Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Genetic toxicity in vitro: Negative. Three studies negative (Haddouk HG, 2003) Genetic toxicity in bacteria (OECD 471), (Brient A., 2016) Chromosome aberration (OECD 473), (Brient A., 2016) Mammalian gene mutation (OECD 476).

Link to relevant study records

Referenceopen allclose all

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:
From 27 may 2003 to 27 august 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from rat liver cells
Test concentrations with justification for top dose:
Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.
Experiments without S9 mix:
The selected treatment-levels were:
• 31.3, 62.5, 125, 250 and 500 μg/plate, for the TA 102 in both experiments,
• 7.81, 15.63, 31.3, 62.5 and 125 μg/plate for the TA 100, TA 98 and TA 1535 strains in both experiments and for the TA 1537 strain in the first experiment,
• 1.95, 3.9, 7.81, 15.63 and 31.25 μg/plate for the TA 1537 strain in the second experiment.
Experiments with S9 mix:
The selected treatment-levels were:
• 62.5, 125, 250, 500 and 1000 μg/plate, for the TA 102 in both experiments,
• 31.3, 62.5, 125, 250 and 500 μg/plate, for the TA 1535, TA 1537 and TA 100 strains in the first experiment and for the TA 98 strain in both experiments,
• 15.63, 31.3, 62.5, 125 and 250 μg/plate in the second experiment with the TA 1535, TA 1537 and TA 100 strains.
Vehicle / solvent:
Vehicle used: The vehicle was dimethylsulfoxide (DMSO), batch No. K30379650 214 (Merck Eurolab, Fontenay-Sous-Bois, France).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see details on test system and conditions
Details on test system and experimental conditions:
Positive controls:
Without S9 mix
. sodium azide (NAN3) 1 µg/plate for TA 1535 - TA 100
. 9-Aminoacridine (9AA) 50 µg/plate for TA 1537
. 2-Nitrofluorene (2NF) 0.5 µg/plate for TA 98
. Mitomycin C (MMC) 0.5 µg/plate for TA 102

With S9 mix
. 2-Anthramine (2AM) 2 µg/plate for TA 1535 - TA 1537 - TA 98 - TA 100 and 10 µg/plate for TA 102

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation method.
Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C).

DURATION
- Preincubation period: 60 min at 37°C
- Exposure duration: 48 to 72 hours
- Expression time (cells in growth medium): the number of revertants is determined at the end of the exposure time.

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: all revertants per plates are counted
Evaluation criteria:
Acceptance criteria
This study is considered valid if the following criteria are fully met:
- the number of revertants in the vehicle controls is consistent with the historical data of the testing facility,
- the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with the historical data of the testing facility.

Evaluation criteria
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
Statistics:
no data
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary toxicity test:
The test item was freely soluble in the vehicle (DMSO) at 50 mg/mL.
Consequently, with a treatment volume of 100 μL/plate, the dose-levels were 10, 100, 500, 1000, 2500 and 5000 μg/plate.
A moderate to marked emulsion was observed in the Petri plates when scoring the revertants at all dose-levels ≥ 1000 μg/plate.
Without S9 mix, moderate to strong toxicity was induced at dose-levels ≥ 100 μg/plate (TA 98 and TA 100 strains) or ≥ 500 μg/plate (TA 102 strain).
With S9 mix, moderate to strong toxicity was induced at dose-levels ≥ 500 μg/plate (TA 98 and TA 100 strains) or ≥ 1000 μg/plate (TA 102 strain).

see details on results in attached document

Conclusions:
Interpretation of results: negative with and without metabolic activation

Under our experimental conditions, the test item Isobornylcyclohexanol (Rhodiantal IBCH) did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.
Executive summary:

This study was performed to investigate the potential of the test item, Isobornylcyclohexanol (Rhodiantal IBCH), to induce reverse mutation in Salmonella typhimurium. The study was performed according to OECD guideline no. 471 and EC guideline n° B13/14 and in compliance with the Principles of Good Laboratory Practice. 

A preliminary toxicity test was performed to define the dose-levels of Isobornylcyclohexanol (Rhodiantal IBCH) to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C).

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test item Isobornylcyclohexanol was dissolved in dimethylsulfoxide (DMSO).

Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

Experiments without S9 mix:

The selected treatment-levels were:

• 31.3, 62.5, 125, 250 and 500μg/plate, for the TA 102 in both experiments,

• 7.81, 15.63, 31.3, 62.5 and 125μg/plate for the TA 100, TA 98 and TA 1535 strains in both experiments and for the TA 1537 strain in the first experiment,

• 1.95, 3.9, 7.81, 15.63 and 31.25μg/plate for the TA 1537 strain in the second experiment.

 

Experiments with S9 mix:

The selected treatment-levels were:

• 62.5, 125, 250, 500 and 1000μg/plate, for the TA 102 in both experiments,

• 31.3, 62.5, 125, 250 and 500μg/plate, for the TA 1535, TA 1537 and TA 100 strains in the first experiment and for the TA 98 strain in both experiments,

• 15.63, 31.3, 62.5, 125 and 250μg/plate in the second experiment with the TA 1535, TA 1537 and TA 100 strains.

 

Without S9 mix, moderate to marked toxicity was noted in the TA 1537 and TA 100 strains at dose-levels ≥ 31.3μg/plate, in the TA 98 and TA 1535 strains at dose-levels ≥ 125μg/plate and in the TA 102 strain at 500μg/plate.

With S9 mix, moderate to marked toxicity was noted in the TA 1535 and TA 100 strains at dose-levels ≥ 125μg/plate, in the TA 98 and TA 1537 strains at dose-levels ≥ 250μg/plate and in the TA 102 strain at dose-levels ≥ 500μg/plate.

The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.

Under our experimental conditions, the test item Isobornylcyclohexanol (Rhodiantal IBCH) did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

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 10 June 2015 to 02 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
5 march 2015
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
primary culture, other: human lymphocytes
Details on mammalian cell type (if applicable):
Cultures of human lymphocytes were prepared from whole blood samples obtained from young, healthy, non-smoking donors and collected into heparinized sterile tubes.
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction (Moltox, Molecular Toxicology, INC, Boone, USA), obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by intraperitoneal route, was used as the metabolic activation system.
Test concentrations with justification for top dose:
Based on available solubility data, the highest dose-level selected for the first experiment was 4000 μg/mL since it was expected to produce precipitation in the culture medium. This dose-level was obtained using a test item concentration of 400 mg/mL and a treatment volume of 55 μL/5.5 mL culture medium.
The dose-levels used for treatment were as follows:
- 31.3, 62.5, 125, 250, 500, 1000, 2000 and 4000 μg/mL for the first experiment with S9 mix,
- 56.9, 62.5, 125, 250, 500, 1000, 2000 and 4000 μg/mL for the first experiment without S9 mix,
- 3.13, 6.25, 12.5, 25, 50 and 100 μg/mL for the second experiment with and without S9 mix and for the third experiment with S9 mix,
- 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 μg/mL for the third experiment without S9 mix.
Vehicle / solvent:
- Vehicle used: According to available solubility data, the vehicle was dimethylsulfoxide (DMSO), batch Nos. K44917950345 and K46301950506.
- Justification for choice of solvent/vehicle: based on available solubility data.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
(MMC) without metabolic activation: at 2 and 3 µg/mL for the 3-h treatment and at 0.2 and 0.3 µg/mL for the continuous treatment
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
(CPA) with metabolic activation: at 12.5 and 25 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: no
- Exposure duration: 3 hours (first experiment, second experiment with and without S9-mix and third experiment with S9-mix), 20 hours (third experiment without S9-mix).
- Expression time (cells in growth medium): 20 hours
- "Selection" time: 3 h before harvesting
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid solution (three hours before harvest).

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: two

NUMBER OF CELLS EVALUATED: Analysis of 300 metaphases / dose level was made, with 150 metaphases/culture.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index [MI] (number of cells in mitosis/number of cells examined)

OTHER:
- Scoring method: The following structural aberrations were recorded for each metaphase: gaps, chromatid and chromosome breaks and exchanges, and others (multiple aberrations and pulverizations). In addition, the following numerical aberrations were recorded when encountered: polyploidy, endoreduplication and hyperdiploidy.
Evaluation criteria:
ACCEPTANCE CRITERIA
The study was considered valid if all the following criteria were met:
- the frequency of cells with structural chromosome aberration in the vehicle controls was consistent with (but not necessary within) the historical data. In any case, this frequency should be ≤ 5%,
- the frequency of cells with structural chromosome aberration in the positive controls was statistically higher than that of the vehicle controls (p ≤ 0.05) and consistent with (but not necessary within) the historical data.

EVALUATION CRITERIA
- Evaluation of a positive response: a test item is considered clearly positive for inducing chromosomal aberrations if, in any of the experimental conditions examined:
. a statistically significant increase in the frequency of cells with structural chromosome aberration is observed at one or more dose-levels,
. a dose-response relationship is demonstrated by a statistically significant trend test,
. any of the results are above the historical data range of the corresponding vehicle control.
- Evaluation of a negative response: a test item is considered clearly negative for inducing chromosomal aberrations if, in all experimental conditions:
. no statistically significant increase is observed in the frequency of cells with structural chromosome aberration at any of the analyzed dose-levels,
. no dose-response relationship is demonstrated by a statistically significant trend test,
. all results are within or below the historical data range of the corresponding vehicle control.
Statistics:
For each condition of the cytogenetic experiment, the frequency of cells with structural chromosome aberration (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. This comparison was performed using the χ2 test unless treated culture data were lower than or equal to the vehicle control data. P = 0.05 was used as the lowest level of significance. This statistical analysis was performed using a validated Excel sheet.
To assess the dose-response trend, a linear regression was performed between the frequencies of cells with structural chromosome aberration (excluding gaps) and the dose-levels. This statistical analysis was performed using SAS Enterprise Guide software.
Species / strain:
primary culture, other: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
see in "any other information on results incl. tables" below for details
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see in "any other information on results incl. tables" below for details
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
At the highest tested dose-level (i.e. 4000 μg/mL), the pH of the culture medium was approximately 6.8 (as for the vehicle control) and the osmolality equal to 388 mOsm/kg H2O (466 mOsm/kg for the vehicle control). Therefore, none of the tested dose-levels was considered to produce extreme culture conditions.
A precipitate was observed in the culture medium at the end of the treatment periods, at dose-levels ≥ 62.5 μg/mL in the first experiment and at 100 μg/mL in the second and third experiments.
The frequencies of cells with structural chromosome aberrations for the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered to be valid.

see details on results in attached document

Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Under the experimental conditions of this study, the test item, Rhodiantal original IBCH, did not induce chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a rat liver metabolizing system.
Executive summary:

The objective of this study was to evaluate the potential of the test item, Rhodiantal original IBCH, to induce chromosome aberrations in cultured human lymphocytes. The study was performed according to the international guidelines (OECD 473 and Commission Directive B10) and in compliance with the Principles of Good Laboratory Practice Regulations.

The test item, dissolved in dimethylsulfoxide (DMSO), was tested in three independent experiments, both with and without a liver metabolizing system (S9 mix), obtained from rats previously treated with Aroclor 1254. The highest dose-level selected for the first experiment was chosen on the basis of available solubility data, since precipitate was expected to be observed in the culture medium. The dose-levels selection for the second and third experiments was based on the level of cytoxicity observed in the first experiment, indicated by the reduction of Mitotic Index (MI). For each culture, heparinized whole blood was added to culture medium containing a mitogen (phytohemagglutinin) and incubated at 37°C, for about 48 hours. In the first and second experiments both with and without S9 mix, as well as in the third experiment with S9 mix, lymphocyte cultures were exposed to the test or control items for 3 hours and then rinsed. Then, the cultures were incubated in fresh medium at 37°C until harvest. Harvest time was 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles. The third experiment without S9 mix exposed the cells continuously to the test or control items until harvest. Harvest time was 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles. Three hours before harvest, each culture was treated with a Colcemid® solution (10 μg/mL) to block cells at the metaphase-stage of mitosis. After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring. The MI was first of all evaluated on each slide. Then, structural and numerical aberrations were recorded for cultures treated in the absence and presence of S9 mix with three dose-levels of the test item, the vehicle and positive controls.

The frequencies of cells with structural chromosome aberration of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid. Based on available solubility data, the highest dose-level selected for the first experiment was 4000μg/mL since it was expected to produce precipitation in the culture medium. This dose-level was obtained using a test item concentration of 400 mg/mL and a treatment volume of 55 μL/5.5 mL culture medium.

The dose-levels used for treatment were as follows:

- 31.3, 62.5, 125, 250, 500, 1000, 2000 and 4000 μg/mL for the first experiment with S9 mix,

- 56.9, 62.5, 125, 250, 500, 1000, 2000 and 4000 μg/mL for the first experiment without S9 mix,

 - 3.13, 6.25, 12.5, 25, 50 and 100 μg/mL for the second experiment with and without S9 mix and for the third experiment with S9 mix,

- 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 μg/mL for the third experiment without S9 mix.

At the highest tested dose-level (i.e. 4000 μg/mL), the pH of the culture medium was approximately 6.8 (as for the vehicle control) and the osmolality equal to 388 mOsm/kg H2O (466 mOsm/kg for the vehicle control). Therefore, none of the tested dose-levels was considered to produce extreme culture conditions.

A precipitate was observed in the culture medium at the end of the treatment periods, at dose-levels ≥ 62.5 μg/mL in the first experiment, and at 100 μg/mL in the second and third experiments.

Experiments without S9 mix

Cytotoxicity

In the first experiment, a marked to severe cytotoxity was observed at dose-levels ≥ 125 μg/mL, as shown by a 76 to 100% decrease in the MI.

In the second experiment, a moderate cytotoxity was observed at dose-levels of 6.25 and 100 μg/mL, as shown by a 42 to 58% decrease in the MI.

In the third experiment, a marked cytotoxicity was noted at the highest tested dose-level of 100 μg/mL, as shown by a 63% decrease in the MI.

Metaphase analysis

As only two dose-levels remained available for metaphase analysis in the first experiment instead of three, no metaphase analysis was done for this experiment.

The dose-levels selected for metaphase analysis in the other experiments were as follows:

- 25, 50 and 100 μg/mL for the 3-hour treatment (second experiment), the latter showing precipitate in the culture medium at the end of treatment,

- 12.5, 25 and 50 μg/mL for the 20-hour treatment (third experiment), the latter induced only 22% decrease in the MI but the higher dose-level was too cytotoxic.

No noteworthy increase in the frequency of cells with structural chromosomal aberrations was noted at any of the analyzed dose-levels, either after the 3- or 20-hour treatments.

Experiments with S9 mix

Cytotoxicity

In the first experiment, a severe cytotoxity was observed at dose-levels ≥ 125 μg/mL, as shown by a 100% decrease in the MI.

In the second experiment, a moderate to severe cytotoxity was observed at dose-levels ≥ 50 μg/mL, as shown by a 53 to 100% decrease in the MI.

In the third experiment, a slight to severe cytotoxity was observed at dose-levels ≥ 50μg/mL, as shown by a 25 to 100% decrease in the MI.

Metaphase analysis

As only two dose-levels remained available for metaphase analysis in the first experiment instead of three, no metaphase analysis was done for this experiment.

The dose-levels selected for metaphase analysis were:

- 12.5, 25 and 50 μg/mL for the second experiment, the latter inducing a 53% decrease in the MI,

- 12.5, 25 and 50 μg/mL for the third experiment, the latter induced only 25% decrease in the MI but the higher dose-level was too cytotoxic.

No noteworthy increase in the frequency of cells with structural chromosomal aberrations was noted at any of the analyzed dose-levels, in either experiment.

Under the experimental conditions of this study, the test item, Rhodiantal original IBCH, did not induce chromosome aberrations in cultured human lymphocytes, either in the presence or the absence of a rat liver metabolizing system.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 10 June 2015 to 21 April 2016
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
5 march 2015
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from rat liver cells
Test concentrations with justification for top dose:
Without S9 mix:
- 1.25, 2.5, 5, 10, 20 and 10 µg/mL for the first experiment (3-hour treatment),
- 1.25, 2.5, 5, 10, 20, 40 and 60 µg/ml for the second experiment (24-hour treatment).

With S9 mix:
- 2.5, 5, 10, 20, 40 and 80 µg/mL for the first experiment,
- 2.5, 5, 10, 20, 40, 60 and 80 µg/ml for the second experiment
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethylsulfoxide (DMSO), batch Nos. K44917950345 and K46301950506
- Justification for choice of solvent/vehicle: based on solubility data obtained at CiToxLAB France
Untreated negative controls:
yes
Remarks:
solvent served as negative control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Remarks:
With S9-mix: CPA: 3 µg/mL; Without S9-mix: MMS: 25 (3-h treatment) or 5 µg/mL (24-h treatment)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;
The culture medium was RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL).
This medium (RPMI 0) was supplemented by heat inactivated horse serum at 10%, v/v (RPMI 10) or 20%, v/v (RPMI 20).
RPMI 10 was diluted with RPMI 0 (1:1, v/v) in order to obtain RPMI 5 which was used for the 3 hour treatment.


DURATION
- Preincubation period: 48 hours at 37°C
- Exposure duration: 3 or 24 hours
- Expression time (cells in growth medium): For the 3-hour treatment, cultures of 20 mL at 5 x 10E5 cells/mL in RPMI 5 were treated in 50-mL tubes with the test or control items for 3 hours, in a 37°C, 5% CO2 humidified incubator.
For the 24-hour treatment, cultures of 50 mL at 2 x 10E5 cells/mL in RPMI 10 were treated in flasks with the test or control items for 24 hours, in a 37°C, 5% CO2 humidified incubator (flasks were gently shaken at least once).


DETERMINATION OF CYTOTOXICITY
other: Preliminary toxicity test. The cytotoxicity was evaluated by cloning efficiency.


OTHER:
At the end of the treatment periods, the cells were rinsed. After centrifugation and removal of the supernatant, the pellets were suspended in RPMI 10 and the cells were counted using a hemocytometer. When sufficient cells survived, the cell concentrations were adjusted to 2 x 105 cells/mL and then, to enable the expression of the mutant phenotype, the cells were incubated in RPMI 10 medium for 48 hours, in a 37°C, 5% CO2 humidified incubator.
During the mutant phenotype expression period, the cultures were maintained at the density of approximately 2 x 105 cells/mL, whenever possible.
At the end of this expression period, the cell density of each culture was determined using a hemocytometer and the cells were seeded after serial dilutions as follows.

Viability plates
To define the number of viable cells (CE2: Cloning Efficiency at the end of the expression period), an average of 1.6 cells/well were seeded in two 96-well plates/culture (four plates/dose-level). After at least 7 days of incubation in a 37°C, 5% CO2 humidified incubator, the clones were counted.

Mutant plates
To select the TFTR (trifluorothymidine resistant) mutant cells (for the determination of CEmutant), 2000 cells/well were seeded in four 96-well plates/culture (eight plates/dose-level). After 11-12 days of incubation in a 37°C, 5% CO2 humidified incubator in the presence of 4 µg TFT/mL of culture medium, the clones were counted, differentiating small and large colonies:
- size of small colonies: < 25% of the diameter of the well,
- size of large colonies: > 25% of the diameter of the well.

The following parameters were used for the scoring of colonies in mutant plates:
- well containing mutant colony (small or large),
- well not containing mutant colony,
- when both small and large colonies are present in the same well both mutant colonies were counted (one small and one large).
Evaluation criteria:
IWGT recommendations were followed for the determination of a positive result, which should fulfill the following criteria:
- at least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the Global Evaluation Factor (GEF) of 126 x 10E-6,
- a dose-response relationship is demonstrated by a statistically significant trend test.

Unless an effect is considered as clearly positive, the reproducibility of a positive effect should be confirmed.

Noteworthy increases in the mutation frequency observed only at high-levels of cytotoxicity (Adj. RTG lower than 10%), but with no evidence of mutagenicity at dose-levels with Adj. RTG between 10 and 20%, are not considered as positive results.
A test item may be considered as non-mutagenic when there is no culture showing an Adj. RTG value between 10 and 20% if :
- there is at least one negative data point between 20 and 25% Adj. RTG and no evidence of mutagenicity in a series of data points between 100 and 20% Adj. RTG,
- there is no evidence of mutagenicity in a series of data points between 100 and 25% and there is also a negative data point between 10 and 1% Adj. RTG.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TEST:
Based on available solubility data, the highest dose-level selected for the preliminary test was 4000 μg/mL since it was expected to produce precipitation in the culture medium. This dose-level was obtained using a test item concentration of 400 mg/mL and a treatment volume of 1% (v/v) in the culture medium (i.e. 200 μL/20 mL culture medium for the 3-hour treatments and 500 μL/50 mL culture medium for the 24-hour treatment).
The dose-levels selected for the treatment of the first preliminary test were 31.3, 62.5, 125, 250, 500, 1000, 2000 and 4000 μg/mL.

At the highest tested dose-level (i.e. 4000 μg/mL), the pH of the culture medium was approximately 6.8 (as for the vehicle control) and the osmolality was equal to 371 mOsm/kg H2O (434 mOsm/kg H2O for the vehicle control). Thus, none of the tested dose-levels was considered to produce extreme culture conditions.

At the end of the 3-hour treatment periods, a precipitate was observed in the culture medium at all tested dose-levels.
At the end of the 24-hour treatment period, a precipitate was observed in the culture medium at dose-levels ≥ 125 μg/mL.

Following the 3-hour treatment without S9 mix, a slight to severe toxicity was induced at all tested dose-levels, as shown by a 57 to 100% decrease in the Adj. RTG.
Following the 24-hour treatment (without S9 mix), a moderate to severe toxicity was induced at all tested dose-levels, as shown by a 77 to 100% decrease in the Adj. RTG.
Following the 3-hour treatment with S9 mix, a moderate to severe toxicity was induced at dose-levels ≥ 62.5 μg/mL, as shown by a 77 to 100% decrease in the Adj. RTG.

As all the tested dose-levels in this preliminary test revealed precipitate in the culture medium at the end of the treatment periods and/or cytoxicity, the selection of dose-levels for the main experiments was not apparent. Consequently, in agreement with the Sponsor, a second preliminary test was implemented under the same experimental conditions, but using a lower range of dose-levels as follows: 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 μg/mL.

At the end of the 3- and 24-hour treatment periods, a precipitate was observed in the culture medium at dose-levels ≥ 25 μg/mL.

Following the 3-hour treatment without S9 mix, a severe toxicity was induced at dose-levels ≥ 50 μg/mL, as shown by a 100% decrease in the Adj. RTG.
Following the 24-hour treatment without S9 mix, a slight to severe toxicity was induced at dose-levels ≥ 25 μg/mL, as shown by a 53 to 100% decrease in the Adj. RTG.
Following the 3-hour treatment with S9 mix, a severe toxicity was induced at the highest tested dose-level of 100 μg/mL, as shown by a 100% decrease in the Adj. RTG.


see details on results in attached document

Conclusions:
Interpretation of results: negative with and without metabolic activation

Under the experimental conditions of this study, the test item Rhodiantal original IBCH did not show any mutagenic activity in the mouse lymphoma assay, in the presence or in the absence of a rat metabolizing system.
Executive summary:

The objective of this study was to evaluate the potential of the test item, Rhodiantal original IBCH, to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK+/- mouse lymphoma cells. The study was performed according to the international guidelines (OECD 476 and Commision Directive B17) and in compliance with Principles of Good Laboratory Practice.

After two preliminary toxicity tests, the test item, Rhodiantal original IBCH, dissolved in dimethylsulfoxide (DMSO), was tested in two independent main experiments, with and without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Cultures of 20 mL at 5 x 105 cells/mL (3-hour treatments) or cultures of 50 mL at 2 x 105 cells/mL (24-hour treatment) were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment periods, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% (3-hour treatment) or 10% (24-hour treatment) in a 37°C, 5% CO2 humidified incubator. For the 24-hour treatment, flasks were gently shaken at least once.

Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2). The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

With one exception which was not considered to have compromised the integrity or validity of the study, the cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria.

For the positive control cultures, the increase in the mutation frequencies met also the acceptance criteria. In addition, the upper limit of cytotoxicity observed in the positive control cultures had an Adj. RTG greater than 10%. The study was therefore considered to be valid.

Since the test item was found poorly soluble and cytotoxic in the preliminary tests, the selection of the highest dose-level for the main experiments was based on the level of precipitate in the culture medium and cytotoxicity, according to the criteria specified in the international guidelines.

A precipitate remaining in the culture medium was observed at dose-levels ≥ 20μg/mL at the end of the treatments of the first main experiment (3-hour treatments with and without S9 mix), and at dose-levels ≥ 40μg/mL at the end of the treatments of the second main experiment (3-hour treatment with S9 mix and 24-hour treatment without S9 mix). This precipitate did not prevent any scoring.

Experiments without S9 mix

The selected dose-levels were as follows:

-1.25, 2.5, 5, 10, 20 and 40μg/mL for the 3-hour treatment,

-1.25, 2.5, 5, 10, 20, 40 and 60μg/mL for the 24-hour treatment.

Cytotoxicity

In the 3-hour treatment, a severe cytoxicity was induced at the highest tested dose-level of 40μg/mL, as shown by a 100% decrease in the Adj. RTG.

In the 24-hour treatment, a slight to severe cytoxicity was induced at dose-levels ≥ 10μg/mL, as shown by a 51 to 100% decrease in the Adj. RTG.

Mutagenicity

In the 3-hour treatment, no increase in the mutation frequency was observed at any of the tested dose-levels inducing acceptable level of cytotoxicity (i.e.Adj. RTG greater than 10%).

In the 24-hour treatment, slight increases in the mutation frequency were observed at the two lowest tested dose-levels. However, these increases were not dose-related (p > 0.05), and the highest IMF (Induced Mutation Frequency) remained substantially below the GEF (Global Evaluation Factor) of 126 x 10-6 mutants.

No dose-level showing an Adj. RTG between 10 and 20% was observed. However, there was no evidence of mutagenicity in a series of data points between 20 and 100%, and there was also a negative data point at 40μg/mL, showing an Adj. RTG < 10%.

These results were thus considered to meet the criteria of a negative response.

Experiments with S9 mix

The selected dose-levels were as follows:

.2.5, 5, 10, 20, 40 and 80μg/mL for the first experiment,

.2.5, 5, 10, 20, 40, 60 and 80μg/mL for the second experiment.

Cytotoxicity

In the first experiment, a severe toxicity was induced at the highest tested dose-level of 80μg/mL, as shown by a 98% decrease in the Adj. RTG.

In the second experiment, a moderate to severe toxicity was induced at dose-levels ≥ 60μg/mL, as shown by a 74 to 100% decrease in the Adj. RTG.

Mutagenicity

In the first experiment, no increase in the mutation frequency was noted at dose-levels up to 40μg/mL, inducing acceptable level of cytotoxicity (i.e.Adj. RTG greater than 10%).

Furthermore, a statistical analysis performed at dose-levels up to 40μg/mL did not demonstrate any linear trend between the mutation frequency and the dose (p > 0.05).

An increase in the mutation frequency exceeding the GEF was noted at the highest tested dose-level of 80μg/mL. However this dose-level induced excessive cytotoxicity, with more than 90% decrease in the Adj. RTG. This increase was thus not considered to be biologically relevant.

In the second experiment, slight increases in the mutation frequency were observed up to the dose-level of 60μg/mL, inducing acceptable level of cytotoxicity (i.e.Adj. RTG greater than 10%). However, these increases were not dose-related (p > 0.05), and the highest IMF remained substantially below the GEF.

These results were considered to meet the criteria of a negative response.

 

Under the experimental conditions of this study, the test item, Rhodiantal original IBCH, did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro

Bacterial gene mutation: One fully reliable study is available (CitoxLab, 2003), conducted according to OECD TG 471 and GLP (bacterial reverse mutation assays (Ames test)). The test item did not induce any noteworthy increase in the number of revertants, either with or without S9 mix, in any of the five strains.

Therefore, Phenol, 2-methoxy-, reaction products with 2,2- dimethyl-3-methylenebicyclo[2.2.1]heptane, hydrogenated is considered not to be mutagenic in this Salmonella typhimurium assay.

Mammalian gene mutation: One fully reliable study is available (CitoxLab, 2016), conducted according to OECD TG 476 and GLP (mouse lymphoma assay, 1.25 – 80 µg/mL, with and without liver microsomal activation, 3 and 24 h of treatment). The test item did not induce mutations in the thymidine kinase locus assay using the mouse lymphoma cell line L5178Y up to the highest tested concentrations.

Therefore,Phenol, 2-methoxy-, reaction products with 2,2- dimethyl-3-methylenebicyclo[2.2.1]heptane, hydrogenated is considered not to be mutagenic in this mouse lymphoma assay.

Mammalian chromosome aberration: One fully reliable study is available (CitoxLab, 2016) conducted according to OECD TG 473 and GLP (chromosome aberrations assay in cultured human lymphocytes, with and without liver microsomal activation, 3-, and 20- hour treatments).

The dose-levels used for treatment were as follows:

- 31.3, 62.5, 125, 250, 500, 1000, 2000 and 4000μg/mL for the first experiment with S9 mix,

- 56.9, 62.5, 125, 250, 500, 1000, 2000 and 4000μg/mL for the first experiment without S9 mix,

 - 3.13, 6.25, 12.5, 25, 50 and 100μg/mL for the second experiment with and without S9 mix and for the third experiment with S9 mix,

- 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100μg/mL for the third experiment without S9 mix.

No noteworthy increase in the frequency of cells with structural chromosomal aberrations was noted in both experiments, at both harvest times and in both the absence and presence of S9 mix.

Therefore, Phenol, 2-methoxy-, reaction products with 2,2- dimethyl-3-methylenebicyclo[2.2.1]heptane, hydrogenated is considered not to be clastogenic in this in vitro mammalian chromosome aberration assay.

In vivo

No data available. Based on REGULATION (EC) No 1907/2006 as at July 2011 and the absence of positive results in the three above mentioned in vitro tests no additional testing for genetic toxicity in vivo is necessary.  

 

Conclusion: 

For each endpoint (bacterial gene mutation, mammalian gene mutation, & mammalian chromosome aberration) reliable and GLP compliant in vitro studies are available that all gave negative results.

Therefore it can be concluded that Phenol, 2-methoxy-, reaction products with 2,2- dimethyl-3-methylenebicyclo[2.2.1]heptane, hydrogenated is neither clastogenic or aneugenic nor mutagenic. Accordingly it can be concluded that Phenol, 2-methoxy-, reaction products with 2,2- dimethyl-3-methylenebicyclo[2.2.1]heptane, hydrogenated is not genotoxic.

Justification for classification or non-classification

Harmonized classification:

No harmonized classification is available according to the Regulation (EC) No 1272/2008.

Self classification:

Three in vitro genotoxicity tests are available. None of the tests showed evidence of genotoxicity. The substance is therefore not regarded to have genotoxic effects and does not require classification for genetic toxicity according to the Regulation (EC) 1272/2008 and the Directive 67/548/EEC..