Nerol

Genetic toxicity: in vitro

Administrative data

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.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Nerol

EC Number:

203-378-7

EC Name:

Nerol

Cas Number:

106-25-2

Molecular formula:

C10H18O

IUPAC Name:

3,7-dimethylocta-2,6-dien-1-ol

impurity 1

Reference substance name:

Geraniol

EC Number:

203-377-1

EC Name:

Geraniol

Cas Number:

106-24-1

Molecular formula:

C10H18O

IUPAC Name:

(2E)-3,7-dimethylocta-2,6-dien-1-ol

impurity 2

Reference substance name:

(R)-3,7-dimethyloct-6-en-1-ol

EC Number:

214-250-5

EC Name:

(R)-3,7-dimethyloct-6-en-1-ol

Cas Number:

1117-61-9

Molecular formula:

C10H20O

IUPAC Name:

(3R)-3,7-dimethyloct-6-en-1-ol

impurity 3

Reference substance name:

(-)-3,7-dimethyloct-6-en-1-ol

EC Number:

231-415-7

EC Name:

(-)-3,7-dimethyloct-6-en-1-ol

Cas Number:

7540-51-4

Molecular formula:

C10H20O

IUPAC Name:

(3S)-3,7-dimethyloct-6-en-1-ol

Test material form:

liquid

Details on test material:

Batch No. : 122207
Purity : 98.7%
Name of test material (as cited in study report): NEROL (CAS 106-25-2)
Physical state: colourless - slightly yellow liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry Date: 21 July 2013

Method

Target gene:

Histidine gene

Species / strain

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.

Controls

Controls 1

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

Controls 2

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).

Results and discussion

Test results

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'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.