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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

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

Test material

Test material information

1

Reference substance name:

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

EC Number:

212-216-4

EC Name:

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

Cas Number:

770-05-8

Molecular formula:

C8H11NO2.ClH

IUPAC Name:

4-(2-amino-1-hydroxyethyl)phenol hydrochloride

Test material form:

solid: particulate/powder

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)

Method

Target gene:

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

Species / strain

Species / strain 1

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 2

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

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.