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Genetic toxicity in vitro

Description of key information

- Ames test: non mutagenic (OECD 471, GLP, rel. 1)


- hprt/ mouse lymphoma L5178Y cells: non mutagenic (OECD 476, GLP, K, rel. 1 - Read across.


- Micronucleus test: non clastogenic, non aneugenic (OECD 487, GLP, K, rel. 1)- Read-across.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From February 24 to April 18, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 471 without any deviation.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Compliance Program (inspected on March 12 to 14, 2014 / Signed on May 12, 2014)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and tryptophan.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10% S9: S9-mix from the livers of male rats treated with phenobarbitone/β-naphthoflavone (80/100 mg/kg bw/day by oral route).
Test concentrations with justification for top dose:
Experiment 1 – Range-finding test (Plate Incorporation Method):1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix
Experiment 2 - Main Test (Pre-Incubation Method): 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.
- Preparation of test materials: The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. All formulations were used within four hours of preparation and were assumed to be stable for this period.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Exposure duration: Plates were incubated at 37 °C ± 3 °C for approximately 48 hours

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: The plates were viewed microscopically for evidence of thinning (toxicity).

OTHERS:
After incubation, the plates were assessed for numbers of revertant colonies using an automated colony counting system.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:

- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
- Fold increases greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: The test material was fully miscible in dimethyl sulphoxide at 50 mg/mL.
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test.

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and positive controls. The comparison was made with the historical control ranges for 2012 and 2013 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the second mutation test (pre-incubation method).

OTHERS:
- The test material formulation, amino acid supplemented top agar and S9-mix used in this experiment were shown to be sterile.

See the attached document for information on tables of results

Conclusions:
Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA-.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA- were exposed to test material diluted in dimethyl sulphoxide both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors). The dose range for the range-finding test (Experiment 1) was predetermined and was 1.5 to 5000 µg/plate (nominal values). The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of the range-finding test and was 50 to 5000 µg/plate (nominal values). Negative, vehicle (dimethyl sulphoxide) and positive control groups were also included in mutagenicity tests.

 

The vehicle control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation in any of the experiments. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 (plate incorporation method) or Experiment 2 (pre-incubation method). 

 

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA- .

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

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 07, 2014 to February 05, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD TG 487 without any deviation.
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2010
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Remarks:
US EPA GLP Compliance Programme
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
- Storage condition of test material: The test item was stored at room temperature, protected from light
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: human peripheral blood lymphocyte
- Suitability of cells: The donor had no recent history of radiotherapy, viral infection or the administration of drugs, and who had abstained from alcohol for at least 12 hours prior to blood donation
- Normal cell cycle time (negative control): 10-16 hours

For lymphocytes:
- Sex, age and number of blood donors: healthy non-smoking 27-year-old adult male
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: not applicable
- Mitogen used for lymphocytes: phytohemagglutinin (2%)

MEDIA USED
- Type and composition of media: RPMI-1640 containing 15% fetal bovine serum, 2mM L-glutamine, 100 units penicillin, 100 µg/mL streptomycin
- CO2 concentration: 5 +/- 1 %
- Humidity level: humidified atmosphere
- Temperature: approximately 37 +/- 1 ºC
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
cytochalasin B (6 μg/mL)
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: Molecular Toxicology Inc. (Boone, NC)
- method of preparation of S9 mix: the S9 was thawed and mixed with a cofactor pool to contain 2 mM magnesium chloride, 6 mM potassium chloride, 1 mM glucose-6-phosphate, 1 mM nicotinamide adenine dinucleotide phosphate (NADP) and 20 µL S9 per milliliter medium (RPMI 1640 serum-free medium supplemented with 100 units penicillin/mL and 100 µg streptomycin/mL and 2 mM L-glutamine).
- concentration or volume of S9 mix and S9 in the final culture medium: 4 mL culture medium + 1 mL of S9 cofactor pool
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): yes, Each bulk preparation of S9 was assayed by the supplier 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:
Preliminary Toxicity Test: from 0.226 to 2260 µg/mL
4 h exposure without S9: 7, 15, 30, 35, 40, 45, 50, 60, 70, 80 μg/mL.
4 h exposure with S9 (2%): 15, 30, 50, 100, 150, 200, 225, 250 μg/mL.
24 h continuous exposure without S9: 7, 15, 25, 27.5, 30, 32.5, 35, 37.5, 40, 45, 50 μg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxyde (DMSO)
- Justification for choice of solvent/vehicle:Dimethyl sulfoxide (DMSO) was used as the vehicle based on the solubility of the test substance and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
vinblastine
Remarks:
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:
cyclophosphamide
Remarks:
Presence of S9-mix
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): duplicate
- Number of independent experiments: 2

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

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h (± S9) and 24 h continuous exposure (-S9) in preliminary toxicity test; 4 h (± S9) and 24 h continuous exposure (-S9) in main experiment
- Harvest time after the end of treatment (sampling/recovery times): At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 h in the presence of Cytochalasin B before harvest. For the 24 hour treatment in the non-activated study, cytoB was added at the beginning of the treatment.

FOR MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure: Cytochalasin B, 6 μg/mL, 24 hour
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Cells were collected by centrifugation, swollen with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 25:1 v/v), capped and stored overnight or longer at 2-8°C, or the slides were prepared immediately after harvest. To prepare slides, the cells were collected by centrifugation and if necessary, the cells were resuspended in fresh fixative. The suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with acridine orange and identified by the BioReliance study number, treatment condition, dose level, test phase, harvest date, activation system, and replicate tube design.

- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): The slides from at least three test substance treatment groups were coded using random numbers by an individual not involved with the scoring process and scored for the presence of micronuclei based on cytotoxicity. A minimum of 2000 binucleated cells from each concentration (1000 binucleated cells from each culture) were examined and scored for the presence of micronuclei.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
1/ the micronucleus should have the same staining characteristics as the main nucleus.
2/ the micronuclei should be separate from the main nuclei or just touching (no cytoplasmic bridges).
3/ the micronuclei should be of regular shape and approximately 1/3 or less than the diameter of the main nucleus.
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): not necessary and not performed in cases where no significant increase in micronucleus frequency is observed.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis block proliferation index (CBPI).
For the preliminary toxicity test, at least 500 cells were evaluated to determine the CBPI at each dose level and the control. For the micronucleus assay, at least 1,000 cells (500 cells per culture) were evaluated to determine the CBPI at each dose level and the control. The CBPI was determined using the following formula:
CBPI = 1X (Mononucleated cells + 2 x Binucleated cells + 3 x Multinucleated cells) / Total number of cells scored
% Cytostasis (cytotoxicity) = 100 -100 {(CBPIt-1) /(CBPIc-1)}
t = test substance treatment culture
c = vehicle control culture

ACCEPTABILITY CRITERIA:
The frequency of cells with micronucleus induction in the vehicle control must be within the historical control range. The percentage of cells with micronucleus induction must be statistically increased (p ≤ 0.05, Fisher's exact test) in the positive control condition relative to the vehicle control.
Evaluation criteria:
Toxicity induced by treatment was based upon CBPI and was reported for the cytotoxicity and micronucleus portions of the study. The percent frequency of micronucleated binucleated (MNBN) cells was determined out of at least 2000 total binucleated cells per dose levels, when possible, and reported for each treatment group.
Statistics:
Statistical analysis of the percentage of micronucleated cells was performed using the Fisher's exact test. The Fisher's test was used to compare pairwise the percent micronucleated cells of each treatment group with that of the vehicle control. Due to negative results, CochranArmitage test was not required to measure dose-responsiveness.
The test substance would be considered positive if it induced a statistically significant and dose-dependent increase the frequency of MN-BN cells (p ≤ 0.05). If only one criterion was met (statistically significant OR dose-dependent increase), the result was considered equivocal. If neither criterion was met, the results were considered to be negative.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH of the highest dose level in the treatment medium was 7.5
- Data on osmolality: The osmolality of the test substance dose levels in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 20%.
- Possibility of evaporation from medium: Not applicable
- Water solubility: not applicable
- Precipitation and time of the determination:
Visible precipitate was observed in treatment medium at dose levels ≥ 150 µg/mL, while dose levels ≤ 100 µg/mL were soluble in treatment medium at the beginning of the treatment period.

RANGE-FINDING/SCREENING STUDIES (if applicable):
A preliminary toxicity test was conducted to observe the cytotoxicity profile of the test substance and to select suitable dose levels for the definitive micronucleus assay. HPBL cells were first exposed to nine concentrations of Norlimbanol ranging from 0.226 to 2260 µg/mL, as well as vehicle controls, in both the absence and presence of an Aroclor-induced S9 activation system for 4 hours, or continuously for 24 hours in the absence of S9 activation.
The test substance was soluble in DMSO at all concentrations tested. Visible precipitate was observed in treatment medium at > 226 µgmL.
Substantial cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at dose levels ≥ 67.8 µg/mL in the non-activated 4 and 24-hour exposure groups, and at dose levels ≥ 226 µg/mL in the S9-activated 4-hour exposure group.

STUDY RESULTS : Cf. Table of results (attached background material)
- non-activated 4-hour exposure group : The dose levels selected for analysis of micronucleus were 15, 35, and 50 µg/mL. At the highest test concentration, 50 µg/mL, cytotoxicity was 53% relative to the vehicle control. The percentage of cells with micronuclei in the test substance-treated group was not significantly increased relative to the vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of micronucleated cells in the MMC (positive control) group (2.1%) was statistically significant (p ≤ 0.01, Fisher's Exact test).
- S9-activated 4-hour exposure group: The dose levels selected for analysis of micronucleus were 50, 100, and 150 µg/mL. At the highest test concentration,
150 µg/mL, cytotoxicity was 55% relative to the vehicle control. The percentage of cells with micronuclei in the test substance-treated group was not significantly increased relative to the vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of micronucleated cells in the CP (positive control) group (0.9%) was statistically significant (p ≤ 0.01, Fisher's Exact test).
- non-activated 24-hour exposure group: The dose levels selected for analysis of micronucleus were 15, 25, and 32.5 µg/mL. At the highest test concentration, 32.5 µg/mL, cytotoxicity was 51% relative to the vehicle control. The percentage of cells with micronuclei in the test substance-treated group was not significantly increased relative to the vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of micronucleated cells in the VB (positive control) group (1.1%) was statistically significant (p ≤ 0.01, Fisher's Exact test).


HISTORICAL CONTROL DATA: Cf. Table of results (attached background material)
- Positive historical control data: all criteria for a valid assay were met.
- Negative (solvent/vehicle) historical control data: all criteria for a valid assay were met.
Conclusions:
Based on the findings of this study, the test item was concluded to be negative for the induction of micronuclei in both non-activated and S9-activated test systems in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes.
Executive summary:

The test item was tested in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an Aroclor-induced S9 activation system,according to OECD Guideline 487 and in compliance with GLP. A preliminary toxicity was performed to establish the dose range for testing in the micronucleus test. The micronucleus assay was used to evaluate the aneugenic and clastogenic potential of the test substance. In the preliminary toxicity and the micronucleus assays, HPBL cells were treated for 4 and 24 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 24 hours after treatment initiation. Dimethyl sulfoxide

(DMSO) was used as the vehicle based on the solubility of the test substance and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.

In the preliminary toxicity assay, the doses tested ranged from 0.226 to 2260 µg/mL (10 mM). Substantial cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at dose levels ≥ 67.8 µg/mL in the non-activated 4 and 24-hour exposure groups, and at dose levels ≥ 226 µg/mL in the S9-activated 4-hour exposure group. Based on these findings, the doses chosen for the micronucleus assay ranged from 7 to 80 µg/mL for the non-activated 4-hour exposure group, from 15 to 250 µg/mL for the S9-activated 4-hour exposure group, and from 7 to 50 µg/mL for the non-activated 24-hour exposure group.

In the micronucleus assay, substantial cytotoxicity was observed at dose levels ≥ 50 µg/mL in the non-activated 4-hour exposure group, at dose levels ≥ 150 µg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥ 32.5 µg/mL in the non-activated 24-hour exposure group.

The highest dose analyzed under each treatment condition produced 50 to 60% reduction in CBPI, which met the dose limit as recommended by testing guidelines for this assay. A minimum of 1000 binucleated cells from each culture were examined and scored for the presence of micronuclei.

The percentage of cells with micronucleated binucleated cells in the test substance-treated groups was not statistically significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher’s Exact test). The results for the positive and negative controls indicate that all criteria for a valid assay were met.

Based on the findings of this study, the test item was concluded to be negative for the induction of micronuclei in both non-activated and S9-activated test systems in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes.

This study is considered as acceptable and satisfies the requirement for in vitro micronucleus endpoint.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 17 to September 26, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 476 and in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
HPRT locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells
- Absence of Mycoplasma contamination: yes
- Methods for maintenance in cell culture: For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated at 37±1ºC. When the cells were growing well, subcultures were established in an appropriate number of flasks.

MEDIA USED
- Type and composition of media:: RPMI 1640 media supplied containing L-glutamine and HEPES
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Molecular Toxicology Incorporated, USA
- method of preparation of S9 mix : prepared from male Sprague Dawley rats induced with Aroclor 1254. The S-9 was supplied as lyophilized S-9 mix (MutazymeTM), stored frozen at <-10°C, and thawed and reconstituted with purified water to provide a 10% S-9 mix just prior to use
- concentration or volume of S9 mix and S9 in the final culture medium : final S9-volume = 1 % (v/v)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).
Test concentrations with justification for top dose:
Mutation Experiment 1 (-S9): 0, 20, 30, 32, 34, 36, 38, 40, 45 µg/mL
Mutation Experiment 1 (+S9): 0, 40, 53, 59, 65, 68, 71, 74 µg/mL
Mutation Experiment 2 (-S9): 0, 20, 30, 35, 37.5, 40, 42 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Norlimbanol was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at concentrations up to at least 510.3 mg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2 in the absence of S9 / 1 in the presence of S9

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: at least 10E07
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours (+ / - S9), 24 hours (- S9)
- Harvest time after the end of treatment (sampling/recovery times): 5 minutes

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 3 or 24 hours
- Method used: microwell plates
- If a selective agent is used: 6-thioguanine (1.5 µmg/mL), at the end of the expression period for 12-14 days
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2 x 10E05

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative survival (RS)
Rationale for test conditions:
Tested up to cytotoxic concentrations.
Evaluation criteria:
For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF at one or more concentrations was significantly greater than that of the negative control (p≤0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05)
3. If both of the above criteria were fulfilled, the results should exceed the upper limit of the last 20 studies in the historical negative control database (mean MF
+/- 2 standard deviations).
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF)
was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Key result
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:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No marked changes in pH were observed in the Range-Finder at the highest concentration tested (200 µg/mL) as compared to the concurrent vehicle controls
- Data on osmolality: No marked changes in osmolality were observed in the Range-Finder at the highest concentration tested (200 µg/mL) as compared to the concurrent vehicle controls
- Possibility of evaporation from medium: not applicable
- Water solubility: not soluble in water
- Precipitation and time of the determination: Upon addition of the test article to the cultures precipitate was observed at all concentrations tested in both the absence and presence of S-9 (156.3 to 5000 µg/mL
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES (if applicable):
In the initial cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 156.3 to 5000 µg/mL (the maximum concentration for test articles of this type as recommended by regulatory test guidelines). Upon addition of the test article to the cultures precipitate was observed at all concentrations tested in both the absence and presence of S-9 (156.3 to 5000 µg/mL). Following the 3 hour treatment incubation period, precipitate was observed at all concentrations tested in the absence of S-9 (156.3 to 5000 µg/mL) and at the highest five concentrations tested in the presence of S-9 (312.5 to 5000 µg/mL).
Complete toxicity for Norlimbanol treated cultures was noted in the cell count data obtained post treatment and this experiment was therefore terminated without plating for survival. A second Range-Finder Experiment was conducted using lower concentrations.
In the second cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 0.02 to 200 µg/mL (limited by toxicity). Upon addition of the test article to the cultures, precipitate was observed at the highest two concentrations tested in the absence and presence of S-9 (63.25 and 200 µg/mL). Following the 3 hour treatment incubation period, precipitate was observed at the highest concentration tested in the presence of S-9 only (200 µg/mL).
Excessive toxicity (<10% RS) was observed at the highest two concentrations tested in the absence and presence of S-9 (63.25 and 200 µg/mL). The highest concentration to give >10% RS was 20 µg/mL which gave 81% and 64% RS in the absence and presence of S-9 respectively

STUDY RESULTS (Cf Attached background materials)

Gene mutation tests in mammalian cells: (Cf Attached background materials)
- Results from cytotoxicity measurements:
In Mutation Experiment 1, twelve concentrations were tested in the absence of S-9 ranging from 5 to 50 µg/mL and fifteen concentrations were tested in the presence of S-9 ranging from 30 to 90 µg/mL. No precipitation was observed in the absence or presence of S-9, either at the time or at the end of treatment. Seven days after treatment, the highest four concentrations tested in the presence of S-9 (77 to 90 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition, the lowest three concentrations tested in the absence of S-9 (5 to 15 µg/mL) and concentrations of 30, 50, 56 and 62 µg/mL tested in the presence of S-9 were not selected as there were sufficient non-toxic concentrations. All other concentrations were selected in the absence and presence of S-9. However, the highest concentration tested in the absence of S-9 (50 µg/mL) was later excluded from analysis due to excessive toxicity. The highest concentrations analysed were 45 µg/mL in the absence of S-9 and 74 µg/mL in the presence of S-9 which gave 31% and 12% RS, respectively.
The results for Mutation Experiment 1 indicated that Norlimbanol showed sporadic, statistically significant increases in mutant frequency when tested in the absence of S-9. These increases were not concentration related and were thought to be of questionable biological relevance. In order to confirm the lack of biological relevance a confirmatory experiment (designated Mutation Experiment 2) was performed in the absence of S-9 only.
In Mutation Experiment 2, fifteen concentrations were tested in the absence of S-9 ranging from 20 to 60 µg/mL. No precipitation was observed either at the time or at the end of treatment. On the day of treatment, the highest four concentrations tested (52.5 to 60 µg/mL) were considered too toxic for determination of relative survival.
Seven days after treatment, the highest four concentrations remaining (44 to 50 µg/mL) were considered too toxic for selection to determine viability and 6TG
resistance. In addition, a concentration of 32.5 µg/mL was not selected as there were sufficient non-toxic concentrations. All other concentrations were selected. The highest concentration analysed was 42 µg/mL which gave 26% RS.
- Results for mutation experiments:
Following the 3 hour treatment in the absence of S-9 in Experiment 1, statistically significant increases in MF, compared to the vehicle control, were observed at the highest and lowest concentrations analysed (20 and 45 µg/mL). The MF value at both concentrations (8.66 and 8.62, respectively) exceeded the historical range generated by the last twenty experiments performed in this laboratory (0.09 to 8.33 in the absence of S-9 at the time of Experiment 1) and there was a statistically significant linear trend.
However, there was no clear evidence of a true concentration related effect and the MF values only marginally exceeded the historical range. A confirmatory experiment (Mutation Experiment 2) was therefore conducted in order to determine biological relevance.
Following the 3 hour treatment in the absence of S-9 in Experiment 2, the maximum concentration analysed for viability and 6TG resistance (42 µg/mL) gave 26% RS. Although marginally above the desired 10-20% limit recommended by the guideline, the toxicity profile was extremely steep (a concentration of 44 µg/mL gave 7% RS) and it was therefore considered that an appropriate maximum concentration had been achieved.
No statistically significant increases in MF, compared to the vehicle control, were observed at any concentration analysed and there was no significant linear trend.
Furthermore, all MF values were within the historical range generated by the last twenty experiments performed in this laboratory (0.15 to 8.04 in the absence of S-9 at the time of Experiment 2). Overall, the observations in Mutation Experiment 1 were not reproduced in Mutation Experiment 2. According to data interpretation strategies proposed by Thybaud et al., 2007, non-reproducible or marginal responses may be considered of very low or no toxicological concern and no further testing should be needed. Based on the lack of reproducibility between experiments, the increases noted in Mutation Experiment 1 were considered of no biological relevance.
Following the 3 hour treatment in the presence of S-9, no statistically significant increases in MF, compared to the vehicle control, were observed at any concentration analysed and there were no statistically significant linear trends. All MF values were within the historical range generated by the last twenty experiments performed in this laboratory (0.00 to 8.45 in the presence of S-9 at the time of this Experiment).

HISTORICAL CONTROL DATA (Cf Attached background materials)
- Positive historical control data: inside
- Negative (solvent/vehicle) historical control data: All MF values were within the historical range generated by the last twenty experiments performed in this
laboratory (0.00 to 8.45 in the presence of S-9 at the time of this Experiment).
Conclusions:
It is concluded that the test material did not induce biologically relevant increases in mutant frequency at the hprt locus in mouse lymphoma L5178Y cells when tested up to toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9).
Executive summary:

The test substance was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol according to the OECD TG No. 476 and in compliance with GLP. The study consisted of a two cytotoxicity Range-Finder Experiments followed by two Mutation Experiments. The Range-Finder Experiments and initial Mutation Experiment were conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The second Mutation Experiment was conducted in the absence of S-9 only. The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

A 3 hour treatment period at 37° was used for each experiment.

In the initial cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 156.3 to 5000 µg/mL (the latter being the maximum concentration for test articles of this nature as recommended by regulatory test guidelines). Post treatment precipitate was observed at all concentrations tested in both the absence and presence of S-9 (156.3 to 5000 µg/mL). Complete toxicity for Norlimbanol treated cultures was noted in the cell count data obtained post treatment and this experiment was therefore terminated without plating for survival. A second Range-Finder Experiment was conducted using lower concentrations.

In the second cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 0.02 to 200 µg/mL (limited by toxicity). Excessive toxicity (10% RS was 20 µg/mL, which gave 81% and 64% RS in the absence and presence of S-9, respectively. In Mutation Experiment 1, twelve concentrations were tested in the absence of S-9 ranging from 5 to 50 µg/mL and fifteen concentrations were tested in the presence of S-9 ranging from 30 to 90 µg/mL. Seven days after treatment, the highest four concentrations tested in the presence of S-9 (77 to 90 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition, the lowest three concentrations tested in the absence of S-9 (5 to 15 µg/mL) and concentrations of 30, 50, 56 and 62 µg/mL tested in the presence of S-9 were not selected as there were sufficient non-toxic concentrations. All other concentrations were selected in the absence and presence of S-9. However, the highest concentration tested in the absence of S-9 (50 µg/mL) was later excluded from analysis due to excessive toxicity. The highest concentrations analysed were 45 µg/mL in the absence of S-9 and 74 µg/mL in the presence of S-9, which gave 31% and 12% RS, respectively.

The results for Mutation Experiment 1 indicated that Norlimbanol showed sporadic, statistically significant increases in mutant frequency when tested in the absence of S-9. These increases were not concentration related and were thought to be of questionable biological relevance. In order to confirm the absence of biological relevance a confirmatory experiment (designated Mutation Experiment 2) was performed in the absence of S-9 only.

In Mutation Experiment 2, fifteen concentrations were tested in the absence of S-9 ranging from 20 to 60 µg/mL. On the day of treatment, the highest four concentrations tested (52.5 to 60 µg/mL) were considered too toxic for determination of relative survival. Seven days after treatment, the highest remaining four concentrations (44 to 50 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition, a concentration of 32.5 µg/mL was not selected as there were sufficient non-toxic concentrations. All other concentrations were selected for plating. The highest concentration analysed was 42 µg/mL which gave 26% RS.

Vehicle and positive control treatments were included in the Mutation Experiments in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore, the study was accepted as valid.

Following the 3 hour treatment in the absence of S-9 in Experiment 1, statistically significant increases in MF, compared to the vehicle control, were observed at the highest and lowest concentrations analysed (20 and 45 µg/mL). The MF value at both concentrations (8.66 and 8.62, respectively) exceeded the historical range generated by the last twenty experiments performed in this laboratory (0.09 to 8.33 in the absence of S-9 at the time of Experiment 1) and there was a statistically significant linear trend. However, the effects were not clearly concentration related and the MF values only marginally exceeded the historical range. A confirmatory experiment (Mutation Experiment 2) was therefore conducted in order to determine biological relevance.

Following the 3 hour treatment in the absence of S-9 in Experiment 2, the maximum concentration analysed for viability and 6TG resistance (42 µg/mL) gave 26% RS. Although marginally above the desired 10-20% limit recommended by the guideline, the toxicity profile was extremely steep (a concentration of 44 µg/mL gave 7% RS) and it was therefore considered that an appropriate maximum concentration had been achieved. No statistically significant increases in MF, compared to the vehicle control, were observed at any concentration analysed and there was no significant linear trend. Furthermore, all MF values were within the historical range generated by the last twenty experiments performed in this laboratory (0.15 to 8.04 in the absence of S-9 at the time of Experiment 2). Overall, the observations in Mutation Experiment 1 were not reproduced in Mutation Experiment 2. According to data interpretation strategies proposed by Thybaud et al., 2007, non-reproducible or marginal responses may be considered of very low or no toxicological concern and no further testing should be needed. Based on the lack of reproducibility between experiments, the increases noted in Mutation Experiment 1 were considered of no biological relevance.

Following the 3 hour treatment in the presence of S-9, no statistically significant increases in MF, compared to the vehicle control, were observed at any concentration analysed and there were no statistically significant linear trends. All MF values were within the historical range generated by the last twenty experiments performed in this laboratory (0.00 to 8.45 in the presence of S-9 at the time of this Experiment).

It is concluded that the test material did not induce biologically relevant increases in mutant frequency at the hprt locus in mouse lymphoma L5178Y cells when tested up to toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9). Small, non-concentration related increases were observed following treatment in the absence of S-9. However, these increases were not reproducible and, according to current data interpretation strategies, may be considered of very low or no toxicological concern and were therefore considered not biologically relevant under the experimental conditions described.

This study is classified as acceptable as it satisfies the requirement for OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.  

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Cf. IUCLID section 13.2

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar Physico-Chemical, and Toxicological properties because of their structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and source substances are both multi-constituents, as reaction mass of stereoisomers of the same substance.
The target and source substances are structurally related, in that both are a reaction mass of stereoisomers of 1-(2,2,6-Trimethylcyclohexyl)hexan-3-ol. They differ by the number of constituents. The two trans 1R,6S diastereoisomers, with both 3R and 3S hydroxyl group, of the source substance are constituents of the target substance, which contains also the trans 1S,6R pair (i.e. 2 pairs of enantiomers).

3. ANALOGUE APPROACH JUSTIFICATION
Based on structural similarity (constituents of the source and the target substance are from the same pool of substance, i.e stereoisomers of 1-(2,2,6-Trimethylcyclohexyl)hexan-3-ol) it is considered appropriate and scientifically justified to read-across the data from the source to the target substance.
The study design (OECD 476, GLP) is adequate and reliable for the purpose of the prediction based on read-across. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the result of the in vitro gene mutation study conducted with the source substance is highly likely to predict the properties of the target substance and is considered as adequate to fulfil the information requirement of Annex VIII, 8.4.2.

4. DATA MATRIX
Cf. IUCLID section 13.2
Reason / purpose for cross-reference:
read-across: supporting information
Reason / purpose for cross-reference:
read-across source
Key result
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:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No marked changes in pH were observed in the Range-Finder at the highest concentration tested (200 µg/mL) as compared to the concurrent vehicle controls
- Data on osmolality: No marked changes in osmolality were observed in the Range-Finder at the highest concentration tested (200 µg/mL) as compared to the concurrent vehicle controls
- Possibility of evaporation from medium: not applicable
- Water solubility: not soluble in water
- Precipitation and time of the determination: Upon addition of the test article to the cultures precipitate was observed at all concentrations tested in both the absence and presence of S-9 (156.3 to 5000 µg/mL
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES (if applicable):
In the initial cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 156.3 to 5000 µg/mL (the maximum concentration for test articles of this type as recommended by regulatory test guidelines). Upon addition of the test article to the cultures precipitate was observed at all concentrations tested in both the absence and presence of S-9 (156.3 to 5000 µg/mL). Following the 3 hour treatment incubation period, precipitate was observed at all concentrations tested in the absence of S-9 (156.3 to 5000 µg/mL) and at the highest five concentrations tested in the presence of S-9 (312.5 to 5000 µg/mL).
Complete toxicity for Norlimbanol treated cultures was noted in the cell count data obtained post treatment and this experiment was therefore terminated without plating for survival. A second Range-Finder Experiment was conducted using lower concentrations.
In the second cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 0.02 to 200 µg/mL (limited by toxicity). Upon addition of the test article to the cultures, precipitate was observed at the highest two concentrations tested in the absence and presence of S-9 (63.25 and 200 µg/mL). Following the 3 hour treatment incubation period, precipitate was observed at the highest concentration tested in the presence of S-9 only (200 µg/mL).
Excessive toxicity (<10% RS) was observed at the highest two concentrations tested in the absence and presence of S-9 (63.25 and 200 µg/mL). The highest concentration to give >10% RS was 20 µg/mL which gave 81% and 64% RS in the absence and presence of S-9 respectively

STUDY RESULTS (Cf Attached background materials)

Gene mutation tests in mammalian cells: (Cf Attached background materials)
- Results from cytotoxicity measurements:
In Mutation Experiment 1, twelve concentrations were tested in the absence of S-9 ranging from 5 to 50 µg/mL and fifteen concentrations were tested in the presence of S-9 ranging from 30 to 90 µg/mL. No precipitation was observed in the absence or presence of S-9, either at the time or at the end of treatment. Seven days after treatment, the highest four concentrations tested in the presence of S-9 (77 to 90 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition, the lowest three concentrations tested in the absence of S-9 (5 to 15 µg/mL) and concentrations of 30, 50, 56 and 62 µg/mL tested in the presence of S-9 were not selected as there were sufficient non-toxic concentrations. All other concentrations were selected in the absence and presence of S-9. However, the highest concentration tested in the absence of S-9 (50 µg/mL) was later excluded from analysis due to excessive toxicity. The highest concentrations analysed were 45 µg/mL in the absence of S-9 and 74 µg/mL in the presence of S-9 which gave 31% and 12% RS, respectively.
The results for Mutation Experiment 1 indicated that Norlimbanol showed sporadic, statistically significant increases in mutant frequency when tested in the absence of S-9. These increases were not concentration related and were thought to be of questionable biological relevance. In order to confirm the lack of biological relevance a confirmatory experiment (designated Mutation Experiment 2) was performed in the absence of S-9 only.
In Mutation Experiment 2, fifteen concentrations were tested in the absence of S-9 ranging from 20 to 60 µg/mL. No precipitation was observed either at the time or at the end of treatment. On the day of treatment, the highest four concentrations tested (52.5 to 60 µg/mL) were considered too toxic for determination of relative survival.
Seven days after treatment, the highest four concentrations remaining (44 to 50 µg/mL) were considered too toxic for selection to determine viability and 6TG
resistance. In addition, a concentration of 32.5 µg/mL was not selected as there were sufficient non-toxic concentrations. All other concentrations were selected. The highest concentration analysed was 42 µg/mL which gave 26% RS.
- Results for mutation experiments:
Following the 3 hour treatment in the absence of S-9 in Experiment 1, statistically significant increases in MF, compared to the vehicle control, were observed at the highest and lowest concentrations analysed (20 and 45 µg/mL). The MF value at both concentrations (8.66 and 8.62, respectively) exceeded the historical range generated by the last twenty experiments performed in this laboratory (0.09 to 8.33 in the absence of S-9 at the time of Experiment 1) and there was a statistically significant linear trend.
However, there was no clear evidence of a true concentration related effect and the MF values only marginally exceeded the historical range. A confirmatory experiment (Mutation Experiment 2) was therefore conducted in order to determine biological relevance.
Following the 3 hour treatment in the absence of S-9 in Experiment 2, the maximum concentration analysed for viability and 6TG resistance (42 µg/mL) gave 26% RS. Although marginally above the desired 10-20% limit recommended by the guideline, the toxicity profile was extremely steep (a concentration of 44 µg/mL gave 7% RS) and it was therefore considered that an appropriate maximum concentration had been achieved.
No statistically significant increases in MF, compared to the vehicle control, were observed at any concentration analysed and there was no significant linear trend.
Furthermore, all MF values were within the historical range generated by the last twenty experiments performed in this laboratory (0.15 to 8.04 in the absence of S-9 at the time of Experiment 2). Overall, the observations in Mutation Experiment 1 were not reproduced in Mutation Experiment 2. According to data interpretation strategies proposed by Thybaud et al., 2007, non-reproducible or marginal responses may be considered of very low or no toxicological concern and no further testing should be needed. Based on the lack of reproducibility between experiments, the increases noted in Mutation Experiment 1 were considered of no biological relevance.
Following the 3 hour treatment in the presence of S-9, no statistically significant increases in MF, compared to the vehicle control, were observed at any concentration analysed and there were no statistically significant linear trends. All MF values were within the historical range generated by the last twenty experiments performed in this laboratory (0.00 to 8.45 in the presence of S-9 at the time of this Experiment).

HISTORICAL CONTROL DATA (Cf Attached background materials)
- Positive historical control data: inside
- Negative (solvent/vehicle) historical control data: All MF values were within the historical range generated by the last twenty experiments performed in this
laboratory (0.00 to 8.45 in the presence of S-9 at the time of this Experiment).
Conclusions:
It is concluded that the source substance did not induce biologically relevant increases in mutant frequency at the hprt locus in mouse lymphoma L5178Y cells when tested up to toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9). The same conclusion applies to the target substance.
Executive summary:

The source substance was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol according to the OECD TG No. 476 and in compliance with GLP. The study consisted of a two cytotoxicity Range-Finder Experiments followed by two Mutation Experiments. The Range-Finder Experiments and initial Mutation Experiment were conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The second Mutation Experiment was conducted in the absence of S-9 only. The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).


A 3 hour treatment period at 37° was used for each experiment.


 


In the initial cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 156.3 to 5000 µg/mL (the latter being the maximum concentration for test articles of this nature as recommended by regulatory test guidelines). Post treatment precipitate was observed at all concentrations tested in both the absence and presence of S-9 (156.3 to 5000 µg/mL). Complete toxicity for Norlimbanol treated cultures was noted in the cell count data obtained post treatment and this experiment was therefore terminated without plating for survival. A second Range-Finder Experiment was conducted using lower concentrations.


 


In the second cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 0.02 to 200 µg/mL (limited by toxicity). Excessive toxicity (10% RS was 20 µg/mL, which gave 81% and 64% RS in the absence and presence of S-9, respectively. In Mutation Experiment 1, twelve concentrations were tested in the absence of S-9 ranging from 5 to 50 µg/mL and fifteen concentrations were tested in the presence of S-9 ranging from 30 to 90 µg/mL. Seven days after treatment, the highest four concentrations tested in the presence of S-9 (77 to 90 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition, the lowest three concentrations tested in the absence of S-9 (5 to 15 µg/mL) and concentrations of 30, 50, 56 and 62 µg/mL tested in the presence of S-9 were not selected as there were sufficient non-toxic concentrations. All other concentrations were selected in the absence and presence of S-9. However, the highest concentration tested in the absence of S-9 (50 µg/mL) was later excluded from analysis due to excessive toxicity. The highest concentrations analysed were 45 µg/mL in the absence of S-9 and 74 µg/mL in the presence of S-9, which gave 31% and 12% RS, respectively.


 


The results for Mutation Experiment 1 indicated that Norlimbanol showed sporadic, statistically significant increases in mutant frequency when tested in the absence of S-9. These increases were not concentration related and were thought to be of questionable biological relevance. In order to confirm the absence of biological relevance a confirmatory experiment (designated Mutation Experiment 2) was performed in the absence of S-9 only.


 


In Mutation Experiment 2, fifteen concentrations were tested in the absence of S-9 ranging from 20 to 60 µg/mL. On the day of treatment, the highest four concentrations tested (52.5 to 60 µg/mL) were considered too toxic for determination of relative survival. Seven days after treatment, the highest remaining four concentrations (44 to 50 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition, a concentration of 32.5 µg/mL was not selected as there were sufficient non-toxic concentrations. All other concentrations were selected for plating. The highest concentration analysed was 42 µg/mL which gave 26% RS.


 


Vehicle and positive control treatments were included in the Mutation Experiments in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore, the study was accepted as valid.


 


Following the 3 hour treatment in the absence of S-9 in Experiment 1, statistically significant increases in MF, compared to the vehicle control, were observed at the highest and lowest concentrations analysed (20 and 45 µg/mL). The MF value at both concentrations (8.66 and 8.62, respectively) exceeded the historical range generated by the last twenty experiments performed in this laboratory (0.09 to 8.33 in the absence of S-9 at the time of Experiment 1) and there was a statistically significant linear trend. However, the effects were not clearly concentration related and the MF values only marginally exceeded the historical range. A confirmatory experiment (Mutation Experiment 2) was therefore conducted in order to determine biological relevance.


 


Following the 3 hour treatment in the absence of S-9 in Experiment 2, the maximum concentration analysed for viability and 6TG resistance (42 µg/mL) gave 26% RS. Although marginally above the desired 10-20% limit recommended by the guideline, the toxicity profile was extremely steep (a concentration of 44 µg/mL gave 7% RS) and it was therefore considered that an appropriate maximum concentration had been achieved. No statistically significant increases in MF, compared to the vehicle control, were observed at any concentration analysed and there was no significant linear trend. Furthermore, all MF values were within the historical range generated by the last twenty experiments performed in this laboratory (0.15 to 8.04 in the absence of S-9 at the time of Experiment 2). Overall, the observations in Mutation Experiment 1 were not reproduced in Mutation Experiment 2. According to data interpretation strategies proposed by Thybaud et al., 2007, non-reproducible or marginal responses may be considered of very low or no toxicological concern and no further testing should be needed. Based on the lack of reproducibility between experiments, the increases noted in Mutation Experiment 1 were considered of no biological relevance.


 


Following the 3 hour treatment in the presence of S-9, no statistically significant increases in MF, compared to the vehicle control, were observed at any concentration analysed and there were no statistically significant linear trends. All MF values were within the historical range generated by the last twenty experiments performed in this laboratory (0.00 to 8.45 in the presence of S-9 at the time of this Experiment).


 


It is concluded that the source substance did not induce biologically relevant increases in mutant frequency at the hprt locus in mouse lymphoma L5178Y cells when tested up to toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9). Small, non-concentration related increases were observed following treatment in the absence of S-9. However, these increases were not reproducible and, according to current data interpretation strategies, may be considered of very low or no toxicological concern and were therefore considered not biologically relevant under the experimental conditions described.


The same conclusion applies to the target substance. Refer to Iuclid section 13 for read-across justification.


 


This study is classified as acceptable as it satisfies the requirement for OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.  

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Cf. IUCLID section 13.2

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar Physico-Chemical, and Toxicological properties because of their structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and source substances are both multi-constituents, as reaction mass of stereoisomers of the same substance.
The target and source substances are structurally related, in that both are a reaction mass of stereoisomers of 1-(2,2,6-Trimethylcyclohexyl)hexan-3-ol. They differ by the number of constituents. The two trans 1R,6S diastereoisomers, with both 3R and 3S hydroxyl group, of the source substance are constituents of the target substance, which contains also the trans 1S,6R pair (i.e. 2 pairs of enantiomers).

3. ANALOGUE APPROACH JUSTIFICATION
Based on structural similarity (constituents of the source and the target substance are from the same pool of substance, i.e stereoisomers of 1-(2,2,6-Trimethylcyclohexyl)hexan-3-ol) it is considered appropriate and scientifically justified to read-across the data from the source to the target substance.
The study design (OECD 487, GLP) is adequate and reliable for the purpose of the prediction based on read-across. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the result of the in vitro micronucleus study conducted with the source substance is highly likely to predict the properties of the target substance and is considered as adequate to fulfil the information requirement of Annex VIII, 8.4.2.

4. DATA MATRIX
Cf. IUCLID section 13.2
Reason / purpose for cross-reference:
read-across: supporting information
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH of the highest dose level in the treatment medium was 7.5
- Data on osmolality: The osmolality of the test substance dose levels in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 20%.
- Possibility of evaporation from medium: Not applicable
- Water solubility: not applicable
- Precipitation and time of the determination:
Visible precipitate was observed in treatment medium at dose levels ≥ 150 µg/mL, while dose levels ≤ 100 µg/mL were soluble in treatment medium at the beginning of the treatment period.

RANGE-FINDING/SCREENING STUDIES (if applicable):
A preliminary toxicity test was conducted to observe the cytotoxicity profile of the test substance and to select suitable dose levels for the definitive micronucleus assay. HPBL cells were first exposed to nine concentrations of Norlimbanol ranging from 0.226 to 2260 µg/mL, as well as vehicle controls, in both the absence and presence of an Aroclor-induced S9 activation system for 4 hours, or continuously for 24 hours in the absence of S9 activation.
The test substance was soluble in DMSO at all concentrations tested. Visible precipitate was observed in treatment medium at > 226 µgmL.
Substantial cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at dose levels ≥ 67.8 µg/mL in the non-activated 4 and 24-hour exposure groups, and at dose levels ≥ 226 µg/mL in the S9-activated 4-hour exposure group.

STUDY RESULTS : Cf. Table of results (attached background material)
- non-activated 4-hour exposure group : The dose levels selected for analysis of micronucleus were 15, 35, and 50 µg/mL. At the highest test concentration, 50 µg/mL, cytotoxicity was 53% relative to the vehicle control. The percentage of cells with micronuclei in the test substance-treated group was not significantly increased relative to the vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of micronucleated cells in the MMC (positive control) group (2.1%) was statistically significant (p ≤ 0.01, Fisher's Exact test).
- S9-activated 4-hour exposure group: The dose levels selected for analysis of micronucleus were 50, 100, and 150 µg/mL. At the highest test concentration,
150 µg/mL, cytotoxicity was 55% relative to the vehicle control. The percentage of cells with micronuclei in the test substance-treated group was not significantly increased relative to the vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of micronucleated cells in the CP (positive control) group (0.9%) was statistically significant (p ≤ 0.01, Fisher's Exact test).
- non-activated 24-hour exposure group: The dose levels selected for analysis of micronucleus were 15, 25, and 32.5 µg/mL. At the highest test concentration, 32.5 µg/mL, cytotoxicity was 51% relative to the vehicle control. The percentage of cells with micronuclei in the test substance-treated group was not significantly increased relative to the vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of micronucleated cells in the VB (positive control) group (1.1%) was statistically significant (p ≤ 0.01, Fisher's Exact test).


HISTORICAL CONTROL DATA: Cf. Table of results (attached background material)
- Positive historical control data: all criteria for a valid assay were met.
- Negative (solvent/vehicle) historical control data: all criteria for a valid assay were met.
Conclusions:
Based on the findings of this study, the source substance was concluded to be negative for the induction of micronuclei in both non-activated and S9-activated test systems in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes. The same conclusion applies to the target substance.
Executive summary:

The source substance was tested in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an Aroclor-induced S9 activation system,according to OECD Guideline 487 and in compliance with GLP. A preliminary toxicity was performed to establish the dose range for testing in the micronucleus test. The micronucleus assay was used to evaluate the aneugenic and clastogenic potential of the test substance. In the preliminary toxicity and the micronucleus assays, HPBL cells were treated for 4 and 24 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 24 hours after treatment initiation. Dimethyl sulfoxide


 


(DMSO) was used as the vehicle based on the solubility of the test substance and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.


 


In the preliminary toxicity assay, the doses tested ranged from 0.226 to 2260 µg/mL (10 mM). Substantial cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at dose levels ≥ 67.8 µg/mL in the non-activated 4 and 24-hour exposure groups, and at dose levels ≥ 226 µg/mL in the S9-activated 4-hour exposure group. Based on these findings, the doses chosen for the micronucleus assay ranged from 7 to 80 µg/mL for the non-activated 4-hour exposure group, from 15 to 250 µg/mL for the S9-activated 4-hour exposure group, and from 7 to 50 µg/mL for the non-activated 24-hour exposure group.


 


In the micronucleus assay, substantial cytotoxicity was observed at dose levels ≥ 50 µg/mL in the non-activated 4-hour exposure group, at dose levels ≥ 150 µg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥ 32.5 µg/mL in the non-activated 24-hour exposure group.


 


The highest dose analyzed under each treatment condition produced 50 to 60% reduction in CBPI, which met the dose limit as recommended by testing guidelines for this assay. A minimum of 1000 binucleated cells from each culture were examined and scored for the presence of micronuclei.


 


The percentage of cells with micronucleated binucleated cells in the test substance-treated groups was not statistically significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher’s Exact test). The results for the positive and negative controls indicate that all criteria for a valid assay were met.


 


Based on the findings of this study, the source substance was concluded to be negative for the induction of micronuclei in both non-activated and S9-activated test systems in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes.


The same conclusion applies to the target substance. Refer to Iuclid section 13 for read-across justification.


 


This study is considered as acceptable and satisfies the requirement for in vitro micronucleus endpoint.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Table 7.6/1: Summary of genotoxicity tests


 










































Test n°



Test / Guideline


Reliability



Focus



Strains tested



Metabolic activation



Test concentration



Statement



1


 


Harlan, 2014



Ames Test


(OECD 471)


K, rel. 2



Gene mutation (bacteria)



TA 1535


TA 1537


TA 98


TA 100


E. coli WP2



-S9


+S9



Up to 5000 µg/plate


(limit concentration)



-S9 : non mutagenic


+S9 : non mutagenic



2


 


Covance, 2020



hprt (OECD 476)


K, rel. 1



Gene mutation (mammalian cells)



Mouse Lymphoma L5178Ycells



-S9


+S9



Up to toxic concentrations)



-S9 : non mutagenic


+S9 : non mutagenic



3


 


BioReliance, 2015



MNT


(OECD 490)


K, rel. 1



Chromosomal aberration



Human Lymphocytes



 -S9


+S9



Up toxic concentrations)



-S9 : non clastogenic


+S9 : non clastogenic



 


Gene mutation Assays (Test n° 1 -2):


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


 


Inability to produce gene mutation was confirmed in mammals using an in vitro forward mutation assay in Mouse Lymphoma L5178Y cells (hprt test) performed on the source substance (Test n°2). None of the dose levels up to toxic concentrations, either in the presence or absence of metabolic activation, induced significant mutant frequency increases in the initial or repeat tests. The substance does not induce forward mutations at the hprt locus in L5178Y cells under activation and non activation conditions whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. The source substance, and by analogy the target substance, is therefore considered as negative for inducing forward mutations at the hprt locus in L5178Y cells under activation and non-activation conditions used in this assay. This result confirms the results of the Ames test and extends the non-mutagenic effect of the substance to mammalian cells.


 


 


Chromosomal aberration (Test n°3)


The clastogenic potential of the substance was determined using an in vitro micronucleus test in human lymphocytes (OECD 487) performed on the source substance, which measures the potential of a substance to increase the incidence of micronuclei in cultured human lymphocytes. The test item was cytotoxic to human lymphocytes but it did not induce any statistically significant increases in the frequency of cells with micronuclei, in any of the exposure groups, using a dose range that included a dose level that induced a sufficient reduction in the cytokinesis block proliferation index (CBPI). Both positive and negative controls validated the sensitivity of the assay. Therefore, the source substance, and by analogy the target substance, is considered as non-clastogenic and non-aneugenic under the conditions used in this assay.

Justification for classification or non-classification

Harmonised classification:


The substance has no harmonised classification for genetic toxicity according to the Regulation (EC) No. 1272/2008 (CLP).


 


Self-classification:


Based on the available data, no self-classification is proposed according to the CLP or the GHS.