L-Histidine Hydrochloride Monohydrate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-10-11 to 2018-02-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

L-Histidine Hydrochloride Monohydrate

EC Number:

611-821-4

Cas Number:

5934-29-2

Molecular formula:

C6-H9-N3-O2.Cl-H.H2-O

IUPAC Name:

L-Histidine Hydrochloride Monohydrate

Test material form:

solid: crystalline

Details on test material:

- Name: L-Histidine Hydrochloride Monohydrate (HHM)
- Appearance: White crystals or crystalline powder
- Batch: P817T011
- CAS no.: 5934-29-2
- Purity/Composition: 100.1%
- Molecular weight: 209.63 g/mol
- Test item storage: room temperature
- Expiry date: 31 October 2020

Method

Target gene:

- Salmonella typhimurium: histidine
- Escherichia coli: tryptophan

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:
Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames (TA1535: 2016, TA1537: 2015, TA98: 2017, TA100: 2016; and Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK (WP2uvrA: 2008)]

MEDIA USED:
AGAR PLATES:
Agar plates (ø 9 cm) contained 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.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and the WP2uvrA, both with and without 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 assay was 5000 µg/plate. At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain in the absence and presence of S9-mix. The first experiment was a direct plate assay and the second experiment was a pre-incubation assay. The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix. To verify the responses observed in the dose-range finding test, an additional dose range finding test was performed with strain TA100 in the absence and presence of S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested again in triplicate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Milli-Q water

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA1535 (5 µg/plate), -S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

TA1537 (2.5 µg/plate), -S9, only direct plate assay

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA1537 (15 µg/plate), -S9, only pre-incubation assay

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 (10 µg/plate), -S9

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

TA100 (650 µg/plate), -S9

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

WP2uvrA (10 µg/plate), -S9

Controls 7

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

All strains, +S9; TA1535 and TA1537 (2.5 µg/plate), TA98 (1 µg/plate); TA100 (1 µg/plate direct plate assay, 5 µg/plate pre-incubation assay); WP2uvrA (15 µg/plate)

Details on test system and experimental conditions:

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 the WP2uvrA, both with and without 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 L-Histidine Hydrochloride Monohydrate (HHM) used in the subsequent mutation assay was 5000 µg/plate or the level at which the test item inhibited bacterial growth. At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain in the absence and presence of S9-mix. The first experiment was a direct plate assay and the second experiment was a pre-incubation assay. The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix. To verify the responses observed in the dose-range finding test, an additional dose range finding test was performed with strain TA100 in the absence and presence of S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested again in triplicate.

First Experiment: Direct Plate Assay:
The above mentioned dose-range finding study with two tester strains is reported as a part of the direct plate assay. In the second part of this experiment, the test item was tested both in the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98. 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.

Second Experiment: Pre-Incubation Assay
The test item was tested both in the absence and presence of S9-mix in all tester strains. Top agar in top agar tubes was melted by heating to 45 ± 2 °C. The following solutions were pre-incubated for 30 ± 2 minutes by 70 rpm at 37 ± 1 °C, 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), 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. After the pre-incubation period the solutions were added to 3 mL molten top agar. 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.

Evaluation criteria:

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 tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent 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 tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat 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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: TA1535, TA1537, TA98, TA100 and WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

only TA100

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

except for tester strain WP2uvrA and TA1535 (absence of S9-mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

other: TA1535, TA1537, TA98, TA100 and WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

only TA100

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

only TA1535, TA98 and TA100

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation in Experiment I: Precipitation of the test item on the plates was not observed at the start of the incubation period. Precipitation of the test item on the plates was observed at the end of the incubation period at concentrations of 164 µg/plate and upwards in tester strains TA98, TA100, TA1535 and TA1537. No precipitate was observed in tester strain WP2uvrA.
- Precipitation in Experiment II: Precipitation of the test item on the plates was not observed at the start of the incubation period. At the end of the incubation period, the test item precipitated on the plates at dose levels of 164 μg/plate and upwards in the tester strains TA1535, TA1537, TA98 and TA100. No precipitate was observed in tester strain WP2uvrA.

RANGE-FINDING/SCREENING STUDIES:
L-Histidine Hydrochloride Monohydrate (HHM) was initially tested in the tester strains TA100 and WP2uvrA as a dose-range finding test with 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 mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 5.4, 17, 52, 164 and 512 μg/plate. To obtain more information about the possible mutagenicity of the test item, a pre-incubation experiment was performed in the absence and presence of S9-mix. Based on the results of the first mutation assay, L-Histidine Hydrochloride Monohydrate (HHM) was tested up to the dose level of 512 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and up to the dose level of 5000 µg/plate in tester strain WP2uvrA.

TOXICITY:
Experiment I: Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in all tester strains in the absence and presence of S9-mix except for tester strain WP2uvrA and TA1535 (absence of S9-mix) where no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
Experiment II: Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strains TA1535, TA98 and TA100. In tester strains TA1537 and WP2uvrA, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

MUTAGENICITY:
Experiment 1: In tester strain TA100, L-Histidine Hydrochloride Monohydrate (HHM) induced up to 2.0- and 1.9-fold, dose related increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively.
No increase in the number of revertants was observed upon treatment with the test item under all conditions tested in all other tester strains. In the second experiment only TA10o was tested to obtain more information about the possible mutagenicity of the test item. In this test, L-Histidine Hydrochloride Monohydrate (HHM) induced up to 1.9- and 2.1-fold, dose related increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively.

Experiment II: In the absence of S9-mix, L-Histidine Hydrochloride Monohydrate (HHM) induced an up to 2.1-fold, dose related increase in the number of revertant colonies compared to the solvent control in tester strain TA100. In the presence of S9-mix, L-Histidine Hydrochloride Monohydrate (HHM) induced an up to 2.8-fold, dose related increase in the number of revertant colonies compared to the solvent control in tester strain TA100. In the absence of S9-mix, L-Histidine Hydrochloride Monohydrate (HHM) induced an up to 3.1-fold, dose related increase in the number of revertant colonies compared to the solvent control in tester strain TA1535. However, since the number of revertants is within the historical control data range, this increase is considered to be not biologically relevant.

Remarks on result:

other: Experiment 1: Direct plate

Any other information on results incl. tables

Table 1: Experiment 1: Mutagenic Response of L-Histidine Hydrochloride Monohydrate (HHM) in the Salmonella typhimurium Reverse Mutation Assay (Direct Plate)

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium.
	TA1535	TA1537	TA98	TA100
Without S9-mix
Positive control	1173 ± 56	994 ± 30	1196 ± 34	947 +/- 122
Solvent control	8 ± 4	6 ± 2	15 ± 4	99 +/- 10
5.4	14 ± 7	6 ± 2	18 ± 2	118 +/-7
17	18 ± 2	9 ± 2	21 ± 2	128 +/- 5
52	19 ± 4^NP	0 ± 1^NP	20 ± 11^NP	190 +/- 21
164	3 ± 6^MP	0 ± 0^MP	0 ± 0^MP	48 +/- 32
512	10 ± 5^n MP	3 ± 1^n MP	4 ± 2^n MP	0 +/- 0
With S9-mix
Positive control	335 ± 34	442 ± 87	1170 ± 114	959 +/- 261
Solvent control	12 ± 4	6 ± 3	22 ± 6	84 +/- 5
5.4	17 ± 8	7 ± 1	26 ± 5	106 +/- 17
17	16 ± 6	8 ± 2	29 ± 2	116 +/- 9
52	24 ± 8^NP	9 ± 4^NP	32 ± 8^NP	179 +/- 18
164	0 ± 0^MP	3 ±^MP	0 ± 0^MP	59 +/- 23
512	5 ± 2^nMP	2 ± 2^nMP	4 ± 2^nMP	2 +/- 2

Table 2: Experiment 2: Mutagenic Response of L-Histidine Hydrochloride Monohydrate (HHM) 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 different strains ofSalmonella typhimuriumand oneEscherichia colistrain.
	TA1535	TA1537	TA98	TA100	WP2uvrA
Without S9-mix
Positive control	1154 ± 60	217 ± 38	1368 ± 58	870 ± 93	257 ± 19
Solvent control	8 ± 2	5 ± 5	12 ± 3	106 ± 24	26 ± 8
1.7	10 ± 3	6 ± 4	16 ± 4	121 ± 23
5.4	10 ± 5	8 ± 6	20 ± 9	127 ± 21
17	18 ± 5	7 ± 2	22 ± 6	153 ± 30	25 ± 5
52	25 ± 8^NP	4 ± 1^NP	16 ± 10^NP	224 ± 19^NP	28 ± 9
164	0 ± 1^MP	5 ± 6^MP	0 ± 1^MP	88 ± 32^MP	24 ± 2
512	3 ± 2^n MP	3 ± 1^n MP	3 ± 2^n MP	5 ± 7^n MP	19 ± 4
1600					22 ± 11
5000					25 ± 8^n NP
With S9-mix
Positive control	245 ± 28	164 ± 44	739 ± 70	1708 ± 76	431 ± 20
Solvent control	13 ± 1	5 ± 2	17 ± 4	83 ± 5	24 ± 9
1.7	13 ± 4	4 ± 1	20 ± 2	102 ± 11
5.4	11 ± 4	6 ± 2	22 ± 3	114 ± 17
17	20 ± 6	7 ± 5	28 ± 3	137 ± 6	26 ± 4
52	25 ± 12^NP	6 ± 5^NP	32 ± 7^NP	236 ± 4^NP	40 ± 4
164	1 ± 1^MP	3 ± 2^MP	0 ± 0^MP	229 ± 84^MP	26 ± 6
512	2 ± 1^nMP	4 ± 2^nMP	4 ± 2^nMP	8 ± 3^nMP	27 ± 8
1600					34 ± 7
5000					37 ± 9^nNP

MP= moderate precipitate

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 L-Histidine Hydrochloride Monohydrate (HHM) is mutagenic in the Salmonella typhimurium assay.

Executive summary:

In a bacterial reverse gene mutation assay conducted according to OECD guideline 471, strains TA98, TA100, TA1535 and TA1537 of Salmonella typhimurium and E. coli strain WP2uvrA were exposed to L-Histidine Hydrochloride Monohydrate (HHM) (100.1 % purity) in water at concentrations of up to 5000 µg/plate in the presence and absence of mammalian metabolic activation. 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 tester strain TA100, L-Histidine Hydrochloride Monohydrate (HHM) induced up to 2.1- and 2.8-fold, dose related increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively. Since the increases in the number of revertant colonies were observed in three independent experiments, dose-related and outside the historical control data range, these increases are considered to be biologically relevant and L-Histidine Hydrochloride Monohydrate (HHM) is considered to be mutagenic.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.51001; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.