1100 mg Na,HP0x7H,0 290 mg KH,PO, 150 mg
pH was adjusted to 7.2
Exposure period 18 and 28 hours
The culture medium of exponentially growing cell cultures was replaced with complete medium (10 % FCS) containing different concentrations of the test item without S9 mix. The medium was not changed until preparation of the cells.
All cultures were incubated at 37° C in a humidified atmosphere with 4.5 % CO, (95.5 % air).

Preparation of the Cultures
16 h and 26 h, respectively after the start of the treatment colcemid was added (0.2 ug/ml culture medium) to the cultures. 2 h later, the cells on the slides were treated 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 3 + 1 methanol + glacial acetic acid. Per experiment both slides per group were prepared. After preparation the cells were stained with Giemsa (E. Merck, D-64293 Darmstadt).
Additionally, two cultures per test item and solvent control treatment group, not treated with Colcemid, were set up in parallel. These cultures were stained in order to determine microscopically the cell number within 10 defined fields per slide. The toxicity of the test item is given as reduction of % cells as compared to the solvent control.

Analysis of Metaphase Cells
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik" (9)) 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. 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome numbers of 22 + 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

Data Recording
The data generated were recorded in the raw data file. The results are presented in tabular form, including experimental groups with the test item, negative, solvent and positive controls.

Evaluation criteria:

A test item is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are 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 are observed

A test item is classified as mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are not in the range of our historical control data ( 0.0 - 4.0% aberrant cells exclusive gaps).
- either a concentration-related or a significant increase of the number of structural chromosome aberrations are observed

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

In experiment I, in the absence of S9 mix, reduced mitotic indices were observed after 4 h treatment with 20 µg/ml (35 % of control) and after  18  h continuous  treatment  with 10 µg/ml (54 % of control).

In the presence of S9 mix after 4 h treatment with 45 µg/ml reduced mitotic indices (37% of control) and cell numbers (39 % of control) were observed.

In experiment II the mitotic indices were reduced after 28 h continuous treatment with  15 µg/ml (56 % of control) in the absence of S9 mix. In addition the cell numbers were reduced at this experimental point (41 % of control). In the presence of S9 mix reduced cell numbers were observed after 4 h treatment with 45 µg/ml (38 % of control).

In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. In the absence and in the presence of S9 mix, the aberration rates of the cells after treatment with the test item (exp.I:0.0 % - 2.0 %; exp. II: 0.5 % - 3.0 %) near to the range of the solvent control values (exp. I: 0.0 % - 1.5 %; exp. II: 1.5 % - 2.0 %) and within the range of our historical control data: 0.0% -4.0%. In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (exp.I:1.8 % - 4.6 %; exp. II: 1.8 % - 5.1 %) as compared to the rates of the solvent controls (exp.I: 3.3 % - 4.9 %; exp. II: 2.7 % - 3.0 %).

In both experiments, EMS (600 and 1000 µg/ml, respectively) and CPA (0.71 µg/ml) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Conclusions:

Under the test conditions, the test item did not induce statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of S9. Javanol was therefore considered to be non-clastogenic in this chromosomal aberration test.

Executive summary:

In an in vitrochromosome aberration test performed according to OECD Guideline 473 and in compliance with GLP, cultured V79 Chinese hamster cells were exposed to Javanol both the absence and the presence of a metabolic activation system (S9-mix) in two independent experiments. Two independent chromosome aberration assays were conducted. The substance was dissolved in DMSO prior to testing.Following study design was performed:

 

without S9-mix				with S9-mix
	exp. I		exp. 1I	exp. I	exp. 1I
Exposure period	4h	18 h	28 h	4h	4h
Recovery	14 h	-	-	14 h	24 h
Preparation interval	18 h	18 h	28 h	18 h	28 h

 

Doses applied were:

Without S9

Experiment 1: 4hr- 5, 10, 20, 30, 40, 50 µg/mL; 18hr- 2.5, 5, 10, 20, 30, 40 µg/ml

Experiment 2: 28hr- 1.4, 2.5, 5, 7.5, 10, 15 µg/mL

With S9

Experiment 1: 4hr- 7.5, 15, 30, 45, 60, 75 µg/mL

Experiment 2: 4hr- 3.8, 7.5, 15, 30, 45, 60 µg/mL

 

Toxic effects indicated by clearly reduced cell numbers and/or mitotic indices were observed in all experimental parts of the cytogenecity study. At the highest concentration which was evaluated for cytogenetic damage, clear toxicity was observed. In both independent experiments, neither a significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls and they induced statistically significant increases in cells with structural chromosome aberrations indicating the validity of the test.

Based on these results, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells in vitro.

Therefore, Javanol is considered to be non-clastogenic in this chromosome aberration test.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 4 September to 6 October 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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): Javanol
- Physical state: Pale yellow viscous liquid

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 97

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : obtained from the livers of male rats (BRL Ltd Füllinsdorf, Switzerland); Mixed function oxidase systems in the livers of rats were stimulated by combined treatment with phenobarbital / beta-napthoflavone.
The animals were killed b decapitation. The livers were removed under aseptic conditions. They were chopped and homogenized with a Potter Elvehjem apparatus with additon of a volume of ice cold 0.15 M potassium chloride equivalent to 3 times the weight of the liver (3 ml KCI per 1 g liver). The homogenate was pooled and centrifugated at 4°C for 10 minutes at 9000 g. The supernatant fraction was collected and dispensed in 2 mL cryotubes (Nunc, Denmark). The tubes were rapidly cooled and kept in the deep freezer (-80°C +/- 20°C) until use. At the end of the preparation about 1 ml of the S9 fraction was dispensed on complete medium plates and incubated at 37°C (and/or 28°C) for 2-5 days to verifiy the sterilty.

exogenous metabolic activation system (S9 mixture)
The S9 mixture was freshly prepared, kept in an ice bath. Its composition was:
Potassium chloride 0.165 M 0.2 ml perml mix Magnesium chloride 0.04 M 0.2 ml per mi mix Sodium phosphate buffered saline 0.2 M, pH 7.4 0.5 ml per ml mix NADP (Boehringer) 3.2 mg permi mix Glucose-6-phosphate (Boehringer) 1.53 mg per mi mix S9 fraction 0.1 ml perm! mix
In order to test the activity of the S9 mix used in the experiments positive controls requiring metabolic activation were tested concurrently.
The protein content of the S9 fraction was determined by the method of Lowry (Lowry et al., 1951) with bovine serum albumin (purity: at least 92 %) from SERVA (Control D) used as the internal standard.
The protein contents of the batches used for the main tests were 40.2 and 34.3 mg/ml (Batches K1/96 and 12/96) and 34.3 mg/ml (batch 11/96) for the toxicity test.

Test concentrations with justification for top dose:

In the preliminary experiment: 50µg/plate javanol
In the standard plate incorporation assay: 1.6 to 158 micrograms/plate (-S9) and 5 to 500 micrograms/plate (+S9)
In the preincubation assay : 0.5 to 50 micrograms/ plate (-S9) and 1.6 to 158 micrograms/plate (+S9)

Dose selection was made based on the range finder assay result.

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

other: ICR 191 and 2-aminoanthracene

Remarks:

Sensitivity of strains were verified with the following: sodium azide (TA1535, TA100; -S9) ICR 191 (TA 97; -S9) 2-nitrofluorene (TA98; -S9) MMC (TA 102; -S9) 2-aminoanthracene (all strains; -/+ S9) Significant increases in mutant frequencies observed

Details on test system and experimental conditions:

Test procedure:
Bacterial strains
The Salmonella typhimurium strains TA1535, TA97, TA98, TA100, and TA102 were obtained from B.N. Ames and are described elsewhere (Ames et al., 1975; Levin et al., 1982a; 1982b; Maron and Ames, 1983).
Nutrient broth cultures of each strain, supplemented with 9 % DMSO were stored in liquid nitrogen. Subcultures with a normal spontaneous frequency were stored in NB + 9 % DMSO at -80°C+ 20°C. The strain identities and characteristics were periodically checked by the recommended procedures (De Serres and Shelby, 1979; Zeiger et al., 1981; Maron and Ames, 1983).
For use in tests, cultures of the strains were grown overnight at 37°C in a shaking water bath in a NB liquid medium. The strain TA102 was incubated with 0.3 pg tetracycline per m! NB medium in order to ensure the presence of an adequate number of plasmids (Albertini and Gocke, 1988). The growth of the overnight cultures was controlled by measuring the optical density ona photometer (Lumetron Colorimeter) at 650 nm. Each bacterial strain was diluted 10° in 0.85 % sodium chloride, and 100 ul of the last dilution step was plated on a NB complete medium. Two replicate plates were incubated at 37°C, upside down, for two days. The number of colonies was registered and the number of cells plated on VB medium was calculated.
The sensitivity of the Salmonella typhimurium strains was verified using the following positive controls: sodium azide with strains TA1535 and TA100, ICR 191 with strain TA97, 2-nitrofluorene with strain TA98 and MMC with strain TA102. Moreover, 2-aminoanthracene was used with all strains with and without metabolic activation to examine the activity of the S9 mix.

Toxicity prescreen and dose selection
Toxicity of the test substance was assessed in a preliminary toxicity assay by evaluating the growth on Vogel-Bonner minimal agar plates (determination of the growth of the background lawn and/or frequency of spontaneous revertants). Each test substance dose, as well as the appropriate solvent control, was evaluated in duplicate in strain TA100 in the plate incorporation version of the assay. The highest test dose for the main experiments is chosen to either produce signs of toxicity (reduction in the revertant colony number and/or observation of thinning or absence of the background lawn) or to be evidently insoluble in the aqueous medium.
There is much debate about selection of highest dose for badly soluble compounds (Kirkland, 1994; Gatehouse et al., 1994). Homogeneous suspensions are included and only particulate aggregation is considered for limiting the dose range to be evaluated. If the compound is soluble and non toxic, then 5 mg/plate is chosen in general as the highest dose level. The solubility of the compound in the solvent, the occurrence of precipitation in the test tube after addition of the soft agar and on the agar plates after the incubation period are noted in the toxicity test with TA100. Since precipitation under the conditions of the preincubation version might differ from the plate incorporation E. Gocke, Report 167’801 -14-
version an examination of precipitation after addition of the phosphate buffer (as in the preincubation test) is included in the prescreen. In this case cells are not added to the mixture. The basis for dose selection is described in results and discussion.

Standard Ames test procedure (Ames et al., 1975; Maron and Ames, 1983)
Test tubes containing 2 ml of 0.7 % agar medium were autoclaved and kept in a prewarmed water bath at 42°C to 45°C. The following solutions were added in order:
- 0.2 ml of the histidine/biotin mixture corresponding to 21 ug L-histidine and 24.4 ug biotin
- 0.1 ml of the test compound at different concentrations or of the solvent or 0.05 ml of the different reference substances which were thawed shortly before use
- 0.1 ml of the overnight cultures of the bacterial strain
- 0.5 mi of the S9 mixture where metabolic activation was needed. The S9 mixture was replaced by 0.5 ml sodium phosphate buffered saline 0.2 M, pH 7.4 in the treatment without metabolic activation.
The contents of the tubes were mixed and poured immediately onto Vogel-Bonner minimal agar plates. Three replicate plates for the test compound and negative control or two replicate plates for the positive controls were incubated at 37°C, upside down, for 2 days.

Liquid preincubation assay (Yahagi et al., 1975; Matsushima et al., 1980)
In the modified procedure the following solutions are added in order:
- 0.1 ml of the test compound solutions or of the solvent or 0.05 ml of the different reference substances which were thawed shortly before use
- 0.5 ml of sodium phosphate buffered saline 0.2 M, pH 7.4 or 0.5 ml of the S9 mixture
- 0.1 ml of the overnight cultures of the bacterial strain.
The test tubes are incubated and shaken for 30 minutes at 37°C. 2.2 ml soft agar supplemented with 21 ug L-histidine and 24.4 ug biotin was added afterwards and the content of the tubes were mixed and poured on Vogel-Bonner minimal agar plates. Three replicate plates for the test compound and negative control or two replicate plates for the positive controls were incubated at 37°C, upside down, for 2 days.

Data reporting
Colonies are usually counted electronically using a DOMINO automatic image analysis system (Perceptive Instruments, Haverhill, Suffolk, England) after having inspected the E. Gocke, Report 167’801 -15-
background lawn for signs of toxicity. Plates exhibiting precipitate or contamination may be counted manually (for details see TABLES).
The microscopic examination of the bacterial background lawn, resulting from the trace of histidine added to the plates, is an aid to determine the toxicity of the test compound and is essential to the interpretation of results. The absence of a confluent lawn of bacteria was reported as BGR and the absence of bacterial lawn and/or presence of microcolonies as BGO. Inhibition of the growth was attributed to toxic effects by the substance (reported as t or T in the summary tables).

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 Salmonella typhimurium strains TA1535 and TA98. For strains 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.
Since it is impossible to define criteria that would apply to every configuration of data generated by the mutation assay, the study director is responsible for the ultimate decision in the evaluation of the results. The factors considered in making the decision are discussed in results and discussion.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

No increase in the number of revertant colonies was apparent for any of the five tester strains after treatment with Javanol.
It can be concluded that neither Javanol per se, nor any of the metabolites formed under the described experimental conditions used is mutagenic in the Ames test.

Executive summary:

In an in vitro reverse gene mutation assay in bacteria, performed according to OECD 471 (183, 1994) the EU Method B.14 (1992) and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA102) were treated with Javanol. A standard plate incorporation and pre-incubation modification assay were performed in absence and in presence of an exogenous metabolic activation system (S9). The activity of the S9 -mix and the responsiveness of the tester strains were verified by including appropriate controls into each experiment.

 

Javanol was dissolved in DMSO. A range finder study with strain TA100 was performed and toxic effects were observed in a at concentrations ≥50 micrograms/plate and ≥158 micrograms/plate in the absence and presence of S9, respectively. Based on the range finder, the concentrations selected for the standard plate incorporation assay were in the range of 1.6 to 158 micrograms/plate (-S9) and 5 to 500 micrograms/plate (+S9). For the pre-incubation test, concentration ranges of 0.5 to 50 micrograms/plate (-S9) and 1.58 to 158 micrograms/plate (+S9) were selected. Some turbidity was apparent at the two highest test concentrations. Toxicity of the compound was generally observed at the two highest test concentrations, with some variation depending on strain and test method.

 

No increase in the number of revertant colonies was apparent for any of the five tester strains after treatment with Javanol.

Thus it can be concluded that neither Javanol, nor any of the metabolites formed under the described experimental conditions used is mutagenic in the Ames test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 5 February 2020 to 28 February 2020

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 using the Hprt and xprt genes)

Deviations:

no

GLP compliance:

yes

Type of assay:

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

Specific details on test material used for the study:

Identification: Javanol
Description: Clear viscous colorless liquid

Target gene:

hypoxanthine-guanine phosphoribosyl transferase HPRT locus (hprt

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

in the presence and absence of an exogenous metabolic activation system, as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr). Dimethyl sulfoxide (DMSO) was used as the vehicle.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:

- source of S9 : The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 was purchased commercially.

- method of preparation of S9 mix :
The S9 mix was prepared on the day of use. The final concentrations of the components in the test system were as indicated below.
Final Concentration in Cultures
NADP (sodium salt): 0.8 mM
Glucose-6-phosphate: 1 mM
Calcium chloride: 2 mM
Potassium chloride: 6 mM
Magnesium chloride: 2 mM
Sodium Phosphate: 10 mM
S9 homogenate: 20 μL/mL

- quality controls of S9 : Each lot of S9 was assayed for sterility and its ability to metabolize at least two pro-mutagens to forms mutagenic to Salmonella typhimurium TA100.

Test concentrations with justification for top dose:

Definitive test:
Without activation: 1, 2, 3, 4, 6, 7, 8, 9, 10 and 12 μg/mL
With S9 mix activation: 25, 35, 45, 55, 65, 70 and 75 μg/mL

In the mutagenicity assay without S9, the average adjusted relative survival was 28.30% at concentration of 8 μg/mL without S9. Cultures treated at concentrations of 2, 3, 4, 6, 7 and 8 μg/mL without S9 were chosen for mutant selection.

In the mutagenicity assay with S9, the average adjusted relative survival was 20.98% at concentration of 55 μg/mL with S9. Cultures treated at concentrations of 25, 35, 45 and 55μg/mL with S9 were chosen for mutant selection.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Dimethyl sulfoxide (CAS 67-68-5) was used as the vehicle

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

In the absence of S9 Mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

In presence of S9 mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
Treatment: ~8 x 106 in 30 mL Complete Ham’s F12

Subculture for Phenotypic Expression and Initial Survival
After 5± 0.5 hours treatment and each 2-3 days : Cells were subcultured at ~2.4 x 10^6 cells/225-cm2 flask in 30 mL Complete Ham’s F12;
Culture for initial survival : 200 cells/60-mm plate in 5 mL Complete Ham’s F12


Mutant selection:
6 x 10^5 cells/150-mm plate in 30 mL; cloning efficiency: 200 cells/60-mm plate in 5 mL

TREATMENT AND HARVEST SCHEDULE:

- Exposure duration: 5 ± 0.5 hours


FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 days
- Selection time (if incubation with a selective agent): 7 days
- selective agent: 6-thioguanine (TG) at 10 μM 7 days exposure at the end of the phenotypic expression period.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2.4 x 10^6 cells from each culture were plated at a density of 6 x 10^5 cells/150-mm plate (4 plates total) in 30 mL Complete Ham’s F12-Hx containing 10 μM TG.
Three 60-mm plates also were plated, at 200 cells/plate in 5 mL Complete Ham’s F12-Hx in triplicate, to determine the cloning efficiency at the time of selection.
The plates were incubated under standard conditions for 7 days.
After the 7-day incubation period, the colonies were fixed with methanol, stained with crystal violet and counted. Mutant frequencies were expressed as the number of TGr mutants/106 clonable cells. The number of clonable cells was determined from the triplicate 60-mm plates.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method : the cytotoxicity was expressed as the adjusted relative survival (ARS)

Adjusted Relative Survival (ARS) = [(CE (treated) x cell density (treated)) / (CE (control) x cell density (control))] x 100%

Cloning Efficiency (CE) = (number of colonies / number of cells plated) x 100%

METHODS FOR MEASUREMENTS OF GENOTOXICIY

Mutant frequency = (number of mutant colonies / number of cells selected)/CE

Rationale for test conditions:

Seven or ten test substance concentrations, as well as the appropriate positive and vehicle controls, were tested in duplicate cultures with and without S9. The pH of the treatment medium was measured, and no pH adjustment was necessary to maintain neutral pH. Precipitation was assessed at the beginning and end of treatment.

Evaluation criteria:

The test substance was considered to have produced a positive response if it induced a dose-related increase in mutation frequency and an increase exceeding the 95% historical vehicle control limits in at least one test dose level(s) as compared with the concurrent vehicle control (p<0.01). If only one criterion was met (a statistically significant or dose-dependent increase or an increase exceeding the 95% historical control confidence interval), the result was considered equivocal. If none of these criteria were met, the results were considered to be negative.
Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations.

Statistics:

Statistical analyses were performed using the method of Snee and Irr (1981).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Based upon the definitive data generated with Duplicate Cultures, the concentrations selected for the mutagenicity assay were as indicated in the following table:

Treatment condition	Treatment time	Concentartion (μg/mL)
Non-activated	5 ± 0.5 hours	1, 2, 3, 4, 6, 7, 8, 9, 10 and 12
S9-activated	5 ± 0.5 hours	25, 35, 45, 55, 65, 70 and 75

In the mutagenicity assay without S9, no visible precipitate was observed at the beginning of treatment; however, visible precipitate was observed at concentrations beginning at 9 μg/mL by the end of treatment.

In the mutagenicity assay with S9, no visible precipitate was observed at the beginning of treatment; however, visible precipitate was observed at concentrations beginning at 65 μg/mL by the end of treatment. The test substance did not have an adverse impact on the pH of the cultures (pH 7.5 at the top dose).

In the mutagenicity assay without S9, the average adjusted relative survival was 28.30% at concentration of 8 μg/mL without S9. Cultures treated at concentrations of 2, 3, 4, 6, 7 and 8 μg/mL without S9 were chosen for mutant selection. Cultures treated at other concentrations were discarded prior to selection because a sufficient number of higher concentrations were available or due to excessive toxicity (RS<10%). No statistically significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated without S9 (p > 0.01). All responses were within 95% historical control limits.

In the mutagenicity assay with S9, the average adjusted relative survival was 20.98% at concentration of 55 μg/mL with S9. Cultures treated at concentrations of 25, 35, 45 and 55μg/mL with S9 were chosen for mutant selection. Cultures treated at higher concentrations were discarded prior to selection due to excessive toxicity (RS<10%). No statistically significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with S9 (p > 0.01). All responses were within 95% historical control limits. The positive controls induced significant increases in mutant frequency (p < 0.01).

All positive and vehicle control values were within acceptable ranges.

Conclusions:

Under the conditions of the assay described in this report, Javanol was concluded to be negative for the induction of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system.

Executive summary:

In an in vitro mammalian cell gene mutation assay performed according to the OECD test guideline No. 476 (2016) and in compliance with GLP, Chinese hamster ovary cells (CHO-K₁-BH₄) were exposed to the test material diluted in duplicate in the presence and absence of metabolic activation (S9) for 5 hrs. Based upon the definitive data, the concentrations selected for the mutagenicity assay were 25, 35, 45, 55, 65, 70 and 75 μg/mL with S9 and 1, 2, 3, 4, 6, 7, 8, 9, 10 and 12 μg/mL without S9. In the mutagenicity assay without S9, no visible precipitate was observed at the beginning of treatment; however, visible precipitate was observed at concentrations beginning at 9 μg/mL by the end of treatment. The average adjusted relative survival was 28.30% at concentration of 8 μg/mL without S9. Cultures treated at concentrations of 2, 3, 4, 6, 7 and 8 μg/mL without S9 were chosen for mutant selection. Cultures treated at other concentrations were discarded prior to selection because a sufficient number of higher concentrations were available or due to excessive toxicity (RS<10%). No statistically significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated without S9 (p > 0.01). All responses were within 95% historical control limits. The positive controls induced significant increases in mutant frequency (p < 0.01). In the mutagenicity assay with S9, no visible precipitate was observed at the beginning of treatment; however, visible precipitate was observed at concentrations beginning at 65 μg/mL by the end of treatment. The average adjusted relative survival was 20.98% at concentration of 55 μg/mL with S9. Cultures treated at concentrations of 25, 35, 45 and 55μg/mL with S9 were chosen for mutant selection. Cultures treated at higher concentrations were discarded prior to selection due to excessive toxicity (RS<10%). No statistically significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with S9 (p > 0.01). All responses were within 95% historical control limits. The positive controls induced significant increases in mutant frequency (p < 0.01). These results indicate Javanol was negative for the ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

No mutagenic properties was demonstrated in any of the studies available on Javanol.

Additional information

Ames

In an in vitro reverse gene mutation assay in bacteria, performed according to OECD 471 (183, 1994) the EU Method B.14 (1992) and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA102) were treated with Javanol. A standard plate incorporation and pre-incubation modification assay were performed in absence and in presence of an exogenous metabolic activation system (S9). The activity of the S9 -mix and the responsiveness of the tester strains were verified by including appropriate controls into each experiment.

 

Javanol was dissolved in DMSO. A range finder study with strain TA100 was performed and toxic effects were observed in a at concentrations ≥50 micrograms/plate and ≥158 micrograms/plate in the absence and presence of S9, respectively. Based on the range finder, the concentrations selected for the standard plate incorporation assay were in the range of 1.6 to 158 micrograms/plate (-S9) and 5 to 500 micrograms/plate (+S9). For the pre-incubation test, concentration ranges of 0.5 to 50 micrograms/plate (-S9) and 1.58 to 158 micrograms/plate (+S9) were selected. Some turbidity was apparent at the two highest test concentrations. Toxicity of the compound was generally observed at the two highest test concentrations, with some variation depending on strain and test method.

 

No increase in the number of revertant colonies was apparent for any of the five tester strains after treatment with Javanol.

Thus it can be concluded that neither Javanol, nor any of the metabolites formed under the described experimental conditions used is mutagenic in the Ames test.

CAT

In anin vitrochromosome aberration test performed according to OECD Guideline 473 and in compliance with GLP, cultured V79 Chinese hamster cells were exposed to Javanol both the absence and the presence of a metabolic activation system (S9-mix) in two independent experiments. Two independent chromosome aberration assays were conducted. The substance was dissolved in DMSO prior to testing.Following study design was performed:

 

without S9-mix				with S9-mix
	exp. I		exp. 1I	exp. I	exp. 1I
Exposure period	4h	18 h	28 h	4h	4h
Recovery	14 h	-	-	14 h	24 h
Preparation interval	18 h	18 h	28 h	18 h	28 h

 

Doses applied were:

Without S9

Experiment 1: 4hr- 5, 10, 20, 30, 40, 50 µg/mL; 18hr- 2.5, 5, 10, 20, 30, 40 µg/ml

Experiment 2: 28hr- 1.4, 2.5, 5, 7.5, 10, 15 µg/mL

With S9

Experiment 1: 4hr- 7.5, 15, 30, 45, 60, 75 µg/mL

Experiment 2: 4hr- 3.8, 7.5, 15, 30, 45, 60 µg/mL

 

Toxic effects indicated by clearly reduced cell numbers and/or mitotic indices were observed in all experimental parts of the cytogenecity study. At the highest concentration which was evaluated for cytogenetic damage, clear toxicity was observed. In both independent experiments, neither a significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls and they induced statistically significant increases in cells with structural chromosome aberrations indicating the validity of the test. Based on these results, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells in vitro. Therefore, Javanol is considered to be non-clastogenic in this chromosome aberration test.

Mammalian gene mutation

In anin vitromammalian cell gene mutation assay performed according to the OECD test guideline No. 476 (2016) and in compliance with GLP, Chinese hamster ovary cells (CHO-K₁-BH₄) were exposed to the test material diluted, in duplicate in the presence and absence of metabolic activation (S9) for 5hrs.  Based upon the definitive data, the concentrations selected for the mutagenicity assay were 25, 35, 45, 55, 65, 70 and 75 μg/mL with S9 and 1, 2, 3, 4, 6, 7, 8, 9, 10 and 12 μg/mL without S9. In the mutagenicity assay without S9, no visible precipitate was observed at the beginning of treatment; however, visible precipitate was observed at concentrations beginning at 9 μg/mL by the end of treatment. The average adjusted relative survival was 28.30% at concentration of 8 μg/mL without S9. Cultures treated at concentrations of 2, 3, 4, 6, 7 and 8 μg/mL without S9 were chosen for mutant selection. Cultures treated at other concentrations were

discarded prior to selection because a sufficient number of higher concentrations were available or due to excessive toxicity (RS<10%). No statistically significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated without S9 (p > 0.01). All responses were within 95% historical control limits. The positive controls induced significant increases in mutant frequency (p < 0.01). In the mutagenicity assay with S9, no visible precipitate was observed at the beginning of treatment; however, visible precipitate was observed at concentrations beginning at 65 μg/mL by the end of treatment. The average adjusted relative survival was 20.98% at concentration of 55 μg/mL with S9. Cultures treated at concentrations of 25, 35, 45 and 55μg/mL with S9 were chosen for mutant selection. Cultures treated at higher concentrations were discarded prior to selection due to excessive toxicity (RS<10%). No statistically significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with S9 (p > 0.01). All responses were within 95% historical control limits. The positive controls induced significant increases in mutant frequency (p < 0.01). These results indicate Javanol was negative for the ability to induce forward mutations at the

hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system.

Under the test conditions, the test material was considered to be non-mutagenic to CHO ells according according to the Regulation (EC) N° 1272-2008 and according to the GHS. This study is considered as acceptable and satisfies the requirement for the mammalian cell gene mutation endpoint. 

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.