Reaction mass of 2,6-Octadien-1-ol, 3,7-dimethyl-, (E) and 2,6-Octadien-1-ol, 3,7-dimethyl-, (Z)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in vitro in bacteria: negative with and without metabolic activation (Ames)
in vitro in mammalian cells: negative with and without metabolic activation (HPRT)

Link to relevant study records

Reference

Reference 1

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

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)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

substrain K3

Metabolic activation:

with and without

Metabolic activation system:

S9 rat

Test concentrations with justification for top dose:

1st Experiment

without S9 mix (4-hour exposure period)
0; 6.3; 12.5; 25.0; 50.0; 100.0; 150.0; 200.0 μg/mL

with S9 mix (4-hour exposure period)
0; 6.3; 12.5; 25.0; 50.0; 100.0; 150.0; 200.0 μg/mL

2nd Experiment

without S9 mix (24-hour exposure period)
0; 25.0; 50.0; 100.0; 150.0; 175.0; 200.0 μg/mL

with S9 mix (4-hour exposure period)
0; 12.5; 25.0; 50.0; 100.0; 150.0; 175.0; 200.0 μg/mL

Vehicle / solvent:

Due to the insolubility of the test substance in water, dimethylsulfoxide (DMSO) was selected
as vehicle, which has been demonstrated to be suitable in the CHO/HPRT assay and for
which historical control data are available.
The final concentration of the vehicle DMSO in the culture medium was 1% (v/v).

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Positive controls:

yes

Positive control substance:

other: methylcholanthrene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium: Ham's F12 + 10%FCS

DURATION
- Preincubation period: 1 week, elimination of spontaneous HPRT-deficient mutants by pretreatment with "HAT" medium
- Exposure duration: 4 h and 24 h
- Expression time (cells in growth medium): about one week
- Selection time (if incubation with a selection agent): about one week
- Fixation time (start of exposure up to fixation or harvest of cells): 16 days

SELECTION AGENT (mutation assays): Hypoxanthine-free Ham's F12 medium supplemented with 6-thioguanine (10 μg/mL), 1% (v/v) L-glutamine (200 mM), and 10% (v/v) fetal calf serum (FCS)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: six flasks from every treatment group

NUMBER OF CELLS EVALUATED:3*10^5 cells seeded per flask at beginning of selection period

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency;

Evaluation criteria:

Cytotoxicity
The cloning efficiency (CE, %) was calculated for each test group as follows:

total number of colonies in the test group
CEabsolute = ————————————————————— x 100
total number of seeded cells in the test group

CEabsolute of the test group
CErelative = —————————————— x 100
CEabsolute of the vehicle/negative control

The number of colonies in every flask was counted and recorded. Using the formula above the values of absolute cloning efficiencies were
calculated. Based on these values the relative cloning efficiencies of the test groups were calculated and given in percentage compared with the
respective CEabsolute value of the corresponding vehicle/negative control (vehicle/negative control = 100%).

Mutant frequency
The number of colonies in every flask was counted and recorded. The sum of the mutant colony counts within each test group was subsequently normalized to 10^6 cells seeded.
The uncorrected mutant frequency (MFuncorr.) per 10^6 cells was calculated for each test group as follows:

total number of mutant colonies
MFuncorr. = —————————————–— x 106
number of seeded cells

The uncorrected mutant frequency was corrected with the absolute cloning efficiency 2 for each test group to get the corrected mutant frequency (MFcorr.):

MFuncorr.
MFcorr. = —–——–— x 100
CE2 absolute

Statistics:

Due to the clearly negative findings, a statistical evaluation was not carried out.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at highest conc. of 200 µg/ml each

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MUTANT FREQUENCY
- no relevant increase in the number of mutant colonies either without S9 mix or after addition of S9 in both experiments (4 and 24 hours treatment)
- positive control substances EMS (without S9 mix; 300 μg/mL) and MCA (with S9 mix; 20 μg/mL) induced clearly increased mutant frequencies

CYTOTOXICITY
- Cytotoxic effects indicated by clearly reduced cloning efficiencies of below 20% of control were observed in both experiments in the absence and presence of S9 mix in the highest applied concentration of 200 μg/mL each.

CELL MORPHOLOGY
- In both experiments, in the absence and presence of S9 mix after 4 and 24 hours treatment the morphology and attachment of the cells was adversely influenced at least at the highest applied concentration.

TREATMENT CONDITIONS
- Osmolarity and pH values were not influenced by test substance treatment.
- In this study, in the absence and the presence of S9 mix no precipitation in culture medium was observed up to the highest applied test substance concentration.

Summary of results

Exp.	Exposure period	Test groups	S9 mix	Prec.*	Genotoxicity** MFcorr. [per 10^6 cells]	Cytotoxicity***
						CE1[%]	CE2[%]
1	4 hrs	Vehicle control^1	-	-	8.17	100.0	100.0
		6.3 μg/mL	-	-	(-)	109.5	(-)
		12.5 μg/mL	-	-	1.67	108.9	104.0
		25.0 μg/mL	-	-	2.95	114.7	105.4
		50.0 μg/mL	-	-	1.69	102.4	97.1
		100.0 μg/mL	-	-	2.65	99.9	102.7
		150.0 μg/mL	-	-	1.45	42.3	93.9
		200.0 μg/mL	-	-	-	0.0	-
		Positive control^2	-	-	164.63	112.5	77.8
2	24 hrs	Vehicle control^1	-	-	3.01	100.0	100.0
		25.0 μg/mL	-	-	3.24	105.8	99.8
		50.0 μg/mL	-	-	5.89	88.6	98.0
		100.0 μg/mL	-	-	1.82	72.7	87.1
		150.0 μg/mL	-	-	7.35	49.7	83.1
		175.0 μg/mL	-	-	0.69	30.6	97.1
		200.0 μg/mL	-	-	-	1.6	-
		Positive control^2	-	-	682.83	55.2	59.8
1	4 hrs	Vehicle control^1	+	-	3.59	100.0	100.0
		6.3 μg/mL	+	-	(-)	91.0	(-)
		12.5 μg/mL	+	-	(-)	95.3	(-)
		25.0 μg/mL	+	-	1.02	86.3	108.2
		50.0 μg/mL	+	-	7.31	71.7	106.2
		100.0 μg/mL	+	-	2.04	94.9	105.2
		150.0 μg/mL	+	-	4.35	71.4	100.8
		200.0 μg/mL	+	-	3.60	5.0	118.6
		Positive control^3	+	-	75.54	96.7	102.1
2	4 hrs	Vehicle control^1	+	-	7.11	100.0	100.0
		12.5 μg/mL	+	-	(-)	83.5	(-)
		25.0 μg/mL	+	-	3.53	85.3	90.3
		50.0 μg/mL	+	-	4.04	79.2	87.6
		100.0 μg/mL	+	-	2.42	100.7	90.8
		150.0 μg/mL	+	-	7.77	76.9	88.2
		175.0 μg/mL	+	-	3.86	34.0	94.0
		200.0 μg/mL	+	-	-	0.0	-
		Positive control^3	+	-	157.17	86.2	96.5

 

*    Precipitation in culture medium at the end of exposure period

**  Mutant frequency MFcorr.: number of mutant colonies per 10^6 cells corrected with the CE2 value

*** Cloning efficiency related to the respective negative/vehicle control

-    Due to strong cytotoxicity the cultures were not continued

(-)  Culture was not continued since a minimum of four concentrations is required by the guidelines

¹DMSO 1% (v/v)              ²EMS 300 μg/mL         ³MCA 20 μg/mL

According to the results of the present in vitro study, the test substance did not lead to a relevant increase in the number of mutant colonies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each

other. The mutant frequencies at any concentration were within the range of the concurrent vehicle control values and within the range of our historical negative control data.

The mutation frequencies of the vehicle control groups were within our historical negative control data range including all vehicles used in our laboratory and, thus, fulfilled the acceptance criteria of this study.

The increase in the frequencies of mutant colonies induced by the positive control substances EMS and MCA clearly demonstrated the sensitivity of the test method and of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study.

Conclusions:

Interpretation of results:
negative

Under the experimental conditions chosen, the conclusion is drawn that the test substance is not a mutagenic substance in the HPRT locus assay using CHO cells in the absence and the presence of metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study according to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Remarks:

Symrise

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate from Aroclor 1254 pretreated male rats

Test concentrations with justification for top dose:

- Prescreen: 5 - 5000 µg/plate
- Test: 5, 15, 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation

Migrated to IUCLID6: 2.5 µg/plate (TA98) in DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation

Migrated to IUCLID6: 0.7 µg/plate (TA100, TA1535) in water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation

Migrated to IUCLID6: 50 µg/plate (TA1537) in water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Migrated to IUCLID6: 0.15 µg/plate (TA102) in water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 0.8 µg/plate (TA98, TA100, TA102, TA1535), 1.7 µg/plate (TA1537) in DMSO

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 - 72 h, 37°C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction in revertant colonies or diminution of background lawn

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without S9, the test article was bacteriotoxic at 1500ug/plate. With S9, the test article was toxic to TA100 at 1500 ug/plate, and TA98, TA1535, and TA1537 at 5000ug/plate.

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without S9, the test article was bacteriotoxic at 1500ug/plate. With S9, the test article was toxic at 1500 ug/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

5 - 5000 μg/plate: no effects

- At 5 ug/plate the test material did not induce any significant increase in the frequency of revertant mutations for strains TA102 with the S9 metabolizing system, and TA98, TA100, and TA102 without S9.

- Negative control plates with vehicle only gave counts of revertant colonies within the normal range.

- All strain specific positive controls, with and without S9, induced numbers of revertant colonies in the expected range.

- No precipitation of the test article was observed on the plates.

- At 15, 50, 150, and 500 ug/plate the test material did not induce any significant increase in the frequency of revertant mutations for strains TA98, TA100, TA102, TA1535, and TA1537 with or without the S9 metabolizing system.

1500 µg/plate:

- At 1500ug/plate the test material did not induce any significant increase in the frequency of revertant mutations for strains TA98, TA1535, and TA1537 with S9 metabolizing system.

- Negative control plates with vehicle only gave counts of revertant colonies within the normal range.

- All strain specific positive controls, with and without S9, induced numbers of revertant colonies in the expected range.

- In the absence of S9, the test article was bacteriotoxic to all strains.

- At this concentration in the presence of S9, the test article was bacteriotoxic to strain TA102 and TA100.

- No precipitation of the test article was observed on the plates

5000 μg/plate:

- The test article at this concentration was found to be bacteriotoxic to all strains of S. typhumurium with or without S9.

- Negative control plates with vehicle only gave counts of revertant colonies within the normal range.

- All strain specific positive controls, with and without S9, induced numbers of revertant colonies in the expected range.

- No precipitation of the test article was observed on the plates

Conclusions:

Interpretation of results (migrated information):
negative

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: acceptable, well documented publication

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

Method according to Ames, McCann & Yamasaki (1975)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium, other: TA 92, TA 94, TA 2637

Metabolic activation:

with and without

Metabolic activation system:

polychlorinated biphenyls induced rat liver microsome S9 mix

Test concentrations with justification for top dose:

up to 0.5 mg/ plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min, 37°C
- Exposure duration: 48 h, 37°C

NUMBER OF REPLICATIONS: 2

Evaluation criteria:

The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated).

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium, other: TA 92, TA 94, TA 2637

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

Interpretation of results: negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

in vivo in mammalian cells: negative (micronucleus test)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Age at study initiation: 5-8 weeks
- Weight at study initiation: 28.15 g
- Assigned to test groups randomly: yes, according to a randomization plan prepared with an appropriate computer program
- Housing: single in Makrolon cages, type M II
- Diet (e.g. ad libitum): standardized pellet feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): drinking water from bottles, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): fully air conditioned rooms with central air conditioning
- Photoperiod (hrs dark / hrs light): 12:12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: DMSO/corn oil (ratio 2:3)
- Justification for choice of solvent/vehicle: limited solubility of test substance in water and better volume for administration
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw

Duration of treatment / exposure:

once

Frequency of treatment:

once

Post exposure period:

24 and 48 hours

Remarks:

Doses / Concentrations:
375 mg/kg bw
Basis:
actual ingested

Remarks:

Doses / Concentrations:
750 mg/kg bw
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1500 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

- cyclophosphamide (CPP): 20 mg/kg bw
- vincristine sulfate (VCR): 0.15 mg/kg bw

Tissues and cell types examined:

- Clinical examinations: After treatment up to the time of sacrifice, the animals were examined for any clinically evident signs of toxicity several times.
- Preparation of the bone marrow: The bone marrow was prepared according to the method described by Schmid and Salamone et al.
- MICROSCOPIC EVALUATION
In general, 2 000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of micronuclei from each animal of every test group, so in total 10 000 PCEs were scored per test group. The normochromatic erythrocytes (= normocytes / NCE) were also scored. The following parameters were recorded:
• Number of polychromatic erythrocytes
• Number of polychromatic erythrocytes containing micronuclei
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the vehicle control group provides an index of a chromosome-breaking (clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the test substance administered.
• Number of normochromatic erythrocytes
• Number of normochromatic erythrocytes containing micronuclei
The number of micronuclei in normochromatic erythrocytes at the early sacrifice interval shows the situation before test substance administration and may serve as a control value. A test substance induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice interval.
• Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicates that the test substance actually reached the bone marrow, means the target determined for genotoxic effects.
• Number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/4)
[d = diameter of micronucleus, D = cell diameter]
The size of micronuclei may indicate the possible mode of action of the test substance, i.e. a clastogenic effect (d < D/4) or a spindle poison effect (d ≥ D/4).
Slides were coded before microscopic analysis.

Details of tissue and slide preparation:

Preparation of the bone marrow
- The animals were anesthetized with isoflurane and afterwards sacrificed by cervical dislocation. Then the two femora were prepared by dissection and removing all soft tissues.
- After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum (FCS) which was preheated up to 37°C (about 2 mL/femur).
- The suspension was mixed thoroughly with a pipette and centrifuged at 300 x g for 5 minutes. The supernatant was removed and the precipitate was resuspended in about 50 μL fresh FCS.
- One drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges. The preparations were dried in the air and subsequently stained.

Staining of the slides
- The slides were stained with eosin and methylene blue (modified May-Grünwald solution or Wrights solution) for about 5 minutes.
- After briefly rinsing in purified water, the preparations were soaked in purified water for about 2 - 3 minutes.
- Subsequently, the slides were stained with Giemsa solution (15 mL Giemsa plus 185 mL purified water) for about 15 minutes.
- After rinsing twice in purified water and clarifying in xylene, the preparations were mounted in Corbit-Balsam.

Evaluation criteria:

Acceptance criteria
The mouse micronucleus test is considered valid if the following criteria are met:
• The quality of the slides must allow the evaluation of a sufficient number of analyzable
cells; i. e. ≥ 2 000 PCEs per animal and a clear differentiation between PCEs and NCEs.
• The ratio of PCEs/NCEs in the concurrent vehicle control animals has to be within the normal range for the animal strain selected.
• The number of cells containing micronuclei in vehicle control animals has to be within the range of the historical vehicle control data both for PCEs and for NCEs.
• The two positive control substances have to induce a distinct increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.

Assessment criteria
A finding is considered positive if the following criteria are met:
• Statistically significant and dose-related increase in the number of PCEs containing micronuclei.
• The number of PCEs containing micronuclei has to exceed both the concurrent vehicle control value and the range of the historical vehicle control data.
A test substance is considered negative if the following criteria are met:
• The number of cells containing micronuclei in the dose groups is not statistically significant increased above the concurrent vehicle control value and is within the range of the historical vehicle control data.

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN (BASF SE). The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there are statistically significant differences between the untreated control group and the treated dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test. Statistical significances were identified as follows:
* p ≤ 0.05
** p ≤ 0.01
However, both biological relevance and statistical significance were considered together.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

piloerection and hunched posture in high dose

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

According to the results of the present study, there are no statistical significances or biologically relevant differences in the frequency of erythrocytes containing micronuclei either between the vehicle control groups and the three dose groups (375 mg/kg, 750 mg/kg and 1 500 mg/kg) or between the two sacrifice intervals (24 and 48 hours). The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d ≥ D/4) did not deviate from the vehicle control values at any of the sacrifice intervals and was within the historical vehicle control data range.
In this study, after single oral administration of the vehicle DMSO/corn oil the ratio of PCEs/NCEs in the vehicle control animals at both sacrifice intervals was within the normal range for the animal strain selected. Besides the number of cells containing micronuclei in these vehicle control animals was within the range of the historical vehicle control data both for PCEs and for NCEs.
In addition, both positive control substances, cyclophosphamide and vincristine sulfate, induced a statistically significant increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.

Summary table – Induction of Micronuclei in bone marrow cells

Test group	Sacrificeinterval [hrs]	AnimalNo.	Micronuclei in PCE		Numberof NCE^c
			total^a[‰]	large^b[‰]
Vehicle control DMSO/corn oil	24	5	2.4	0.1	3 505
Test substance 375 mg/kg bw.	24	5	1.3	0.0	3 490
Test substance 750 mg/kg bw.	24	5	1.3	0.0	3188
Test substance 1 500 mg/kg bw.	24	5	1.4	0.0	3 699
Positive control cyclophosphamide 20 mg/kg bw.	24	5	13.7**	0.1	4 076
Positive control vincristine sulfate 0.15 mg/kg bw.	24	5	47.9**	14.7**	5 879
Vehicle control DMSO/corn oil	48	5	1.3	0.0	4 630
Test substance 1 500 mg/kg bw.	48	5	1.3	0.0	4 078

 

PCE = polychromatic erythrocytes

NCE = normochromatic erythrocytes

bw. = body weight

 

^a= sum of small and large micronuclei

^b= large micronuclei (indication for spindle poison effect)

^c= number of NCEs observed when scoring 10 000 PCEs

 

* = p ≤ 0.05

** = p ≤ 0.01

Conclusions:

Interpretation of results (migrated information): negative
According to the aouthors, under the experimental conditions chosen here, the test substance has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells of NMRI mice in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Mutagenicity to bacteria

Geraniol

The genetic toxicity in vitro was analyzed in several bacterial reverse mutation assays according to the method of as well as in some chromosome aberration tests:

In one Ames test, bacteria strains S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA92, TA94 and TA2637 were tested with concentrations of geraniol up to 0.5 mg/ plate with and without metabolic activation by polychlorinated biphenyls induced rat liver microsome S9 mix (Ishidate, 1984). As a result, no mutagenic effects induced by geraniol were observed in this study.

In another Ames test, also no mutagenic effects were found, when S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 were incubated with 3 µmol/plate (= 0.46 mg/plate) with and without metabolic activation (Florin, 1980).

Only one bacteria strain was tested in two studies, where S. typhimurium TA 100 was tested according the method by Ames with concentrations of 0.01 - 1 ul geraniol /2 ml medium with and without metabolic activation by Aroclor 1254 induced rat liver S9 mix (Eder, 1980, 1982). In both studies, geraniol was found to be negative. The most recent Ames test used unspecified S. typhimurium and E coli strains and found geraniol to be not mutagenic.

Nerol

Genetic toxicity of nerol was analyzed in a bacterial reverse mutation assay performed under GLP according to OECD guideline 471 (Symrise, 2000). Bacteria strains S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were treated with nerol at concentrations of 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation by Aroclor-induced rat liver S9 mix. As a result, cytotoxicity was observed at 1500 µg/plate in TA100 and TA 102, and in TA98, TA1535 and TA1537 at a concentration of 5000ug/plate. However, nerol was found to be not mutagenic under conditions of the study

 

Mutagenicity to mammalian cells

Reaction Mass

The reaction mass of geraniol and nerol (E- and Z-isomers) was tested in a HPRT test in CHO cells with and without metabolic activation (BASF SE, 2013). Cytotoxicity was found in the highest doses of 200 µg/ml for each condition. The test substance did not cause any relevant increase in the mutant frequencies in two independent experiments.

 

Cytogenicity to mammalian cells

Reaction Mass

The reaction mass of geraniol and nerol (E- and Z-isomers) was also tested in a MNT in NMRI mice (BASF SE, 2013). For this purpose, the test substance, dissolved in DMSO and emulsified in corn oil, was administered once orally to male animals at dose levels of 375 mg/kg, 750 mg/kg and 1 500 mg/kg body weight in a volume of 10 mL/kg body weight in each case. The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 1 500 mg/kg body weight and in the vehicle controls. In the test groups of 750 mg/kg and 375 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2 000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2 000 polychromatic erythrocytes were also recorded. As vehicle control, male mice were administered merely the vehicle, DMSO/corn oil (ratio 2:3), by the same route and in the same volume as the animals of the dose groups, which gave frequencies of micronucleated polychromatic erythrocytes within the historical vehicle control data range. Both positive control substances, cyclophosphamide for clastogenicity and vincristine sulfate for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.

According to the results of the study, the single oral administration of the reaction mass of geranion and nerol did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was within the range of the concurrent vehicle control in all dose groups and at all sacrifice intervals and within the range of the historical vehicle control data. Thus, under the experimental conditions of this study, the reaction mass of geraniol and nerol does not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo.

Justification for classification or non-classification

Due to numerous negative results in bacteria and mammalian cells in vitro (mutagenicity and cytogenicity) as well as negative results in vivo, Geraniol 60 does not need to be classified as mutagenic according to Regulation (EC) No 1272/2008.