2,2'-dihydroxy-4,4'-dimethoxybenzophenone

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1980-06-05 - 1980-06-20

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Well-documented study according to OECD 471 with minor deviations: only four strains of S. typhimurium (TA1535, TA1537, TA98, TA100) were used as stipulated by the current version of the guideline, strain TA1538 does not serve as surrogate for the fifth strain, data on E.coli WP2 strains or S. typhimurium TA102 are lacking. However, since these strains were mainly included in the recent version of OECD 471 because the four formerly only recommended S. typhimurium strains TA1535, TA1537, TA98 and TA100 may not detect certain oxidising mutagens, cross-linking agents and hydrazines, and this mode of action is not likely to occur based on the chemical structure of the test item, this restriction is considered to be negligible. Further, according to the guideline, Equivocal results should be clarified by further testing preferably using a modification of experimental conditions. Negative results need to be confirmed on a case-by-case basis. Confirmation was not done here.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

before 1997

Deviations:

no

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-dihydroxy-4,4'-dimethoxybenzophenone

EC Number:

205-027-3

EC Name:

2,2'-dihydroxy-4,4'-dimethoxybenzophenone

Cas Number:

131-54-4

Molecular formula:

C15H14O5

IUPAC Name:

2-(2-hydroxy-4-methoxybenzoyl)-5-methoxyphenol

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Storage condition of test material: at room temperature protected from direct light.

Method

Target gene:

his

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

1000 µg, 500 µg, 100 µg, 10 µg, 1 µg/plate

Vehicle / solvent:

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

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 5 µg N-methyl-N-nitro-N-nitrosoguanidine (MNNG) in DMSO for TA1535

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 5.0 µg/plate N-methyl-N-nitro-N-nitrosoguanidine (MNNG) in DMSO for TA 100

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
NUMBER OF REPLICATIONS: 3

Bacterial Preparation:
Upon receipt of the indicator strains (obtained from Bruce N. Ames), nutrient broth (containing 0.5 % NaCl) cultures were prepared. All strains were checked for the appropriate mutation markers. The cultures were then frozen, 0.8 ml nutrient broth culture with 0.07 ml DMSO, and stored at -80 °C. Each week, new cultures were started from the frozen permanents m 1Dm1 of nutrient broth with 0.5 % NaCl and incubated on a shaker overnight at 37 °C. These cultures were then stored in a refrigerator and checks for the appropriate gentic markers were conducted before each culture was released for use in testing. Such cultures were only used for one week.

Rationale for test conditions:

All plate assays for bacterial mutagenesis were conducted according to the methodology described by B. N. Ames, et al.

Evaluation criteria:

The following criteria were used in the evaluation and reporting of the mutagenic potential of the test material:
a. the spontaneous revertant levels for each strain when used in either the direct plate assay or the activated plate assay must be within the acceptable limits as defined by the historical data shown in Table 1 (below),
b. all sterility controls must be negative,
c. all positive controls must demonstrate that the indicator strains are functional with known mutagens as evidenced by an increase of at least three times the number of revertant colonies per plate as the spontaneous revertant controls,
d. to be considered positive for mutagenic activity, the test material should exhibit a dose reponse effect (increasing numbers of revertant colonies with increased amounts of the test sample),
e. for strains TA 1535, TA 1537, TA 1538, and TA 98, the test sample should produce a positive dose response over the concentrations with the lowest increase in revertants/plate greater than or equal to 3x the solvent control value or the S-9 fraction control value, as applicable, to be considered mutagenic , and
f. to be considered mutagenic for strain TA 100, the test sample should produce a positive dose response over three concentrations with at least one dose producing an increase in revertants/plate greater than or equal to 3.5 x the solvent control value or the S-9 fraction control value, as applicable.
All of the criteria noted above must be met before the results of testing conducted with any test sample can be considered valid.

Statistics:

Least squares linear regression analysis was used to compute the "best fit" regression line of dose reponse on dose level for the test sample. These regression lines represent the strength of dose response obtained against each bacterial indicator strain.
y = mx + b,
the sample regression coefficient, m, was tested for a statistically significant deviation from the value zero (0) which would indicate mutagenic, activity. The null hypothesis and the alternative hypothesis are stated below:
H0: m = 0, the data do not suggest mutagenic activity
HA: m > 0, the data suggest increasing mutagenic activity dose
Significance level = 0.05

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

other: toxicity occurres at test doses 500µg and 1000 µg

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: At test dose 500µg and 1000 µg with metabolic activation toxicity occurres in S. typhimurium strains TA1535, TA1538 and TA98. With strain TA1538 toxicity occurres at 100µg with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Under the conditions of this investigation, Benzophenone-6 was found to be mutagenic to Salmonella strain TA 1537 at concentrations of 100 ug and 10 ug with the metabolic activation system (S-9). Some toxicity resulted at concentrations of 1000 ug and 500 ug with the above sample against strain TA 1537 and the addition of S-9, so no absolutlely clear determination could be made as to the mutagenicity or nonmutagenicity, but non-mutagenicity ist considered most likely.

Executive summary:

Evaluation of the mutagenic activity of Benzophenone-6 was performed in this study equivalent to OECD TG 471 in the Salmonella typhimurium reverse mutation assay (plate incorporation methods). The test substance was tested with five histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA100 and TA98). The study procedures described in this report were based (with some minor defciencies) on the most recent OECD and EC guidelines. The test item was tested up to concentrations of 1000 µg/plate in the strains TA1535, TA1537, TA1538, TA100 and TA98.

The vehicle of the test item was dimethyl sulfoxide. 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.

Under the conditions of this investigation, Benzophenone-6 did not induce a significant dose-related increase in the number of revertant (His+) colonies in the tester strains TA1535, TA1538, TA98 and TA100 both in the absence and presence of S9-metabolic activation. The test substance was found to be mutagenic to Salmonella strain TA 1537 at concentrations of 100 µg and 10 µg with the metabolic activation system (S-9). Some toxicity resulted at concentrations of 1000 ug and 500 ug with the above sample against strain TA 1537 and the addition of S-9, so no determination could be made as to the mutagenicity or nonmutagenicity.