DL-α-(aminomethyl)-p-hydroxybenzylic alcohol hydrochloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

When tested in a bacterial reverse mutation assay (OECD Guideline 471) Octopamine hydrochloride did not induce a significant dose-related increase in the number of revertant (His⁺) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) or in the number of revertant (Trp⁺) colonies in the tester strain WP₂uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. Consequently, Octopamine hydrochloride was considered to be non-mutagenic in this assay.

The possible clastogenicity of Octopamine hydrochloride was tested in two independent experiments in accordance with OECD Guideline 473

Octopamine hydrochloride did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments.

No biologically relevant effects of Octopamine hydrochloride on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. In conclusion, Octopamine hydrochloride was not clastogenic in human lymphocytes under the experimental conditions of the study.

In the mammalian cell gene mutation assay, conducted in accordance with OECD Guideline 490, Octopamine hydrochloride did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor. Consequently, Octopamine hydrochloride is considered to be non-mutagenic in this assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

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 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Appearance: White to off white powder
Batch: D151-1710037
Purity/Composition: 99.8%
Test item storage: At room temperature protected from light
Stable under storage conditions until: 26 October 2019 (retest date)

Target gene:

Histidine Locus- S.typhimurium
Tryptophan operon- E.coli

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: rfa; gal; chl; bio; uvrB

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate), male Sprague Dawley rats, Aroclor 1254

Test concentrations with justification for top dose:

Dose rangefinding : 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
experiment 1: 52, 164, 512, 1600, 5000 µg/plate
experiment 2: 492, 878, 1568, 2800, 5000 µg/plate.
The highest concentration of the test item used in the subsequent mutation assays was 5000 µg/plate or the level at which the test item inhibited bacterial growth

Vehicle / solvent:

The vehicle of the test item was Milli-Q water

Untreated negative controls:

yes

Remarks:

milli-Q water

Negative solvent / vehicle controls:

yes

Remarks:

milli-Q water

True negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191 TA1537 without S9 2.5µg; 2-aminoanthracene - All strains with S9

Details on test system and experimental conditions:

Preparation of bacterial cultures
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1°C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL). Freshly grown cultures of each strain were used for testing.

Agar plates
Agar plates (ø 9 cm) containing 25 mL glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 µg/plate biotin (Merck) and 15 µg/plate histidine (Sigma) and the agar plates for the test with the Escherichia coli strain contained
15 µg/plate tryptophan (Sigma).

Top agar
Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v) sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 mL top agar were transferred into 10 mL glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.

Environmental conditions
All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 34.9 - 39.2°C). The temperature was continuously monitored throughout the experiment. Due to addition of plates (which were at room temperature) to the incubator or due to opening and closing the incubator door, temporary deviations from the temperature may occur. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Metabolic Activation System
Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
Each S9 batch is characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively.

Preparation of S9-Mix
S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per
10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL or 5.0 mL Milli-Q water (first or second experiment respectively) (Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 µm)-sterilized. To 9.5 mL of
S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the
S9-mix in the first experiment and to 9.0 mL of S9-mix components 1.0 mL S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.

Experimental Design
Dose-range Finding Test
Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate. The highest concentration of the test item used in the subsequent mutation assays was 5000 µg/plate or the level at which the test item inhibited bacterial growth.

Mutation Assay
At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain. The above mentioned dose-range finding study with the two tester strains TA100 and WP2uvrA, is reported as a part of the first mutation experiment. In the second part of this experiment, the test item was tested both in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment with additional parameters, the test item was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture
(109 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in Milli-Q water and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Colony Counting
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope

Rationale for test conditions:

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without
S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Evaluation criteria:

No formal hypothesis testing was done.
In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent vehicle control.

b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No formal hypothesis testing was done.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000µg/plate without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Dose-range Finding Test/First Mutation Experiment

Octopamine hydrochloride was tested in the tester strains TA100 and WP₂uvrAat concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of S9-mix. Based on the results of the dose-range finding test, the following dose-range was selected for the first mutation experiment with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate.

The results are shown in Table1and Table2. 

Precipitate

Precipitation of Octopamine hydrochloride on the plates was not observed at the start or at the end of the incubation period in any tester strain. 

Toxicity

To determine the toxicity of Octopamine hydrochloride, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. The definitions are stated inAppendix 2.

No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed, except in tester strain TA1537 in the absence of S9-mix, where a decrease in the number of revertants was observed at the highest dose level tested.

Mutagenicity

No increase in the number of revertants was observed upon treatment with Octopamine hydrochloride under all conditions tested.

 Second Mutation Experiment

To obtain more information about the possible mutagenicity of Octopamine hydrochloride, a second mutation experiment was performed in the absence and presence of 10% (v/v) S9-mix. Based on the results of the first mutation assay, the test item was tested up to the dose level of
5000 µg/plate in strains TA1535, TA1537, TA98, TA100 and WP₂uvrA. The results are shown inTable3.

Precipitate

Precipitation of Octopamine hydrochloride on the plates was not observed at the start or at the end of the incubation period. 

Toxicity

In the second mutation assay, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

Mutagenicity

In the second mutation assay, no increase in the number of revertants was observed upon treatment with Octopamine hydrochloride under all conditions tested.

Table 1: Dose-Range Finding Test: Mutagenic Response of Octopamine hydrochloride in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with oneSalmonella typhimuriumand oneEscherichia colistrain.
	TA100	WP2uvrA

 

Without S9-mix

 

Positive control	947	±	90		1278	±	95
Solvent control	133	±	15		25	±	5
1.7	134	±	10		18	±	7
5.4	118	±	9		22	±	11
17	124	±	8		17	±	10
52	112	±	18		17	±	2
164	104	±	5		20	±	7
512	138	±	12		20	±	4
1600	126	±	5		27	±	7
5000	131	±	3	^n NP	25	±	4	^n NP

 

With S9-mix¹

 

Positive control	1252	±	60		265	±	23
Solvent control	114	±	9		21	±	8
1.7	118	±	11		22	±	11
5.4	131	±	15		25	±	3
17	119	±	9		27	±	6
52	136	±	11		21	±	5
164	130	±	14		32	±	6
512	115	±	9		26	±	2
1600	129	±	8		22	±	10
5000	140	±	13	^n NP	32	±	12	^n NP

 

^1	Plate incorporation assay (5% S9)
NP	No precipitate
n	Normal bacterial background lawn

 

Table2: Experiment 1: Mutagenic Response of Octopamine hydrochloride in the Salmonella typhimurium Reverse Mutation Assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains ofSalmonella typhimurium.
	TA1535	TA1537	TA98

 

Without S9-mix

 

Positive control	962	±	39		996	±	35		769	±	107
Solvent control	10	±	5		4	±	1		9	±	2
52	6	±	2		5	±	1		7	±	3
164	7	±	4		6	±	2		10	±	2
512	6	±	3		5	±	2		8	±	3
1600	10	±	4		4	±	1		13	±	3
5000	9	±	3	^n NP	2	±	1	^n NP	9	±	2	^n NP

 

With S9-mix¹

 

Positive control	359	±	47		436	±	107		1024	±	120
Solvent control	9	±	2		5	±	1		12	±	6
52	8	±	3		4	±	1		16	±	3
164	7	±	2		4	±	2		15	±	5
512	8	±	3		6	±	1		13	±	2
1600	8	±	3		4	±	2		13	±	2
5000	10	±	4	^n NP	5	±	2	^n NP	14	±	2	^n NP

 

^1	Plate incorporation assay (5% S9)
NP	No precipitate
n	Normal bacterial background lawn

 

Table 3: Experiment 2: Mutagenic Response of Octopamine hydrochloride in theSalmonella typhimuriumReverse Mutation Assay and in theEscherichia coliReverse Mutation Assay

 

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains ofSalmonella typhimuriumand oneEscherichia colistrain.
	TA1535	TA1537	TA98	TA100	WP2uvrA

 

Without S9-mix

 

Positive control	877	±	65		998	±	45		1075	±	10		892	±	27		866	±	688
Solvent control	15	±	8		6	±	5		16	±	5		131	±	20		21	±	14
492	13	±	6		4	±	3		20	±	2		127	±	10		28	±	10
878	12	±	6		6	±	2		14	±	2		123	±	2		23	±	5
1568	14	±	3		7	±	5		12	±	2		130	±	3		32	±	8
2800	15	±	6		6	±	2		18	±	2		125	±	16		23	±	7
5000	12	±	5	^n NP	5	±	2	^n NP	16	±	4	^n NP	130	±	12	^n NP	28	±	9	^n NP

 

 

With S9-mix¹

 

Positive control	201	±	16		324	±	42		388	±	44		948	±	303		185	±	138
Solvent control	15	±	4		4	±	4		21	±	6		89	±	6		30	±	15
492	17	±	4		6	±	0		28	±	16		89	±	7		41	±	7
878	10	±	2		4	±	2		16	±	3		86	±	4		42	±	7
1568	9	±	3		7	±	1		19	±	11		85	±	12		37	±	6
2800	17	±	6		6	±	3		28	±	5		96	±	5		38	±	4
5000	17	±	3	^n NP	8	±	4	^n NP	23	±	3	^n NP	85	±	6	^n NP	49	±	12	^n NP

 

 

^1	Plate incorporation assay (10% S9)
NP	No precipitate
n	Normal bacterial background lawn

Conclusions:

In conclusion, based on the results of this study it is concluded that Octopamine hydrochloride is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The objective of this study was to determine the potential of Octopamine hydrochloride and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP₂uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). 

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

Batch D151-1710037 of Octopamine hydrochloride was a white to off white powder. The vehicle of the test item was Milli-Q water.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP₂uvrAin the absence and presence of 5% (v/v)
S9-mix. In the first mutation experiment, the test item was again tested up to concentrations of 5000 µg/plate in the strains TA1535, TA1537 and TA98 in the absence and presence of 5% (v/v) S9-mix. In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 492 to 5000 µg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP₂uvrA. In all three experiments the test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed, except in tester strain TA1537 in the absence of S9-mix in the first experiment, where a decrease in the number of revertants was observed at the highest dose level tested.  

Octopamine hydrochloride did not induce a significant dose-related increase in the number of revertant (His⁺) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp⁺) colonies in the tester strain WP₂uvrAboth in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

In conclusion, based on the results of this study it is concluded that Octopamine hydrochloride is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. 

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Jun 2018 to 23 Sep 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Deviations:

yes

Remarks:

None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Identification: Octopamine hydrochloride
Appearance: White to off white powder (determined by
Charles River Den Bosch)
Batch: D151-1710037
Test item storage: At room temperature protected from light
Stable under storage conditions until: 26 October 2019 (retest date)

Species / strain / cell type:

lymphocytes:

Remarks:

Human

Metabolic activation:

with and without

Metabolic activation system:

Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is
prepared from male Sprague Dawley rats that have been dosed orally with a suspension of
phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).

Test concentrations with justification for top dose:

In the first cytogenetic assay, Octopamine hydrochloride was tested up to 1896 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. This is the highest concentration recommended for testing in the guideline (= 0.01 M). In the second cytogenetic assay, Octopamine hydrochloride was also tested up to 1896 µg/mL for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time, respectively.

Vehicle / solvent:

culture medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Test System
Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in international guideline (OECD). Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2017) are presented below:
Dose-range finding study: age 26, AGT = 14.7 h
First cytogenetic assay: age 26, AGT = 13.5 h
Second cytogenetic assay: age 23, AGT = 13.9 h

Cell Culture
Blood samples
Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One, Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were started.

Culture medium
Culture medium consisted of RPMI 1640 medium (Life technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life technologies), L-glutamine (2 mM) (Life technologies), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) (Life technologies) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).

Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin (Remel, Europe Ltd., Dartford, United Kingdom) was added.

Environmental conditions
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 61 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.1 - 37.2°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Metabolic Activation System
Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).

Preparation of S9-Mix
S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 µmol HEPES (Life technologies). The above solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix. Metabolic activation was achieved by adding 0.2 mL S9-mix to 5.3 mL of a lymphocyte culture (containing 4.8 mL culture medium, 0.4 mL blood and 0.1 mL (9 mg/mL) phytohaemagglutinin). The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).

Experimental Design
Dose-range Finding Test In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose-range finding test. Octopamine hydrochloride was tested in the absence and in the presence of 1.8% (v/v) S9-fraction. Lymphocytes (0.4 mL blood of a healthy donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed to selected doses of Octopamine hydrochloride for 3 h, 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix. A negative control was included at each exposure time. The highest tested concentration was the recommended 0.01 M (1896 µg/mL). After 3 h exposure to Octopamine hydrochloride in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium and incubated for another 20 - 22 h (24 h fixation time). The cells that were exposed for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 h and 48 h fixation time). Cytotoxicity of Octopamine hydrochloride in the lymphocyte cultures was determined using the mitotic index. Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level was the recommended 0.01 M (1896 µg/mL).

Cytogenetic Assay
The cytogenetic assay was carried out as described by Evans, 1984 (2) with minor modifications. Octopamine hydrochloride was tested in the absence and presence of 1.8% (v/v) S9-fraction in duplicate in two independent experiments. First cytogenetic assay Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected doses of Octopamine hydrochloride for 3 h in the absence and presence of S9-mix. After 3 h exposure, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium and incubated for another 20 - 22 h (24 h fixation time). Appropriate negative and positive controls were included in the first cytogenetic assay. Based on the mitotic index of the dose-range finding test and the first cytogenetic assay and on the solubility of Octopamine hydrochloride in the culture medium appropriate dose levels were selected for the second cytogenetic assay. As clear negative results were obtained in the presence of metabolic activation, the repetition of the experiment was not considered necessary. The follow up experiment was performed with the following modifications of experimental conditions. Second cytogenetic assay Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected doses of Octopamine hydrochloride for 24 h and 48 h in the absence of S9-mix. The cells were not rinsed after exposure but were fixed immediately after 24 h and 48 h (24 h and 48 h fixation time). Appropriate negative and positive controls were included in the second cytogenetic assay.

Chromosome Preparation
During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 µg/mL medium) (Acros Organics, Geel, Belgium). Thereafter the cell cultures were centrifuged for 5 min at 365 g and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride (Merck) solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol (Merck): acetic acid (Merck) fixative (3:1 v/v).

Preparation of Slides
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands). 4.7.5. Mitotic Index/Dose Selection for Scoring of the Cytogenetic Assay The mitotic index of each culture was determined by counting the number of metaphases from at least 991 cells (with a maximum deviation of 5%, see study plan deviation). At least three analyzable concentrations were used for scoring of the cytogenetic assay. Octopamine hydrochloride was not cytotoxic, the highest concentration analyzed was the recommended 0.01 M.

Analysis of Slides for Chromosome Aberrations
To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with Charles River Den Bosch study identification number and code was placed over the marked slide. One hundred and fifty metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 38 in 75 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analyzed. The number of cells with aberrations and the number of aberrations were calculated. Since the lowest concentration of MMC-C resulted in a positive response the highest concentration was not examined for chromosome aberrations.

Rationale for test conditions:

Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in international guideline (OECD).
Background of the test system

Whole blood samples obtained from healthy subjects were treated with an anti-coagulant (heparin) and cultured in the presence of a mitogen (phytohaemagglutinin). These stimulated human lymphocytes were used because they are sensitive indicators of clastogenic activity of a broad range of chemicals (1-5). The stimulated lymphocytes were exposed to Octopamine hydrochloride both in the absence and presence of a metabolic activation system (S9-mix). In combination with this metabolic activation system indirect chemical mutagens, i.e. those requiring metabolic transformation into reactive intermediates, can be tested for possible clastogenic effects in vitro. At predetermined intervals after exposure of the stimulated human lymphocytes to Octopamine hydrochloride, cell division was arrested in the metaphase stage of the cell cycle by addition of the metaphase-arresting chemical colchicine. Cells were harvested, stained and metaphase cells were analyzed for the presence of structural chromosome aberrations such as breaks, gaps, minutes, dicentrics and exchange figures. Results from cultures treated with Octopamine hydrochloride were compared with control (vehicle) treated cultures. Chromosome aberrations are generally evaluated in the first post-exposure mitosis (i.e. 24 hours after exposure). However, since the appearance of the first post-exposure mitosis could be considerably delayed due to toxic insult to the cells, cells were also harvested 48 hours after exposure to cover the interval in which maximum aberration frequency was expected. A test item that induces a positive response in this assay is presumed to be a potential mammalian cell clastogenic agent. The design of this study is based on the following study guideline: OECD Guideline 473

Evaluation criteria:

A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the
95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those
generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the number of cells
with chromosome aberrations. The positive control data will be analyzed by the Fisher’s
exact test (one-sided, p < 0.05).

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical
analysis of the data.
A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact
test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-
sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Additional information on results:

Dose-range Finding Test
A concentration of 1896 µg/mL (= 0.01 M) showed no precipitation in the culture medium. Therefore, this concentration was used as the highest concentration of Octopamine hydrochloride. The pH and osmolarity of a concentration of 1896 µg/mL were 7.3 and 322 mOsm/kg respectively (compared to 7.3 and 304 mOsm/kg in the solvent control). In the dose-range finding test blood cultures were treated with 125, 250, 500, 1000 and 1896 µg Octopamine hydrochloride/mL culture medium with and without S9-mix.

First Cytogenetic Assay
Based on the results of the dose-range finding test the following dose levels were selected for the cytogenetic assay: Without and with S9-mix : 500, 1000 and 1896 µg/mL culture medium (3 h exposure time, 24 h fixation time).
All dose levels were selected for scoring of chromosome aberrations. Both in the absence and presence of S9-mix, Octopamine hydrochloride did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.Both in the absence and presence of S9-mix, Octopamine hydrochloride did not show a biologically relevant increase in the number of polyploid cells and cells with endoreduplicated chromosomes.

Second Cytogenetic Assay
To obtain more information about the possible clastogenicity of Octopamine hydrochloride, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to Octopamine hydrochloride in the absence of S9-mix for 24 or 48 hours. The following dose levels were selected for the second cytogenetic assay: Without S9-mix : 500, 1000 and 1896 µg/mL culture medium (24 h and 48 h exposure time, 24 h and 48 h fixation time). All doses were selected for scoring of chromosome aberrations. Octopamine hydrochloride did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. Octopamine hydrochloride did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

Evaluation of the Results
Both in the absence and presence of S9-mix Octopamine hydrochloride did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments. No biologically relevant effects of Octopamine hydrochloride on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Octopamine hydrochloride does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Conclusions:

In conclusion, this test is valid and Octopamine hydrochloride is not clastogenic in human lymphocytes under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate Octopamine hydrochloride for its ability to induce

structural chromosome aberrations in cultured human lymphocytes, either in the presence or

absence of a metabolic activation system (S9-mix).

The possible clastogenicity of Octopamine hydrochloride was tested in two independent

experiments.

The study procedures described in this report are in compliance with the most recent OECD

guideline.

Batch D151-1710037 of Octopamine hydrochloride was a white to off white powder.  The

vehicle of the test item was culture medium.

In the first cytogenetic assay, Octopamine hydrochloride was tested up to 1896 µg/mL for a

3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v)

S9-fraction.  This is the highest concentration recommended for testing in the guideline

(= 0.01 M).  

In the second cytogenetic assay, Octopamine hydrochloride was also tested up to 1896 µg/mL

for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time, respectively.  

The number of cells with chromosome aberrations found in the solvent control cultures was

within the 95% control limits of the distribution of the historical negative control database.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically

significant increase in the incidence of cells with chromosome aberrations.  It was therefore

concluded that the test conditions were adequate and that the metabolic activation system (S9mix)

functioned

properly.

Octopamine hydrochloride did not induce any statistically significant or biologically relevant

increase in the number of cells with chromosome aberrations in the absence and presence of

S9-mix, in either of the two independently performed experiments.

No biologically relevant effects of Octopamine hydrochloride on the number of polyploid

cells and cells with endoreduplicated chromosomes were observed both in the absence and

presence of S9-mix.  Therefore it can be concluded that Octopamine hydrochloride does not

disturb mitotic processes and cell cycle progression and does not induce numerical

chromosome aberrations under the experimental conditions described in this report.  

In conclusion, this test is valid and Octopamine hydrochloride is not clastogenic in human

lymphocytes under the experimental conditions described in this report.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

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 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Identification: Octopamine hydrochloride
Appearance: White to off white powder (determined by Charles River Den Bosch)
Batch: D151-1710037
Purity/Composition: See Certificate of Analysis
Test item storage: At room temperature protected from light
Stable under storage conditions until: 26 October 2019 (retest date)
Additional information
Test Facility test item number: 209460/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Molecular weight: 189.64
Solubility in vehicle: Exposure medium: Not indicated
Stability in vehicle: Exposure medium: Not indicated

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and
ß-naphthoflavone (100 mg/kg body weight).
S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 µmol HEPES (Life technologies). The above solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-mix components 0.5 mL
S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
The concentration of the S9-fraction in the exposure medium was 4% (v/v).

Test concentrations with justification for top dose:

In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating 8 x 106 cells (106 cells/mL for 3 hour treatment) or 6 x 106 cells (1.25 x 105 cells/mL for 24 hour treatment) with a number of test item concentrations increasing by approximately half log steps.
Eight doses of the test item were tested in the mutation assay. The test item was tested in the presence of S9-mix with a 3 hour treatment period and in the absence of S9-mix with 3 and 24 hour treatment periods.
The top concentration was the recommended 0.01 M (1896 µg/mL).
The following dose-range was selected for the mutagenicity tests in the absence and presence of S9-mix: 16, 31, 63, 125, 250, 500, 1000 and 1896 μg/mL exposure medium.

Vehicle / solvent:

The vehicle for the test item was RPMI 1640 (exposure medium (R5) Hepes buffered medium (Dutch modification) (Life Technologies, Bleiswijk, The Netherlands)).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

see below

Evaluation criteria:

see below

Statistics:

In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Octopamine hydrochloride did not precipitate in the exposure medium up to and including the concentration of 1896 μg/mL (= 10 mM). Since testing up to 0.01 M is recommended in the guidelines, this concentration was used as the highest test item concentration in the dose-range finding test.
The pH and osmolarity at a concentration of 1896 μg/mL were 7.54 and 0.280 Osm/kg respectively (compared to 7.58 and 0.265 Osm/kg in the solvent control).

Dose-range Finding Test

In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 125 to 1896 µg/mL in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.

Table 1 shows the cell counts of the cultures after 3 hours of treatment with various concentrations of the test item and after 24 and 48 hours of subculture, the calculated suspension growth and the relative suspension growth. 

Both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test item concentration of 1896 μg/mL compared to the suspension growth of the solvent control.

Table 2 shows the cell counts of the cultures after 24 hours of treatment with various concentrations of the test item and after 24 hours of subculture and the calculated suspension growth and the relative suspension growth.

 The relative suspension growth was 56% at the test item concentration of 1896 μg/mL compared to the relative suspension growth of the solvent control

Mutation Experiment

Table3andTable4show the percentages of cell survival and the mutation frequencies for various concentrations of the test item. Individual colony counts of cloning and selective plates and cell counts during subculturing are listed inTable5toTable9ofAppendix 2.

Based on the results of the dose-range finding test, Octopamine hydrochloride was tested in two mutation assays. The first experiment was performed in the absence and presence of S9-mix with a 3 hour treatment period. The second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period. The following dose-range was selected for the mutagenicity tests in the absence and presence of S9-mix: 16, 31, 63, 125, 250, 500, 1000 and 1896 μg/mL exposure medium. 

Evaluation of toxicity

No significant toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix in both experiments.

Evaluation of the mutagenicity

No biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Conclusions:

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database (Table 10).
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database (see Table 11). It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
The suspension growth over the two-day expression period for cultures treated with exposure medium was between 13 and 21 (3 hour treatment) and 92 and 113 (24 hour treatment) (See Table 5, Table 6 and Table 8).
In the absence of S9-mix, Octopamine hydrochloride did not induce a biologically relevant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment.
In the presence of S9-mix, Octopamine hydrochloride did not induce a biologically relevant increase in the mutation frequency.
In conclusion, Octopamine hydrochloride is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the mutagenic potential of Octopamine hydrochloride by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions.

The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.  

The study procedures described in this report were based on the most recent OECD guideline. 

Batch D151-1710037 of the test item was white to off white powder. The vehicle of the test item was exposure medium.

In the first experiment, Octopamine hydrochloride was tested up to concentrations of
1896 µg/mL (= 0.01 M, recommended in the guidelines) in the absence and presence of
S9-mix. The incubation time was 3 hours. In the second experiment, Octopamine hydrochloride was again tested up to concentrations of 1896 µg/mL in the absence of S9-mix. The incubation time was 24 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. This is the highest concentration recommended in the guidelines.

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. 

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, Octopamine hydrochloride did not induce a biologically relevant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

In the presence of S9-mix, Octopamine hydrochloride did not induce a biologically relevant increase in the mutation frequency.

In conclusion, Octopamine hydrochloride is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based upon the existing in vitro results, which were negative the battery of three studies, no further in vivo studies are required according to the Integrated Testing Strategy. The criteria to classify the substance for Germ Cell Mutagenicity, in accordance with 1272/2008/EC are not met.