Nerol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative results were obtained in all in vitro tests performed with nerol: mutagenicity in bacteria, mutagenicity in mammlian cells and clastogenicity tests.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 April - 6 June 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP study conducted according to OECD Guideline 471 with minor deviations: temperature of the incubator rose to 38.1 °C overnight, just above the 37 ± 1 °C limit.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

temperature of the incubator rose to 38.1 °C overnight, just above the 37 ± 1 °C limit

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: All strains were checked for characteristics (histidine dependence, rfa character, uvrB character and resistance to ampicillin or ampicillin plus tetracycline).

Metabolic activation:

with and without

Metabolic activation system:

10 % S9 mix; S9 fraction prepared from liver homogenates of male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Mutagenicity tests:
- Experiment 1: 5, 15.81, 50, 158.1, 500, 1581 and 5000 μg/plate, with and without S9 mix in all 5 strains (plate incorporation method)
- Experiment 2: 8.192, 20.48, 51.2, 128, 320 and 800 μg/plate, with S9 mix (preincubation method) and without S9 mix (plate incorporation method) in all 5 strains [2000 μg/plate employed for treatments of all strains in the absence of S9 only; 2500 μg/plate employed for treatments of all strains in the presence of S9 only]
- Experiment 3: 5, 10, 25, 50, 75, 100 and 125 μg/plate, with S9 mix in strain TA 102 (preincubation method)
- Experiment 4: 2.344, 4.688, 9.375, 18.75, 37.5, 75 and 150 μg/plate, with S9 mix in strain TA 1537 (preincubation method)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
Formulation procedure:
- Test article stock solutions were prepared by formulating Nerol in DMSO under subdued lighting conditions with the aid of vortex mixing (as required) immediately prior to assay to give the maximum required treatment solution concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 6 h of initial formulation.

Volume addition: 0.1 mL volume additions of vehicle/test article solution were used for all plate-incorporation treatments, 0.05 mL volume additions of vehicle/test article solution were used for pre-incubation treatments.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Remarks:

without metabolic activation

Untreated negative controls:

no

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 metabolic activation

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: Strains TA 98, TA 1535 and TA 1537 were originally obtained from the UK NCTC. Strains TA 100 and TA 102 were derived from cultures originally obtained from Covance Laboratories Inc., USA.

METHOD OF APPLICATION: In agar (plate incorporation and preincubation method)

DURATION
- Preincubation period: 20 minutes at 37 ± 1 °C
- Incubation period: Plates were inverted and incubated at 37 ± 1 °C in the dark for 3 days.

NUMBER OF REPLICATIONS:
- Treatment and positive control groups: 3 plates/dose
- Vehicle control group: 5 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn was inspected for signs of toxicity.

OTHER:
Colony counting: Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as bubbles or splits in the agar affected the accuracy of the automated counter.

Evaluation criteria:

- For valid data, the test article was considered to be mutagenic if:
1. When assessed using Dunnett's test, an increase in revertant numbers gave a significant response (p ≤ 0.01) which was concentration related.
2. The positive trend/effects described above were reproducible.
- Test article was considered positive in this assay if all of the above criteria were met.
- Test article was considered negative in this assay if none of the above criteria were met.

- Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.

Statistics:

- Dunnett's test was used to compare the counts at each concentration with the control. The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 μg/plate).

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: Mean vehicle control counts fell within the normal historical ranges (historical control data of February 2008 - July 2009).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Experiment 1: Toxicity was observed in all strains at 1581 and 5000 μg/plate.
- Experiment 2: Toxicity was observed at 51.2 μg/plate and above in strain TA 1537 in the presence of S9; 128 μg/plate and above in strains TA 98, TA 100 and TA 102 in the presence of S9; 320 μg/plate and above in strains TA 98 and TA 1537 in the absence of S9 and strain TA 1535 in the presence of S9; and 800 μg/plate in strains TA 100, TA 1535 and TA 102 in the absence of S9.
- Experiment 3: Toxicity was observed at 125 μg/plate with S9 mix in strain TA 102.
- Experiment 4: Toxicity was observed at 150 μg/plate with S9 mix in strain TA 1537.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 7.6.1/2: Results of mutagenicity

Group	Concentration (µg/plate)	Mean revertant numbers/plate	Concentration (µg/plate)	Mean revertant numbers/plate
	Experiment 1		Experiment 2
TA 98 -S-9
DMSO		23.4		20.6
Test item	5	28.3	8.192	24.3
	15.81	23.0	20.48	18.3
	50	22.0	51.2	22.7
	158.1	26.7	128	16.3
	500	19.0	320	16.3
	1581	8.0	800	17.0
	5000	T	2000	5.0
2NF	5	816.3	5	1183.0
TA 98 +S-9
DMSO		30.4		37.2
Test item	5	31.3	8.192	21.7
	15.81	32.0	20.48	23.3
	50	36.3	51.2	27.7
	158.1	32.0	128	28.3
	500	29.3	320	28.3
	1581	15.0	800	T
	5000	T	2500	T
B[a]P	10	306.3	10	540.3
TA 100 -S-9
DMSO		94.8		91.6
Test item	5	95.0	8.192	92.0
	15.81	108.7	20.48	94.7
	50	105.3	51.2	99.7
	158.1	100.3	128	89.3
	500	91.3	320	95.0
	1581	45.0	800	78.7
	5000	T	2000	22.7
NaN3	2	670.0	2	828.3
TA 100 +S-9
DMSO		100.4		97.2
Test item	5	110.3	8.192	101.0
	15.81	107.7	20.48	96.0
	50	116.3	51.2	97.0
	158.1	104.0	128	95.3
	500	110.0	320	75.0
	1581	66.3	800	T
	5000	T	2500	T
AAN	5	667.7	5	1343.0
TA 1535 -S-9
DMSO		22.8		18.2
Test item	5	21.0	8.192	20.7
	15.81	27.3	20.48	23.7
	50	27.3	51.2	20.0
	158.1	25.7	128	25.0
	500	24.0	320	23.7
	1581	15.3	800	24.0
	5000	T	2000	15.3
NaN3	2	463.7	2	711.3
TA 1535 +S-9
DMSO		22.2		20.4
Test item	5	22.3	8.192	21.3
	15.81	22.7	20.48	23.3
	50	25.0	51.2	17.7
	158.1	29.0	128	12.3
	500	26.7	320	11.3
	1581	13.0	800	T
	5000	T	2500	T
AAN	5	276.7	5	223.3
TA 1537 -S-9
DMSO		12.0		10.8
Test item	5	18.3	8.192	9.3
	15.81	11.3	20.48	9.7
	50	15.0	51.2	12.0
	158.1	9.0	128	14.0
	500	8.0	320	8.3
	1581	3.0	800	5.0
	5000	T	2000	T
AAC	50	201.7	50	55.3
TA 1537 +S-9
DMSO		12.6		17.2
Test item	5	15.0	8.192	16.3
	15.81	18.0	20.48	12.0
	50	17.3	51.2	12.3
	158.1	15.0	128	13.0
	500	16.7	320	7.7
	1581	6.7	800	T
	5000	T	2500	T
AAN	5	184.7	5	95.3
TA 102 -S-9
DMSO		265.8		241.2
Test item	5	232.3	8.192	241.7
	15.81	278.3	20.48	257.7
	50	274.7	51.2	248.0
	158.1	277.0	128	244.0
	500	259.7	320	232.3
	1581	71.3	800	149.3
	5000	T	2000	10.7
MMC	0.2	743.0	0.2	794.7
TA 102 +S-9
DMSO		231.0		239.8
Test item	5	266.3*	8.192	256.7
	15.81	235.3	20.48	282.0*
	50	261.3	51.2	296.0**
	158.1	222.0	128	231.3
	500	198.3	320	217.3
	1581	202.7	800	T
	5000	T	2500	T
AAN	20	1108.0	20	1042.0
	Experiment 3 (TA 102 +S-9)		Experiment 4 (TA 1537 +S-9)
DMSO		233.6		10.8
Test item	5	212.7	2.344	8.0
	10	239.3	4.688	8.0
	25	242.7	9.375	7.3
	50	242.0	18.75	8.7
	75	242.0	37.5	5.0
	100	218.7	75	7.7
	125	222.3	150	7.0
AAN	20	1012.3	5	103.3

T: Toxic, no revertant colonies; * p ≤ 0.05; ** p ≤ 0.01

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Nerol is not considered as mutagenic in S. typhimurium strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102).

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) were exposed to nerol at the following concentrations.

- Experiment 1: 5, 15.81, 50, 158.1, 500, 1581 and 5000 μg/plate, with and without S9 mix in all 5 strains (plate incorporation method)

- Experiment 2: 8.192, 20.48, 51.2, 128, 320 and 800 μg/plate, with S9 mix (preincubation method) and without S9 mix (plate incorporation method) in all 5 strains [2000 μg/plate employed for treatments of all strains in the absence of S9 only; 2500 μg/plate employed for treatments of all strains in the presence of S9 only]

- Experiment 3: 5, 10, 25, 50, 75, 100 and 125 μg/plate, with S9 mix in strain TA 102 (preincubation method)

- Experiment 4: 2.344, 4.688, 9.375, 18.75, 37.5, 75 and 150 μg/plate, with S9 mix in strain TA 1537 (preincubation method)

Metabolic activation system used in this test was 10% of S9 mix. S9 fraction was prepared from liver homogenates of male Sprague Dawley rats induced with Aroclor 1254. Vehicle control and positive control groups were also included in mutagenicity tests.

In Experiment 1, toxicity was observed in all strains at 1581 and 5000 μg/plate. In Experiment 2, toxicity was observed at 51.2 μg/plate and above in strain TA 1537 in the presence of S9; 128 μg/plate and above in strains TA 98, TA 100 and TA 102 in the presence of S9; 320 μg/plate and above in strains TA 98 and TA 1537 in the absence of S9 and strain TA 1535 in the presence of S9; and 800 μg/plate in strains TA 100, TA 1535 and TA 102 in the absence of S9. In Experiment 3, toxicity was observed at 125 μg/plate with S9 mix in strain TA 102. In Experiment 4, toxicity was observed at 150 μg/plate with S9 mix in strain TA 1537. The positive and vehicle controls induced the appropriate responses in the corresponding strains. Statistically significant increase in revertant numbers was observed in experiment 2 following Nerol treatments of strain TA102 at 51.2 μg/plate in the presence of S9 (Dunnett's Test, 1 % level), however this was not concentration-related or reproducible (Experiment 3) and was of insufficient magnitude to be considered as clear evidence of mutagenic activity in this assay system.

Therefore, nerol is not considered as mutagenic in this bacterial system.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 April - 14 June 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP study conducted according to OECD Guideline 473 with minor deviations: positive control slides were not analysed for mitotic inhibition.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

positive control slides were not analysed for mitotic inhibition

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

None

Species / strain / cell type:

lymphocytes: Blood from three healthy, non-smoking male volunteers were used to prepare cultures for each experiment in this study.

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

2 % S9 mix (final concentration); S9 fraction was obtained from the liver of male Sprague Dawley rats induced with Phenobarbital / 5,6 Benzoflavone

Test concentrations with justification for top dose:

- Range-Finder Experiment: 5.598, 9.330, 15.55, 25.92, 43.19, 71.99, 120, 200, 333.3, 555.5, 925.8 and 1543 μg/mL [3 h treatment + 17 h recovery, -S9; 3 h treatment + 17 h recovery, +S9; 20 h treatment without recovery, -S9]
- Experiment 1: 50, 100, 150, 200, 220, 240, 260, 280, 300, 320, 360, 400 and 500 μg/mL [3 h treatment + 17 h recovery, ±S9]
- Experiment 2: 50, 100, 150, 200, 215, 230, 245, 260, 275, 290, 305, 350 and 400 μg/mL [3 h treatment + 17 h recovery, -S9]
- Experiment 2: 100, 150, 200, 230, 245, 260, 275, 290, 305, 320, 335, 350 and 400 μg/mL [3 h treatment + 17 h recovery, +S9]
- Experiment 2: 10, 20, 40, 60, 70, 80, 90, 100, 110, 120, 130, 150 and 200 μg/mL [20 h treatment without recovery, -S9]

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Test article stock solutions were prepared by formulating Nerol under subdued lighting in DMSO, with the aid of vortex mixing, to give the maximum desired concentrations. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 4 h of initial formulation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without S9 mix: 4-Nitroquinoline 1-oxide (NQO) - 2.50 and 5.00 µg/mL; with S9 mix: Cyclophosphamide (CPA) - 6.25 and 12.50 µg/mL

Details on test system and experimental conditions:

PREPARATION OF CULTURES:
- Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled heparinised blood into 9.0 mL pre-warmed (in an incubator set to 37 ± 1 °C) HEPES-buffered RPMI medium containing 10 % (v/v) heat inactivated foetal calf serum and 0.52 % penicillin / streptomycin, so that the final volume following addition of S9 mix or KCl and the test article in its chosen vehicle was 10 mL. The mitogen Phytohaemagglutinin was included in the culture medium at a concentration of approximately 2 % of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37 ± 1 °C for approximately 48 h.

METHOD OF APPLICATION: In RPMI medium containing 10 % (v/v) heat inactivated foetal calf serum, 0.52 % penicillin / streptomycin and phytohaemagglutinin.

DURATION
- Exposure duration: Range-Finder Experiment - 3 h (±S9) and 20 h (- S9); Experiment 1 - 3 h (±S9); Experiment 2- 3 h (±S9) and 20 h (- S9)
- Fixation time (start of exposure up to harvest of cells): 20 h

SPINDLE INHIBITOR (cytogenetic assays): Mitotic activity was arrested by addition of colchicine at 1 µg/mL (final concentration) for each culture, approximately 2 h before the harvest.

STAIN (for cytogenetic assays): 4 % (v/v) Giemsa in pH 6.8 Gurr’s buffer

NUMBER OF REPLICATIONS:
- Range-Finder Experiment: 2 cultures for vehicle control; single culture per dose for test item group
- Experiment 1 and 2: 4 cultures for vehicle control; 2 cultures per dose for test item and positive control groups

NUMBER OF CELLS EVALUATED:
- Cytotoxicity of the test item was evaluated using the mitotic index, which indicates whether an test item induces mitotic inhibition. At least 1000 cells counted, where possible to calculate the mitotic index.
- Where possible / appropriate, one hundred metaphases from each code were analysed for chromosome aberrations. Where 10 cells with structural aberrations (excluding gaps) were noted on a slide, analysis may have been terminated. Only cells with 44 to 48 chromosomes were considered acceptable for analysis. Any cell with more than 48 chromosomes (i.e., polyploid, hyperdiploid or endoreduplicated cells) observed during this evaluation was noted and recorded separately.

DETERMINATION OF CYTOTOXICITY
Method:
- Mitotic index = (Number of cells in mitosis/Total number of cells observed)*100
- Mitotic inhibition [MIH (%)] = [1 - (mean MIT/mean MIC)] x 100 % (where T = treatment and C = vehicle control)

OTHER EXAMINATIONS:
-Structural aberrations: Gaps, chromosome deletions, chromosome exchanges, chromatid deletions, chromatid exchanges and other structural aberrations (multiple aberrations and pulverizations)
- Numerical aberrations: Polyploid, hyperdiploid and endoreduplication

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic events if:
1. A proportion of cells with structural aberrations at one or more concentrations that exceeded the normal range was observed in both replicate cultures.
2. A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) was observed (p ≤ 0.05).
3. There was a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps).
- Test article was considered positive in this assay if all of the above criteria were met.
- Test article was considered negative in this assay if none of the above criteria were met.
- Results which only partially satisfied the above criteria were to be dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result (Scott et al., 1990). Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.

Statistics:

- Statistical significance of increases in the percentage of cells with structural aberrations for any data set was only taken into consideration if the frequency of aberrant cells in both replicate cultures at one or more concentrations exceeds the normal range. The statistical method used was Fisher's exact test (Richardson et al., 1989). Probability values of p ≤ 0.05 were accepted as significant.
- Proportions of aberrant cells in each replicate were also used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test (Richardson et al., 1989). Probability values of p ≤ 0.05 were accepted as significant.

Key result

Species / strain:

lymphocytes: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
Solubility: Test item was miscible in DMSO at concentrations of at least 167.07 mg/mL.
Precipitation: Solubility limit in culture medium was in the range of 835.35 to 1670.7 μg/mL, as indicated by precipitation at the higher concentrations which persisted for at least 20 h after test article addition.
Effects of pH and osmolality: No marked changes in osmolality or pH were observed at the highest concentration tested (1543 μg/mL) as compared to the concurrent vehicle controls.

RANGE-FINDING EXPERIMENT:
- 3 h treatment + 17 h recovery, -S9: Following treatment of cultures for 3+17 h in the absence of S9, a fairly steep toxicity profile was observed based on MIH. Minimal cytotoxicity was observed at concentrations of 120.0 μg/mL and below, with extreme cytotoxicity at 333.3 μg/mL and above. Precipitate was observed by eye at the end of treatment at 555.5 μg/mL and above concentrations.

- 3 h treatment + 17 h recovery, +S9: Following treatment of cultures for 3+17 h in the presence of S9, a fairly steep toxicity profile was again observed based on MIH. Minimal cytotoxicity was observed at concentrations of 200 μg/mL and below (there were small increases and decreases in MIH at the lower concentrations but due to the differences between MI values of the replicate vehicle cultures, all MI values below 200 μg/mL were considered similar to that of the vehicle), with extreme cytotoxicity at 333.3 μg/mL and above. Precipitate was observed by eye at the end of treatment at 925.8 μg/mL and above concentrations.

- 20 h treatment without recovery, -S9: Following treatment of cultures for 20+0 hours in the absence of S9, minimal cytotoxicity was observed at concentrations of 25.92 μg/mL and below, with extreme cytotoxicity at 120 μg/mL and above. Precipitate was observed by eye at the end of treatment at 555.5 μg/mL and above concentrations.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with the historical data (May 2009 - January 2012) of the laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- See table 7.6.1/1

Remarks on result:

other: other: human lymphocytes

Remarks:

Migrated from field 'Test system'.

Table 7.6.1/1: Range finder experiment

Treatment (μg/mL)	MIH (%)
	3+17 hours, -S-9	3+17 hours, +S-9	20+0 hours, -S-9
Vehicle	-	-	-
5.598	0	0	1
9.33	0	13	16
15.55	2	18	0
25.92	0	19	0
43.19	0	6	20
71.99	0	24	36
120	4	8	73
200	24	17	86
333.3	100 P	84 P	100 P
555.5	PEH T	PH T	PH T
925.8	PEH T	PEH T	PH T
1543	PEH T	PEH T	PH T

P = Precipitation observed at treatment; E = Precipitation observed at the end of treatment incubation; H = Precipitation observed at harvest; T: Toxic

Table 7.6.1/2: Results of chromosomal aberration assay

Concentration (µg/mL)	Cytotoxicity - MIH (%)	% Cells with Chromosome Aberrations (Excluding Gaps)	Historical Control Range (%)	Statistical significance
Experiment 1: 3+17 h treatment, +S9
Vehicle	-	1.00	0-3	-
150	3	0.50		NR
220	23	0.00		NR
280	47	0.50		NR
300	38	0.50		NR
CPA, 12.50	ND	52.63		p ≤ 0.001
Experiment 2: 3+17 h treatment, -S9
Vehicle	-	0.00	0-3	-
50	0	0.50		NR
100	25	0.00		NR
150	23	1.50		NR
200	47	0.00		NR
NQO, 2.50	ND	16.58		p ≤ 0.001
Experiment 2: 3+17 h treatment, +S9
Vehicle	-	2.00	0-3	-
100	0	1.00		NR
200	22	1.50		NR
245	38	0.00		NR
290	61	1.50		NR
CPA, 6.25	ND	28.57		p ≤ 0.001
Experiment 2: 20+0 h treatment, -S9
Vehicle	-	0.00	0-3	-
40	0	1.00		NR
70	18	0.00		NR
100	31	0.00		NR
130	53	0.50		NR
NQO, 2.50	ND	13.02		p ≤ 0.001

NR = Not required as there were no concentrations analysed where both cultures demonstrated aberrant cell frequencies (excluding gaps) that exceeded historical vehicle control ranges; ND = Not determined

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Nerol is not considered as clastogenic in cultured human lymphocytes with and without metabolic activation.

Executive summary:

In an in vitro chromosome aberration test performed according to OECD Guideline 473 and in compliance with GLP, cultured human lymphocytes were exposed to nerol at the following concentrations.

- Range-Finder Experiment: 5.598, 9.330, 15.55, 25.92, 43.19, 71.99, 120, 200, 333.3, 555.5, 925.8 and 1543 μg/mL [3 h treatment + 17 h recovery, -S9; 3 h treatment + 17 h recovery, +S9; 20 h treatment without recovery, -S9]

- Experiment 1: 50, 100, 150, 200, 220, 240, 260, 280, 300, 320, 360, 400 and 500 μg/mL [3 h treatment + 17 h recovery, ±S9]

- Experiment 2: 50, 100, 150, 200, 215, 230, 245, 260, 275, 290, 305, 350 and 400 μg/mL [3 h treatment + 17 h recovery, -S9]

- Experiment 2: 100, 150, 200, 230, 245, 260, 275, 290, 305, 320, 335, 350 and 400 μg/mL [3 h treatment + 17 h recovery, +S9]

- Experiment 2: 10, 20, 40, 60, 70, 80, 90, 100, 110, 120, 130, 150 and 200 μg/mL [20 h treatment without recovery, -S9]

Mitotic activity was arrested by addition of colchicine at 1 µg/mL for each culture, 2 h before the harvest. The cells were then treated with a hypotonic solution, fixed, stained and examined for mitotic indices and chromosomal aberrations. Metabolic activation system used in this test was 2% of S9 mix (final concentration). S9 fraction was obtained from the liver of male Sprague Dawley rats induced with Phenobarbital / 5,6 Benzoflavone.

In both the experiments, test item did not induce chromosome aberrations in the absence and presence of metabolic activation, when tested up to cytotoxic concentrations. The positive control substances, 4-Nitroquinoline 1-oxide (2.50 and 5.00 μg/mL, without S9 mix) and cyclophosphamide (6.25 and 12.50 μg/mL, with S9 mix) gave the expected statistically significant increases in the number of cells with structural chromosome aberrations, which demonstrates the sensitivity of the test system.

Therefore, nerol is not considered as clastogenic in cultured human lymphocytes with and without metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 July - 9 October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well conducted and well described study in accordance with GLP and OECD Guideline 476 without any deviation.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

hprt gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Source: Dr Donald Clive, Burroughs Wellcome Co.
- Type and identity of media: RPMI 1640 medium
RPMI A: Penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL) and pluronic (0.5 mg/mL)
RPMI 10: Horse serum (heat inactivated, 10 % v/v), penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL) and pluronic (0.5 mg/mL)
RPMI 20: Horse serum (heat inactivated, 20 % v/v), penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL)
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Other details: For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5±1 % v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

2 % S9 (final concentration); S9 fraction was prepared from liver homogenates of rats treated with Aroclor 1254.

Test concentrations with justification for top dose:

Range-Finder Experiment:
- 48.22, 96.44, 192.9, 385.8, 771.5 and 1543 μg/mL, with and without S9 mix (3 h exposure)

Experiment 1:
- Without S9 mix (3 h exposure): 30, 60, 90, 120, 140, 160, 180, 200, 225, 250 and 300 μg/mL
- With S9 mix (3 h exposure): 50, 100, 150, 200, 230, 260, 290, 320, 350, 400 and 500 μg/mL

Experiment 2:
- Without S9 mix (3 h exposure): 50, 100, 150, 170, 190, 200, 210, 220, 230, 250, 275 and 300 μg/mL
- With S9 mix (3 h exposure): 50, 100, 150, 200, 230, 260, 270, 280, 290, 300 and 350 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Formulation preparation: Test article solutions were prepared by formulating Nerol in DMSO under subdued lighting (with the aid of vortex mixing, as required) immediately prior to assay to give the maximum required concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within 2.5 h of initial formulation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO diluted 100-fold in the treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

0.15 and 0.20 μg/mL; without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO diluted 100-fold in the treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

2.00 and 3.00 μg/mL; with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: RPMI 1640 medium

DURATION
- Exposure duration: 3 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 12 days
- All incubations were performed at 37 ± 1 °C in a humidified incubator gassed with 5±1 % v/v CO2 in air

SELECTION AGENT (mutation assays): 6-thioguanine (6TG) at a final concentration of 15 μg/mL

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single cultures/dose for test item and vehicle control
- Main test: Two cultures for vehicle control and test item; single culture for positive control

NUMBER OF CELLS EVALUATED: 1.6, 1.6 and 20000 cells per well plated for survival, viability and 6TG resistance respectively.

DETERMINATION OF CYTOTOXICITY
- Method: Percentage Relative Survival
Plating efficiency (PE) = P / No of cells plated per well; and as an average of 1.6 cells/well were plated on all survival and viability plates, PE = P/1.6.
Percentage relative survival (% RS) = [PE (test)/PE (control)] x 100.
Adjusted % RS = % RS x (Post-treatment cell concentration for test article treatment / Post-treatment cell concentration for vehicle control)

OTHER:
Mutant Frequency (MF) per 10^6 viable cells for each set of plates was calculated as: MF = [PE (mutant)/PE (viable)] x 10^6.

Evaluation criteria:

For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
- The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p ≤ 0.05).
- There was a significant concentration-relationship as indicated by the linear trend analysis (p ≤ 0.05).
- The effects described above were reproducible.
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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentration tested (1543 μg/mL), compared to the concurrent vehicle controls.
- Precipitation: Solubility limit in culture medium was approximately 835.4 to 1671 μg/mL, as indicated by precipitation at the higher concentration which persisted for 20 h after test article addition.

RANGE-FINDER EXPERIMENT:
- In the cytotoxicity Range-Finder Experiment, upon addition of the test article to the cultures, precipitate was observed at the highest three concentrations in the absence and presence of S-9 (385.8 to 1543 μg/mL). Following the 3 h treatment incubation period, precipitate was observed at the highest concentration in the absence and presence of S-9 (1543 μg/mL), therefore all cultures were retained during the expression period. The highest concentration to provide ≥10 % RS was 192.9 μg/mL in the absence and presence of S-9, which gave 21 % and 108 % RS, respectively.

MAIN STUDY:
- In Experiment 1, upon addition of the test article to the cultures, precipitate was observed at the highest two concentrations in the presence of S-9 (400 and 500 μg/mL), but no precipitate was observed in the absence and presence of S-9 following the 3 h treatment incubation period. Immediately following treatment, the highest concentration in the presence of S-9 (500 μg/mL) was not plated for survival due to excessive toxicity. Seven days after treatment the highest concentrations selected to determine viability and 6TG resistance were 250 μg/mL in the absence of S-9 and 290 μg/mL in the presence of S-9, which gave 6 % and 5 % RS, respectively. Steep concentration-related toxicity was observed in both the absence and presence of S-9 and although the highest concentrations analysed under both treatment conditions gave below 10 % RS, they were considered acceptable for analysis.
- In Experiment 2, no precipitate was observed in the absence and presence of S-9. Seven days after treatment the highest concentrations selected to determine viability and 6TG resistance were 190 μg/mL in the absence of S-9 and 260 μg/mL in the presence of S-9, which gave 18 % and 2 % RS, respectively. Steep concentration-related toxicity was observed following treatment in the presence of S-9 and although cultures at 260 μg/mL gave 2 % RS, this concentration was analysed due to the toxicity profile and the close concentration spacing.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical control data.

Remarks on result:

other: strain/cell type: L5178Y tk^+/- (3.7.2C)

Remarks:

Migrated from field 'Test system'.

See attached Document for Tables of Results

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Nerol is not considered as mutagenic at the hprt locus of L5178Y mouse lymphoma cells, in the presence and absence of metabolic activation.

Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, L5178Y tk^+/-(3.7.2C) mouse lymphoma cells were exposed to nerol at the following concentrations. 

Range-Finder Experiment:

- 48.22, 96.44, 192.9, 385.8, 771.5 and 1543 μg/mL, with and without S9 mix (3 h exposure)

Experiment 1:

- Without S9 mix (3 h exposure): 30, 60, 90, 120, 140, 160, 180, 200, 225, 250 and 300 μg/mL

- With S9 mix (3 h exposure): 50, 100, 150, 200, 230, 260, 290, 320, 350, 400 and 500 μg/mL

Experiment 2:

- Without S9 mix (3 h exposure): 50, 100, 150, 170, 190, 200, 210, 220, 230, 250, 275 and 300 μg/mL

- With S9 mix (3 h exposure): 50, 100, 150, 200, 230, 260, 270, 280, 290, 300 and 350 μg/mL

 

Vehicle and positive control groups were also included in each mutagenicity test. Metabolic activation system used in this test was 2 % S9 mix (final concentration). S9 fraction was prepared from liver homogenates of rats treated with Aroclor 1254.

 

In the cytotoxicity Range-Finder Experiment, upon addition of the test article to the cultures, precipitate was observed at the highest three concentrations in the absence and presence of S-9 (385.8 to 1543 μg/mL). Following the 3 h treatment incubation period, precipitate was observed at the highest concentration in the absence and presence of S-9 (1543 μg/mL), therefore all cultures were retained during the expression period. The highest concentration to provide ≥10% RS was 192.9 μg/mL in the absence and presence of S-9, which gave 21% and 108% RS, respectively. In Experiment 1, upon addition of the test article to the cultures, precipitate was observed at the two highest concentrations in the presence of S-9 (400 and 500 μg/mL), but no precipitate was observed in the absence and presence of S-9 following the 3 h treatment incubation period. Immediately following treatment, the highest concentration in the presence of S-9 (500 μg/mL) was not plated for survival due to excessive toxicity. Seven days after treatment the highest concentrations selected to determine viability and 6TG resistance were 250 μg/mL in the absence of S-9 and 290 μg/mL in the presence of S-9, which gave 6% and 5% RS, respectively. Steep concentration-related toxicity was observed in both the absence and presence of S-9 and although the highest concentrations analysed under both treatment conditions gave below 10% RS, they were considered acceptable for analysis. In Experiment 2, no precipitate was observed in the absence and presence of S-9. Seven days after treatment the highest concentrations selected to determine viability and 6TG resistance were 190 μg/mL in the absence of S-9 and 260 μg/mL in the presence of S-9, which gave 18% and 2% RS, respectively. Steep concentration-related toxicity was observed following treatment in the presence of S-9 and although cultures at 260 μg/mL gave 2% RS, this concentration was analysed due to the toxicity profile and the close concentration spacing.In Experiments 1 and 2, no statistically significant increases in mutant frequency were observed following treatment with Nerol at any concentration tested in the absence and presence of S-9 and there were no significant linear trends. Mutant frequencies in negative control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals [4-nitroquinoline 1-oxide(without S9 mix) and benzo(a)pyrene (with S9 mix)] indicating the validity of the study.

 

Therefore, nerol is not considered as mutagenic at the hprt locus of L5178Y mouse lymphoma cells, in the presence and absence of metabolic activation.

Additional information

In a reverse gene mutation assay in bacteria, performed according to OECD guideline 471 and in compliance with GLP, S. typhimurium strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102) were exposed to nerol in presence and absence of metabolic activation, up to limit or cytotoxic concentrations, except a statistically significant increase in revertant numbers observed in one experiment following nerol treatments of strain TA102 at 51.2 μg/plate in the presence of S9 (Dunnett's Test, 1 % level). However this was not concentration-related or reproducible and was of insufficient magnitude to be considered as clear evidence of mutagenic activity in this assay system. Therefore, the results were negative.

The results were also negative in a chromosome aberration test performed in cultured human peripheral blood lymphocytes (in presence and absence of metabolic activation) according to OECD guideline 473 and in compliance with GLP.

The results were also negative in a gene mutation test (HPRT) in Mouse Lymphoma L5178Y cells (in presence and absence of metabolic activation) performed according to OECD guideline 476 and in compliance with GLP.

Justification for selection of genetic toxicity endpoint
No robust study summary was chosen for this endpoint because a weight of evidence approach was adopted; therefore, it was not possible to select only one of the studies used for this endpoint.

Short description of key information:
The results were negative in all in vitro mutagenicity tests available (Ames test and HPRT test). The results were also negative in a chromosome aberration test.

Endpoint Conclusion: No adverse effect observed (negative results)

Justification for classification or non-classification

As the results were negative in all in vitro tests performed with nerol (mutagenicity in bacteria, mutagenicity in mammlian cells and clastogenicity tests), it is not classified according to Directive 67/548/EEC and CLP Regulation (EC) No. 1272/2008.