3,3-bis(4-hydroxy-5-isopropyl-o-tolyl)phthalide

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

literature data

Data source

Reference

Reference Type:

publication

Title:

Mutagenicity of arylmethane dyes in salmonella

Author:

Bonin et al.

Year:

1981

Bibliographic source:

Mutation Research, 89 (1981) 21-34

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3,3-bis(4-hydroxy-5-isopropyl-o-tolyl)phthalide

EC Number:

204-729-7

EC Name:

3,3-bis(4-hydroxy-5-isopropyl-o-tolyl)phthalide

Cas Number:

125-20-2

Molecular formula:

C28H30O4

IUPAC Name:

3,3-bis(4-hydroxy-5-isopropyl-o-tolyl)phthalide

Method

Target gene:

his+

Species / strain

Species / strain / cell type:

other: S. typhimurium TA98, TA100, TA1535, TA1537 and TA1538

Details on mammalian cell type (if applicable):

Standard tester strains of Salmonella typhimurium, TA98, TA100, TA1535, TA1537 and TA1538, were obtained from B.N. Ames in the form of soft agar impregnated disc cultures, and were subsequently cultured and employed in mutagenesis testing according to established techniques (Ames et al., 1975).

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

1000, 320, 100, 32 and 0 µg/plate (dose ranges for mutagenesis were determined first by preliminary pour-plate toxicity tests at 10 000,1000,100 and 10 µg/plate)

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Remarks:

pooled data

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

other: β-propiolactone

Details on test system and experimental conditions:

22 arylmethane dyes which have been used as food colours, commercial dyes, laboratory stains and pH indicators were tested in the Salmonella/mammalian microsome mutagenicity assay.

Evaluation criteria:

A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Results and discussion

Test results

Species / strain:

other: S. typhimurium TA98, TA100, TA1535, TA1537 and TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Although not being conducted to recent guidelines, the test was conducted scientifically reasonable with negligible deficiencies. Also, the testing was sufficiently documented, positive and negative controls gave the appropriate response. Hence, the results can be considered as sufficiently reliable to assess the mutagenic potential of the test substance in bacteria.
Interpretation of results: negative with and without metabolic activation

Executive summary:

22 arylmethane dyes which have been used as food colours, commercial dyes, laboratory stains and pH indicators were tested in the Salmonella/mammalian microsome mutagenicity assay (Bonin, 1981). 8 mutagenic dyes were identified, including 5 food colours and 3 common laboratory stains; none of the 11 indicator dyes (incl. thymolphthalein) tested was mutagenic.

The commercial and laboratory dyes Methyl Violet 2B C.I. 42535 and Crystal Violet C.I. 42555 were mutagenic in base-pair substitution mutation detector strain TA1535 in the absence of metabolic activation. By contrast, the food colours Benzyl Violet 4B C.I. 42640, Guinea Green B C.I. 42085, Light Green SF CI. 42095, Lissamine Green B CI. 44090 and Violet BNP CI. 42581 and the bacteriological stain, Basic Fuchsin CI. 42500-42510, were all mutagenic in frameshift mutation detector strains TA98 and/or TA1538 and required metabolic activation. Most of these compounds gave weak mutagenic responses with Salmonella and were positive only within narrow dose ranges. Since conflicting results were obtained using dyes from different sources, minor dye components may have been responsible for their mutagenicity. This suggests the need to improve knowledge about impurities in arylmethane colours still used in food and to review the toxicological role of such impurities.

The toxicology of arylmethane dyes has been reviewed previously (Khera and Munro, 1979; Radomski, 1974) and genetic toxicity testing has been reported (Au et ah, 1979; Auletta et ah, 1977; Brown et ah, 1978; Kada et al., 1972; Price et ah, 1978; Rozenkranz and Carr, 1971; Sankaranarayanan and Murthy, 1979). However, in most cases the dyes studied were those in current use in foods, particularly in the United States. There has been little reference to those food dyes used elsewhere in the world or to arylmethane dyes other than food colours.

For food colours, chronic toxicity studies based on animal feeding experiments are considered the best indicators of safety (Radomski, 1974), yet several food dyes appear not to have been tested by chronic feeding studies in animals (Violet BNP, Lissamine Green B and Patent Blue V) and feeding experiments conducted with other food dyes (Light Green SF and Fast Green FCF) have been considered inadequate for their evaluation (Arrhenius et al., 1978). Because of the controversy surrounding subcutaneous tumour induction by these dyes (Hooson et al., 1973) and because several (Benzyl Violet 4B, Guinea Green B) have clearly given rise to an increased incidence of animal cancers in chronic feeding experiments (Arrhenius et al., 1978), we were encouraged to undertake mutagenic testing of a wide range of arylmethane compounds.