Santicizer 278®

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:

10 November 1981 to 25 January 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to OECD guidelines, with deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Study did not utilise strains to detect cross-linking agents

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

yes

Remarks:

as above

Principles of method if other than guideline:

Method: other: Ames et al. (1975). Mutat. Res. 31:347-364.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Mass of Benzyl (1R,1S) 2,2,4-trimethyl-1-[(2-methylpropanoyl)oxy]pentan-3-yl benzene-1,2-dicarboxylate and Benzyl (3R,3S) 2,2,4-trimethyl-3-[(2-methylpropanoyl)oxy]pentyl benzene-1,2-dicarboxylate

EC Number:

701-008-3

Molecular formula:

C27H34O6

IUPAC Name:

Reaction Mass of Benzyl (1R,1S) 2,2,4-trimethyl-1-[(2-methylpropanoyl)oxy]pentan-3-yl benzene-1,2-dicarboxylate and Benzyl (3R,3S) 2,2,4-trimethyl-3-[(2-methylpropanoyl)oxy]pentyl benzene-1,2-dicarboxylate

Details on test material:

IUCLID4 Test substance: as prescribed by 1.1 - 1.4

- Name of test material (as cited in study report): "texanol" benzyl phthalate
- Substance type: liquid
- Physical state: light yellow liquid
- Analytical purity: 98%
- Lot/batch No.: T810061
- Stability under test conditions: Stable to heat, light, and water
- Storage condition of test material: "Stored in the dark at ambient temperatures"

Method

Species / strain

Species / strain / cell type:

other: S.typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

0.01, 0.04, 0.2, 1.0, 3.0, and 10 ul/plate (i.e. mg/plate)

Vehicle / solvent:

- Vehicle(s)/solvent(s): dimethyl sulfoxide (DMSO)

Controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-N-oxide, 2-acetylaminofluorene, benzo(a)pyrene, sodium nitrite, 2-aminoanthracene, 9-aminoacridine,

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: at least 48 hours

SELECTION AGENT (mutation assays):

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: other: Plates were examined visually for toxicity after at least 48 hours [no further details given on determination of cytotoxicity].

Evaluation criteria:

A positive response is indicated if three or more treatments on the initial test (and retest, if performed) are significantly greater than control (p<0.01) and if there is a significant positive dose-response (p<0.01) for the initial test (or retest, if performed).

Statistics:

Values of revertants/plate were transformed using log (base 10) for further analysis. Bartlett's test was performed to determine if a significant difference existed among treatment variables. Treatments were compared with controls using a one-sided t-test and within-levels pooled variance. Dose response was evaluated for all treatments which were not significantly lower (p<0.01) than controls, and used regression analysis for a log-log straight line. A t-test was used to evaluate significance of dose response.

Results and discussion

Test results

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: levels of 3 mg/plate and higher exceeded the solubility limits of the test material

RANGE-FINDING/SCREENING STUDIES: In the toxicity screen, the test material was not toxic at up to 10 mg/plate in strain TA 100 (with and without S-9)

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

Santicizer(R) 278 showed no evidence of mutagenic activity in S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or TA 1538 after exposure for at least 2 days at up to 10 mg/plate in the presence and absence of a rat liver metabolic activation system (S-9). Levels of 10 mg/plate were not toxic to any of the five strains in the presence or absence of S-9, but levels of 3 mg/plate and higher exceeded the solubility of the test material.

Executive summary:

Santicizer(R) 278 was examined for mutagenic activity in five strains of S. typhimurium using the plate incorporation test. The bacterial strains employed are capable of detecting both induced frameshift (TA 1537, TA 1538 and TA 98) and base-pair substitution (TA 1535 and TA 100) mutations. Tests were conducted in triplicate with the test material in DMSO at levels from 0.01 to 10 mg/plate, both with and without the addition of S-9. Revertant colonies per plate and toxicity were measured after at least 48 hours, and compared to the controls. Due to possible significant increases in revertants/plate over controls, two retests (again in triplicate) were conducted with the test material at 0.5, 1 and 1.5 mg/plate in S.typhimurium TA 100 (with S-9) and in TA 1537 (without S-9).

None of the test strain results had three treatments with revertants/plate significantly greater than controls and a significant dose response. No toxicity was seen in any of the five strains at up to 10 mg/plate in the presence or absence of S-9, although levels of 3 mg/plate and higher exceeded the solubility of the test material. The study authors concluded that the “plate incorporation test results indicated no significant mutagenic activity for this test material".

 

This study does not entirely conform with current OECD guidelines which recommend using S. typhimurium strain TA 102, or Escherichia coli WP2 uvrA to detect cross-linking agents. In additon, the study report does not describe how "toxicity" was determined, although it was probably based on a thinning of the background lawn of non-revertant cells.