3,5-dihydroxy-2,6,6-tris(3-methylbuten-2-yl)-4-(3-methyl-1-oxobutyl)cyclohexa-2,4-dien-1-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

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

Justification for type of information:

Principal key study on "Evaluation of the Mutagenic Activity of Hop Extract in the Salmonella typhimurium Reverse Mutation Assay and the Escherichia coli Reverse Mutation Assay (Plate Incorporation and Pre-Incubation Methods)" peformed in 2017 according to GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Key study: The objective of this study was to determine the potential of Hop Extract 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 design of this study is based on the following study guidelines:
• OECD Guideline 471. Genetic Toxicology: Bacterial Reverse Mutation Test. (Adopted July 21, 1997).
• EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.13/14: "Mutagenicity: Reverse Mutation Test using Bacteria”. Official Journal of the European Union No. L142, 31 May 2008.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Key study: Hop extract. Batch no. 16/4021. Dark green/brown resin; UVCB.

Target gene:

Key study: The objective of this study was to determine the potential of Hop Extract 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).

Species / strain / cell type:

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

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Key study: 1600 ug per plate. Initial range finding showed that 5000 ug per plate caused precipitation of hop extract.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Key study: Hop Extract is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Hop extract contains beta-acids and the closely-related alpha acids. This batch contained 15.6% beta-acids and 42.7% alpha acids.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

Well-documented study from 1991, performed to GLP. This supporting study was performed on hop beta acids.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

The test article, 40% lupulones (i.e. hop beta-acids);40% concentration, was tested in the Salmonella Mutagenicity Assay using tester strains TA98, TA100, TA1535, TA1537 and TA1538 in the presence and absence of Aroclor-induced rat liver microsomal enzymes. Three phases, using the plate incorporation method: 1st: a dose range-finding study; 2nd and 3rd: mutagenicity and confirmatory assays.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine mutations hisG46, hisC3076 and hisD3052, and additional mutations: rfa, uvrB

Species / strain / cell type:

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

Metabolic activation:

with and without

Test concentrations with justification for top dose:

The top dose in range-finding study (100 ug per plate) showed that toxicity was only present in the absence of microsomal enzymes, and no precipitate was observed. In consultation with original sponsor of the work, the maximum dose was 1000 ug per plate in the presence of microsomal enzymes, and up to 333 ug per plate in the absence of microsomal enzymes.

Vehicle / solvent:

Ethanol

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

other: 2-aminoanthracene, ICR-191

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the absence of microsomal enzymes at the highest dose in the dose range-finding study

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Test report shows that all criteria for a valid study were met. The results indicated that the substance did not cause a positive response in any of the tester strains in the presence and absence of microsomal enzymes prepared from Aroclor-induced liver.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

Well-documented study from 1995, performed to GLP. This supporting study was performed on a major derivative of hop iso-alpha acids.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

The test article, RHO isoalpha acids; 89.4% concentration, was tested in the CHO/HGPRT Mutation Assay in two phases.

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Lot no. 1463-F; purity 89.4%; described as thick golden brown, very viscous liquid

Target gene:

HGPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

0.5 - 5000 ug per ml.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Test report shows that all criteria for a valid study were met. The results indicated that the substance did not cause a positive response in the non-activated and activated systems, and was concluded to be negative.
Study performed on major derivative of hop iso-alpha acids. Supporting evidence for the related hop beta acids.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Justification for type of information:

This study is taken from a recently-published scientific article by S. Suzuki et al. on "Genetic, acute and subchronic toxicity studies of matured hop extract produced by extraction from heat-treated hops", published in the Journal of Toxicological Sciences (2018) vol. 43, no. 7, pp. 473-484, available freely as full text in the public domain. Matured hop extract contains oxidised alpha and beta acids. This substance is therefore of direct relevance to hop extract, and to alpha acids, iso-alpha acids, and beta acids, as the oxidised alpha and beta acids are common degradation products of hop alpha and beta acids. This article is useful in that it reports a number of toxicological studies, and the in vivo tests on chromosome aberration are reported in this end-point to add as weight of evidence for the lack of genotoxicity expected for hop extract and its constituents.

Qualifier:

according to guideline

Guideline:

other: In accordance with a previous protocol, viz. Hayashi, M., Sofuni, T. and Ishidate, M. Jr. (1983): An application of Acridine Orange fluorescent staining to the micronucleus test. Mutat. Res., 120, 241-247.

GLP compliance:

not specified

Type of assay:

mammalian germ cell cytogenetic assay

Specific details on test material used for the study:

An aqueous extract of a heat-treated hop extract, designed for production and extraction of oxidised alpha and beta acids.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

Eight-week-old male rats. Body weight was between 289 - 310 g.

Route of administration:

oral: unspecified

Vehicle:

Sterlized water

Details on exposure:

High dose group: 2,000 mg/kg bw/day; middle dose group: 1,000 mg/kg bw/day; low dose group: 500 mg/kg bw/day. Once daily for 2 days.

Duration of treatment / exposure:

2 days

Frequency of treatment:

Once daily

Post exposure period:

24 h

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

High dose group

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Middle dose group

Dose / conc.:

500 mg/kg bw/day

Remarks:

Low dose group

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

MMC. This was dissolved in sterilized water and it was injected intraperitoneally at 2 mg/kg bw. One dose only.

Tissues and cell types examined:

Bone marrow cell smears for erythrocytes.

Details of tissue and slide preparation:

Notes from journal: "Bone marrow cell smears were prepared and stained with acridine orange. Numbers of polychromatic erythrocytes (PCEs) per 200 erythrocytes, and micronucleated polychromatic erythrocytes (MNPCEs) per 2,000 PCEs were counted."

Evaluation criteria:

Notes from journal: "Numbers of polychromatic erythrocytes (PCEs) per 200 erythrocytes, and micronucleated polychromatic erythrocytes (MNPCEs) per 2,000 PCEs were counted."

Key result

Sex:

male

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

These results are part of a larger study reported in the article. Results in brief:

• Ames test – negative, up to 5,000 ug per plate, with and without metabolic activation
• In vitro chromosomal aberration test – positive at 3,330 and 5,000 ug per plate without metabolic activation (i.e. elevated structural aberration above negative control) but no increase in polyploid cells
• In vivo micronucleus test in rat bone marrow – no clastogenic potential at max dose of 2,000 mg/kg bw/day, i.e. negative
• Acute toxicity – No noticeable toxic signs at 2,000 mg/kg bw
• 90 days study – NOAEL considered to be over 3,484 mg/kg bw/day for male and 4,022 mg/kg bw/day for female rats.

Authors’ conclusion is that although the structural chromosomal aberration was positive in high dose group for the in vitro test, Ames test and in vivo micronucleus test suggests that the matured hop extract has no potential for mutagenicity and genotoxicity under in vivo conditions.

Further consideration of results noted that cytotoxicity was observed in the in vitro test.If the toxicity mechanism was to slow cell division or similar, it could look like a chromosome aberration. The in vivo test indicates lack of genotoxicity at appropriate doses. The authors' comment was as follows: "From the results mentioned above, it is judged that MHE does not have clastogenic potential in rats at the maximum dose, which is recommended by OECD guidelines (OECD, 1997)."

Conclusions:

The studies reported in this journal article add weight of evidence to the lack of toxicity of hop extract, in particular for substances derived from hop alpha and beta acids.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)