7-(4-ethyl-1-methyloctyl)quinolin-8-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In accordance with the requirements of Regulation (EC) No.1907/2006, Annex VII, only one study is performed. The study (OECD 471) was performed under the conditions of GLP.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 November 2018 - 06 December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21. Jul. 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

adopted 30. May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor / 1702-18-01/O
- Expiration date of the lot/batch: 25 March 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature (20 ± 5 °C); Keep away from light
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle:

Stability in solvents H2O: unknown; Ethanol: unknown; acetone; unknown; CH3CN: unknown; DMSO: unknown
Solubility H2O: < 0.1 g/L; Ethanol: unknown; acetone: > 1 g/L; CH3CN: unknown; DMSO: unknown

- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid, stock liquid or gel:

In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 mL/L in demineralized water, dimethyl sulfoxide (DMSO) and acetone.
The liquid test item is sufficiently soluble in DMSO and acetone.
Based on the non-GLP pre-test, DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
On the day of the start of the experiment 1a, a stock solution containing 50 mL/L of the test item in DMSO was prepared. The test item solution was not sterile filtrated before use.
The stock solution was used to prepare the geometric series of the concentrations to be tested.
The following nominal concentrations were prepared for the first experiment:
5 μL/plate, 1.5 μL/plate, 0.5 μL/plate, 0.15 μL/plate and 0.05 μL/plate
On the day of the start of the experiment 1b and 2, a stock solution containing 1.5 mL/L of the test item in DMSO was prepared.
The following nominal concentrations were prepared for the experiment 1b:
0.15 μL/plate, 0.05 μL/plate, 0.015 μL/plate, 0.005 μL/plate, 0.0015 μL/plate and 0.0005 μL/plate
The following nominal concentrations were prepared for the experiment 2:
0.15 μL/plate, 0.075 μL/plate, 0.038 μL/plate, 0.019 μL/plate, 0.009 μL/plate, 0.005 μL/plate, 0.002 μL/plate and 0.001 μL/plate

Species / strain / cell type:

S. typhimurium TA 97

Remarks:

TA 97a

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 102

Species / strain / cell type:

S. typhimurium TA 1535

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : S9 was obtained by Trinova Biochem GmbH, Gießen.
Batch nos. 3970, 3833, 3974
Specification produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraper-itoneally.

Test concentrations with justification for top dose:

Experiment 1a: 5, 1.5, 0.5, 0.15, 0.05 µL / plate
Experiment 1b: 0.15, 0.05, 0.015, 0.005, 0.0015, 0.0005 µL / plate - dose levels chosen due to toxicity in Experiment 1a
Experiment 2: 0.15, 0.075, 0.038, 0.019, 0.009, 0.005, 0.002, 0,001 µL / plate

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: Based on the non-GLP pre-test, DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylenediamine

Remarks:

Concentration per plate: 20 μg Solvent: DMSO Strains: TA97a, TA98 and TA102 Metabolic activation: none

Negative solvent / vehicle controls:

yes

Remarks:

Demineralised water

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Concentration per plate: 1 μg Solvent: H2O Strains: TA100 and TA1535 Metabolic activation: none

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-amino anthracene

Remarks:

Concentration per plate: 1 μg Solvent DMSO Strains: TA97a, TA100, TA102 and TA1535. Metabolic activation: S9

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Concentration per plate: 20 μg Solvent: DMSO Strain: TA98 Metabolic activation: S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 3
- Number of independent experiments : 3


TREATMENT AND HARVEST SCHEDULE:
Plate incorporation method:

The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
• 100 μL test solution at each dose level, solvent (negative control) or reference mu-tagen solution (positive control)
• 500 μL S9 mix (see chapter 6.5.18, for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
• 100 μL bacteria suspension (see chapter 6.4.2, test system, culture of the strains)
• 2000 μL overlay agar (top agar)
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ±1 °C.

Pre-incubation method:

The following materials were gently vortexed in a test tube and incubated at 37 ±1°C for 20 minutes:
• 100 μL test solution at each dose level, solvent (negative control) or reference mu-tagen solution (positive control)
• 500 μL S9 mix (see chapter 6.5.18, for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
• 100 μL bacteria suspension (see chapter 6.4.2, test system, culture of the strains)
After the pre-incubation for 20 minutes, 2000 μL top agar was added and the tube was gently vortexed. The mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ±1 °C.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition
Toxicity Control
Performed in experiment 1a only analogously to the titre control with the maximum dose of test item on maximal-soft agar, two replicates with and without metabolic activation, incuba-tion for 48 hours at 37 ±1°C.

- OTHER:

Genotype Confirmation
Genotype confirmation is performed for each batch of lyophilized bacteria before stock culture preparation.

Histidine requirement
Each strain was streaked on a biotin and a histidine-biotin-plate, using a sterilized wire loop. The plates were incubated for 24 hours at 37 ±1 °C.

Ampicillin/Tetracycline-Resistance (pKM101, pAQ1)
Each strain was streaked on an ampicillin agar plate and on an ampicillin-tetracycline agar plate. TA1535 was used as control strain, since it is not ampicillin resistant. The plates were incubated for 24 hours at 37 ±1 °C.

UV-sensitivity (uvrB)
Each strain was streaked on a plate, and one half of the plate covered with aluminium foil so that one half of each streak was protected against light. The plates for the strain TA97a, TA100 and TA102 were irradiated for 8 seconds, the plates for the strain TA98 were irradi-ated for 10 seconds and the plates for the strain TA1535 were irradiated for 6 seconds with a germicidal lamp (254 nm, 30W).
Keeping a distance of 33 cm for the strains TA97a, TA102 and TA1535.
Keeping a distance of 66 cm for the following strains: TA98, TA100.
Incubation for 24 hours at 37 ±1 °C followed.

Crystal violet sensitivity (deep rough/rfa)
For each strain, two plates were used. 0.1 mL of bacteria suspension were mixed with 2 mL Top-Agar and poured on nutrient agar. Sterile paper discs ( 9 mm), each soaked with 10 μL of crystal violet solution (0.1%) were placed into the middle of each plate, followed by incubation for 24 hours at 37 ±1°C.

Spontaneous Revertants
Three replicates, with/without S9, for each solvent which was used in the test, incubation for 48 hours at 37 ±1°C.

Determination of Titre
The titre was determined by dilution of the overnight culture using sodium chloride solution and placing 0.1 mL on maximal-soft agar. Incubation for 48 hours at 37 ±1 °C followed. It should give a density of 109 cells/mL (at the least), two replicates with and without metabolic activation.

Sterility Control
Performed analogously to the test with solvent only and S9 (without adding bacteria) on top agar, incubation for 48 hours at 37 ±1°C, four replicates.

Solubility
Plates were checked for precipitation of test item at the end of the incubation by visual in-spection.

Positive Controls
Using diagnostic mutagens (see chapter 6.3, page 12), three replicates were prepared. The stock solutions of the substances were diluted to achieve an application volume of 0.1 mL/plate, incubation for 48 hours at 37 ±1°C.

Rationale for test conditions:

In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 mL/L in demineralized water, dimethyl sulfoxide (DMSO) and acetone.
The liquid test item is sufficiently soluble in DMSO and acetone.
Based on the non-GLP pre-test, DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
On the day of the start of the experiment 1a, a stock solution containing 50 mL/L of the test item in DMSO was prepared. The test item solution was not sterile filtrated before use.
The stock solution was used to prepare the geometric series of the concentrations to be tested.
The following nominal concentrations were prepared for the first experiment:
5 μL/plate, 1.5 μL/plate, 0.5 μL/plate, 0.15 μL/plate and 0.05 μL/plate
On the day of the start of the experiment 1b and 2, a stock solution containing 1.5 mL/L of the test item in DMSO was prepared.
The following nominal concentrations were prepared for the experiment 1b:
0.15 μL/plate, 0.05 μL/plate, 0.015 μL/plate, 0.005 μL/plate, 0.0015 μL/plate and 0.0005 μL/plate
The following nominal concentrations were prepared for the experiment 2:
0.15 μL/plate, 0.075 μL/plate, 0.038 μL/plate, 0.019 μL/plate, 0.009 μL/plate, 0.005 μL/plate, 0.002 μL/plate and 0.001 μL/plate

Evaluation criteria:

The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard devia-tions of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants less mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of re-vertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Key result

Species / strain:

other: TA 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In highest concentration only

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In top concentration only

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In highest concentration only

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In top concentration only

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In top concentration only

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: low
- Precipitation and time of the determination: no precipitation was observed

RANGE-FINDING/SCREENING STUDIES (if applicable):

See attached study report for full results


STUDY RESULTS
- Concurrent vehicle negative and positive control data
See attached study report for full results

Ames test:
- Signs of toxicity : yes
- Individual plate counts : see below field
- Mean number of revertant colonies per plate and standard deviation : see below field


HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
See study report attached

Experiment 1a

Confirmation of the Criteria and Validity

All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory (historical data of the laboratory see chapter 16, page 49). All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.

Solubility and Toxicity

In this experiment, the test item showed no precipitates on the plates in all tested concentrations.

The test item showed a clear toxicity towards all five bacteria strains. In the three highest concentrations (5, 1.5 and 0.5 μL/plate) no bacteria growth and no bacterial background lawn was visible.

In the next lower concentration (0.15 μL/plate), the bacterial background lawn was present, but a decrease in the number of revertants was observed in all bacteria strains.

In the lowest concentration (0.05 μL/plate) no signs of toxicity were observed.

Mutagenicity

No increase of the number of revertant colonies in the treatments with and without metabolic activation in the non-toxic concentrations could be observed. No concentration-related in-crease over the tested range was found.

Therefore, the test item is stated as not mutagenic under the conditions of this experiment.

Based on the toxicity results, this experiment was repeated under the same conditions with lower concentrations.

According to OECD guideline 471, five non-toxic concentrations must be evaluated for mutagenicity.

Survey of the Findings

The mean revertant values of the three replicates are presented in the following table.

Table 1. Mean Revertants Experiment 1a

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	77	117	45	44	90	93	239	245	12	13
	sd	4.0	7.6	9.0	2.6	11.0	9.0	20.5	52.8	3.5	3.6
DMSO	Mean	87	108	44	41	94	109	207	203	11	12
	sd	7.4	22.5	10.5	4.5	12.5	9.9	43.1	10.1	1.2	2.9
Positive Controls*	Mean	621	497	305	483	717	941	1291	1349	145	313
	sd	128.6	55.2	2.3	120.1	57.2	219.4	101.3	74.3	37.2	68.9
	f(I)	7.14	4.60	6.93	11.78	7.97	8.63	6.24	6.65	12.08	26.08
5 µL/plate	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
1.5 µL/plate	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
0.5 µL/plate	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
0.15 µL/plate	Mean	10	5	5	5	24	14	52	23	2	1
	sd	1.5	1.2	2.1	0.6	6.4	3.5	2.3	3.0	0.6	0.0
	f(I)	0.11	0.05	0.11	0.12	0.26	0.13	0.25	0.11	0.18	0.08
0.05 µL/plate	Mean	81	98	36	35	98	86	267	213	11	9
	sd	12.0	13.1	4.9	4.6	17.6	8.7	25.7	37.2	1.7	1.2
	f(I)	0.93	0.91	0.82	0.85	1.04	0.79	1.29	1.05	1.00	0.75

f(I) = increase factor

* Different positive controls were used

Experiment 1b

Confirmation of the Criteria and Validity

All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory (historical data of the laboratory see chapter 16, page 49). All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and nearly all were within the historical control data ranges.

Solubility and Toxicity

In this experiment, the test item showed no precipitates on the plates in all tested concentrations.

In the highest concentration (0.15 μL/plate), the bacterial background lawn was present, but a decrease in the number of revertants was observed in all bacteria strains.

In the lower concentrations no signs of toxicity were observed.

Mutagenicity

No increase of the number of revertant colonies in the treatments with and without metabolic activation in the non-toxic concentrations could be observed. No concentration-related in-crease over the tested range was found.

Therefore, the test item is stated as not mutagenic under the conditions of this experiment.

To verify the results of the experiment 1a and 1b a further experiment was performed with the pre- incubation method.

Survey of the findings - Experiment 1b

The mean revertant values of the three replicates are presented in the following table.

Table 2. Mean Revertants Experiment 1b

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	90	100	51	46	94	88	297	271	11	11
	sd	10.7	19.9	2.6	4.5	10.8	10.4	67.0	46.4	1.2	1.5
DMSO	Mean	86	97	42	41	90	93	223	239	9	9
	sd	15.9	7.4	1.0	2.3	10.4	11.7	12.2	54.5	1.2	2.3
Positive Controls*	Mean	784	789	293	199	720	s.g.	1145	1440	349	269
	sd	104.6	37.0	48.4	40.5	56.0	--	342.5	24.0	61.1	52.1
	f(I)	9.12	8.13	6.98	4.85	7.66	--	5.13	6.03	31.73	29.89
0.15 µL/plate	Mean	19	9	5	10	31	24	94	78	2	2
	sd	2.6	4.0	4.0	1.0	3.6	6.7	15.3	5.8	0.6	1.2
	f(I)	0.21	0.09	0.10	0.22	0.33	0.27	0.32	0.29	0.18	0.18
0.05 µL/plate	Mean	91	119	44	44	92	99	277	276	10	11
	sd	20.1	11.0	4.0	3.5	13.4	17.2	43.1	18.3	1.7	5.2
	f(I)	1.01	1.19	0.86	0.96	0.98	1.13	0.93	1.02	0.91	1.00
0.015 µL/plate	Mean	93	104	46	41	105	121	339	307	10	10
	sd	14.1	12.5	3.1	2.5	10.4	6.6	36.3	18.0	1.2	2.9
	f(I)	1.03	1.04	0.90	0.89	1.12	1.38	1.14	1.13	0.91	0.91
0.005 µL/plate	Mean	101	79	41	42	111	127	296	320	11	9
	sd	14.7	10.1	5.0	5.0	20.0	19.0	40.0	59.2	2.9	4.0
	f(I)	1.12	0.79	0.80	0.91	1.18	1.44	1.00	1.18	1.00	0.82
0.0015 µL/plate	Mean	95	94	42	45	122	114	337	316	10	10
	sd	11.7	7.5	9.3	6.7	4.5	24.2	46.4	8.0	0.6	1.2
	f(I)	1.06	0.94	0.82	0.98	1.30	1.30	1.13	1.17	0.91	0.91
0.0005 µL/plate	Mean	85	86	40	44	89	81	297	333	12	10
	sd	10.1	10.5	9.0	5.5	15.5	5.5	11.5	68.9	1.7	1.0
	f(I)	0.94	0.86	0.78	0.96	0.95	0.92	1.00	1.23	1.09	0.91

  s.g.= strong growth, too strong for counting of revertants

f(I) = increase factor

* Different positive controls were used

Experiment 2

  

Confirmation of the criteria and validity

All strains met the criterion of at least 10⁹bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory (historical lab data). .All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and nearly all were within the historical control data ranges.

Solubility and Toxicity

In the experiment 2, the test itemshowed no precipitates on the plates in all tested concentrations.

Signs of toxicity towards all the bacteria strains could be observed in the highest concentration (0.15 µL/plate). The number of revertants was decreased and the bacterial background lawn was slightly visible.

In the lower concentrations the bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.

Mutagenicity

No increase of the number of revertant colonies in the treatments with and without metabolic activation in the non-toxic concentrations could be observed. No concentration-related increase over the tested range was found.

 

Therefore, the test item is stated as not mutagenic under the conditions of this experimen

     

Survey of Findings - Experiment 2

The mean revertant values of the three replicates are presented in the following table.

Table 3. Mean Revertants Experiment 2

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	69	84	42	39	96	98	288	300	16	13
	sd	4.5	6.8	2.6	1.5	29.4	21.2	48.5	24.3	1.0	3.0
DMSO	Mean	86	110	49	35	78	93	309	279	13	15
	sd	10.0	4.0	13.3	0.0	7.4	15.0	28.4	68.0	2.9	3.6
Positive Controls*	Mean	371	355	150	133	517	s.g.	1355	1411	379	219
	sd	62.0	26.6	42.5	28.3	106.5	--	16.7	52.1	86.4	36.3
	f(I)	4.31	3.23	3.06	3.80	5.39	--	4.39	5.06	23.69	14.60
0.15 µL/plate	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
0.075 µL/plate	Mean	0	0	42	41	77	114	261	323	4	5
	sd	0.0	0.0	1.7	3.0	4.0	10.0	43.9	28.1	1.2	0.0
	f(I)	0.00	0.00	0.86	1.17	0.99	1.23	0.84	1.16	0.31	0.33
0.038 µL/plate	Mean	0	0	43	42	89	127	255	303	13	11
	sd	0.0	0.0	4.0	1.5	11.5	18.7	32.6	20.1	2.5	1.7
	f(I)	0.00	0.00	0.88	1.20	1.14	1.37	0.83	1.09	1.00	0.73
0.019 µL/plate	Mean	120	87	39	41	89	121	309	302	11	11
	sd	3.2	25.2	0.6	6.4	6.1	3.1	22.0	31.1	2.3	0.6
	f(I)	1.40	0.79	0.80	1.17	1.14	1.30	1.00	1.08	0.85	0.73
0.009 µL/plate	Mean	99	90	41	34	83	83	285	301	16	12
	sd	7.1	11.5	1.7	3.5	3.1	8.3	14.0	20.5	0.0	3.5
	f(I)	1.15	0.82	0.84	0.97	1.06	0.89	0.92	1.08	1.23	0.80
0.005 µL/plate	Mean	83	106	40	36	95	96	259	293	12	14
	sd	21.5	12.7	7.2	4.4	11.0	4.7	12.9	22.0	1.7	1.7
	f(I)	0.97	0.96	0.82	1.03	1.22	1.03	0.84	1.05	0.92	0.93
0.002 µL/plate	Mean	94	112	37	42	95	83	295	245	11	13
	sd	12.0	2.5	3.8	5.1	5.9	15.0	22.0	36.1	2.3	0.6
	f(I)	1.09	1.02	0.76	1.20	1.22	0.89	0.95	0.88	0.85	0.87
0.001 µL/plate	Mean	105	117	37	35	95	93	267	244	11	11
	sd	1.2	17.2	5.8	0.6	9.2	17.0	50.0	27.7	1.2	2.6
	f(I)	1.22	1.06	0.76	1.00	1.22	1.00	0.86	0.87	0.85	0.73

s.g.= strong growth, too strong for counting of revertants

f(I) = increase factor

* Different positive controls were used

Conclusions:

The test item 7-(4-ethyl-1-methyloctyl)quinolin-8-ol showed no increase in the number of revertants in all bacteria strains in all three experiments.
All negative and nearly all strain-specific positive control values were within the laboratory historical con-trol data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that 7-(4-ethyl-1-methyloctyl)quinolin-8-ol is not mu-tagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.

Executive summary:

SUMMARY

Title of Study: Determination of the mutagenic potential of 7-(4-ethyl-1-methyloctyl)quinolin-8-ol with the Bacterial Reverse Muta-tion Test following OECD 471 and EU B.13/14

Findings and Results:

Three valid experiments were performed.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

The test item 7-(4-ethyl-1-methyloctyl)quinolin-8-ol was tested in the Salmonella typhi-murium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535).

The test was performed in three experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

Experiment 1a:

In the experiment 1a, the test item (dissolved in dimethyl sulfoxide) was tested up to con-centrations of 5 μL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

The test item showed a clear toxicity towards all five bacteria strains. In the three highest concentrations (5, 1.5 and 0.5 μL/plate) no bacteria growth and no bacterial background lawn was visible.

In the next lower concentration (0.15 μL/plate), the bacterial background lawn was present, but a decrease in the number of revertants was observed in all bacteria strains.

In the lowest concentration (0.05 μL/plate) no signs of toxicity were observed.

The results of this experiment showed that none of the tested non-toxic concentrations showed an increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Experiment 1b:

Based on the toxicity results of the experiment 1a, the experiment 1a was repeated under the same conditions with lower concentrations.

The test item (dissolved in dimethyl sulfoxide) was tested up to concentrations of 0.15 μL/plate in the absence and presence of S9-mix in all bacteria strains.

The test item showed no precipitates on the plates at any of the concentrations.

In the highest concentration (0.15 μL/plate), the bacterial background lawn was present, but a decrease in the number of revertants was observed in all bacteria strains.

In the lower concentrations no signs of toxicity were observed.

The results of this experiment showed that none of the tested concentrations resulted in an increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Experiment 2:

Based on the results of the experiment 1a and 1b, the test item was tested up to concentrations of 0.15 μL/plate in the absence and presence of S9-mix in all bacteria strains using the pre-incubation method.

The test item showed signs of toxicity in all bacteria strains.

TA97a: 0.15 μL/plate, 0.075 μL/plate, 0.038 μL/plate (no bacteria growth)

TA98: 0.15 μL/plate (no bacteria growth)

TA100: 0.15 μL/plate (no bacteria growth)

TA102: 0.15 μL/plate (no bacteria growth)

TA1535: 0.15 μL/plate (no bacteria growth), 0.075 μL/plate (a decrease in the number of revertants)

The bacterial background lawn was visible in all concentrations.

The results of this experiments showed that the test item caused no increase in the number of revertants in all bacteria strains compared to the solvent control, both in the absence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic acti-vation.

Based on the results of this study it is concluded that 7-(4-ethyl-1-methyloctyl)quinolin-8-ol is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The available studies suggest thatm 7-(4-ethyl-1-methyloctyl)quinolin-8-ol is not mutagenic.