Capsicum annuum, ext.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro bacteria reverse mutation: Weigth of evidence: Two bacterial assays have been reported with contradictory results. The test substance chili pepper oleoresin was found as no mutagenic in a Salmonella / mammalian microsome mutagenicity assay (Buchanan1981) and the test substance red pepper oleoresin showed dose-response effects for the mutagenicity in SD1018 and SD7823 strains (Damhoeri1985).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

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 with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : livers of rats induced with phenobarbital.
- concentration or volume of S9 mix in the final culture medium: 0.5 mL/plate

Test concentrations with justification for top dose:

2.5, 25, 250, 2500 and 25000 µg/plate.

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: N-methyl-N-nitro-N-nitrosoguanidine and picrolinic acid

Remarks:

Without metabolic activation.

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Remarks:

With metabolic activation.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 4
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Not specified.
- Test substance added in agar (plate incorporation) at a level of 0.5 mL/plate.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 hr at 37ºC.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition.

Key result

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:

not applicable

Positive controls validity:

valid

Key result

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:

not applicable

Positive controls validity:

valid

Key result

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:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The test substance chili pepper oleoresin was found as no mutagenic in a Salmonella / mammalian microsome mutagenicity assay.

Executive summary:

The test substance chili pepper oleoresin was tested for in vitro mutagenicity using the Salmonella / mammalian microsome mutagenicity assay similar to OECD Guideline 471. The experiments were performed with at least four replications, using Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1538 in the presence and absence of metabolic activation (S9 fraction prepared from the livers of rats). The test item was formulated in 0.1 mL DMSO as vehicle and concentrations of 2.5, 25, 250, 2500 and 25000 µg/plate were used in the assay. Vehicle control and appropriate positive controls (N-methyl-N-nitro-N-nitrosoguanidine and picrolinic acid without metabolic activation and 2-acetylamino-flourene with metabolic activation) were tested simultaneously. The test substance did not produce an increased reversion rate, while negative and positive controls were within expected ranges. These data indicates that the test substance is not mutagenic.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

- Principle of test: Two streptomycin-dependent (SM (1)) strains, SD1018 and SD7823, were isolated from S. typhimurium TA100 and TA98, respectively, according to the method of Kada et al. (2), and used in spot tests ans plate incorporation tests.
- Short description of test conditions: For plate incorporation tests, two methods were employed, the SA method in which a 1-ml sample solution is mixed with 9ml of molten B2 broth agar (10g Kyokuto beef extract, 10g Daigo-Eiyo polypeptone, 5g NaCl and 15g Difco agar per liter, pH 7.0), and the PI method in which a 0.1 mL sample solution is incorporated into 2 mL of top agar (3). No metabolic activation of the samples was performed throughout this study.

(1) On leave from: Faculty of Agriculture, Tohoku University, Sendai, Japan.
(2) T. Kada, K. Aoki and T. Sugimura, Environ. Mutagenesis, 5, 9 (1983).
(3) D. M. Maron and B. N. Ames, Mutat. Res., 113, 173 (1983).

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: SD1018

Additional strain / cell type characteristics:

other: Streptomycin-dependent strain isolated from S. typhimurium TA100.

Species / strain / cell type:

S. typhimurium, other: SD7823

Additional strain / cell type characteristics:

other: Streptomycin-dependent strain isolated from S. typhimurium TA98.

Metabolic activation:

without

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide

Details on test system and experimental conditions:

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Not specified.
- Test substance added in agar (plate incorporation)

Key result

Species / strain:

S. typhimurium, other: SD1018

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: SD7823

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

With the SA method, RPG values were in the order of around 1E2 for capsicum peppers. Higher RPG values for red pepper (1E3) were obtained by the PI method as compared with the SA method.

The mutagenic effects of red pepper were unaffected by the different sterilization methods used for samples, autoclaving at 121ºC and nitration through a membrane filter, suggesting that the mutagenic factors in this spice are thermostable. On extraction of red pepper oleoresin with hexane, its major mutagenic and bactericidal factors were found to remain in the residues. Capsaicin, a major pungent principle of capsicum pepper, was negative for SD1018 and SD7823 in spot tests. This may suggest that capsaicin is not involved in capsicum pepper mutagenesis.

Conclusions:

The test substance red pepper oleoresin showed obvious dose-response effects for the mutagenicity in SD1018 and SD7823 strains.

Executive summary:

The test substance red pepper oleoresin was tested for in vitro mutagenicity using 2 streptomycin-dependent strains, SD1018 and SD7823, isolated from S. typhimurium TA100 and TA98, respectively, in spot tests and plate incorporation tests. For plate incorporation tests, two methods were employed, the SA method in which a 1-ml sample solution is mixed with 9ml of molten B2 broth agar (10g Kyokuto beef extract, 10g Daigo-Eiyo polypeptone, 5g NaCl and 15g Difco agar per liter, pH 7.0), and the PI method in which a 0.1 mL sample solution is incorporated into 2 mL of top agar (3). No metabolic activation of the samples was performed throughout this study. The test substance showed obvious dose-response effects for the mutagenicity in SD1018 and SD7823 strains.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

According to JECFA2009 (1) extracts of chilli peppers of different levels of purity have been tested, with mixed, inconsistent and often contradictory results.

The EFSA Panel (2) concluded that the limited information available from the open literature does not allow a reliable assessment of the genotoxicity of paprika extracts. Therefore, upon request of EFSA, two new GLP compliant genotoxicity studies using Paprika extract were performed: an OECD TG 471 mutation assay and a OECD TG 487in vitromicronucleus assay. Respective authors concluded that tested paprika extract did not induce mutation in the OECD test TG 471 mutation assay and did not induce micronuclei in cultured human peripheral blood lymphocytes. The Panel agreed with these conclusions.

Based on these results, the EFSA Panel concluded that paprika extracts used as a food colour do not raise a genotoxic concern.

References:

1. JECFA, 2009. Safety evaluation of certain food additives and contaminants. WHO Food Additives Series No. 59, 2008. Available online: http://apps.who.int/iris/bitstream/10665/43823/1/9789241660594_eng.pdf

2. EFSA ANS Panel (EFSA Panel on Food Additives and Nutrient Sources added to Food), 2015. Scientific Opinion on the re-evaluation of paprika extract (E 160c) as a food additive. EFSA Journal 2015;13(12):4320, 52 pp. doi:10.2903/j.efsa.2015.4320.

Justification for classification or non-classification

Based on the available information, the substance is considered to be negative for genetic toxicity, and therefore the substance is not classified in accordance with CLP Regulation (EC) no 1272/2008.