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Toxicological information

Genetic toxicity: in vitro

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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 guidelineopen allclose all
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
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

Constituent 1
Reference substance name:
Reaction mass of benzyl 1-(isobutyryloxy)-2,2,4-trimethylpentan-3-yl phthalate and benzyl 3-(isobutyryloxy)-2,2,4-trimethylpentyl phthalate
EC Number:
701-008-3
Molecular formula:
C27H34O6
IUPAC Name:
Reaction mass of benzyl 1-(isobutyryloxy)-2,2,4-trimethylpentan-3-yl phthalate and benzyl 3-(isobutyryloxy)-2,2,4-trimethylpentyl phthalate
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.