4,4',4''-(ethan-1,1,1-triyl)triphenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In Vitro (Mutagenic effects - bacterial): QSAR. Negative. Reliability = 2.

In Vitro (Mutagenic effects - mammalian): equivalent to OECD 476; Mouse lymphoma assay. Negative. Reliability = 2.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

(Q)SAR

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Remarks:

The scientific validity of the (Q)SAR model has been established in accordance with the OECD Principles for (Q)SAR Model Validation

Justification for type of information:

QSAR prediction

Qualifier:

according to guideline

Guideline:

other: Other guideline: Data generated using Times for Ames mutagenicity v2.26.3

GLP compliance:

no

Type of assay:

other: QSAR

Species / strain / cell type:

other: QSAR prediction

Species / strain:

other: QSAR prediction

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: not applicable

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Supporting documentation is provided in the attached QMRF and QPRF.

Conclusions:

The Times model predicts that the test substance is not a mutagen.

Executive summary:

The mutagenic potential of the test substance was estimated using Times for Ames mutagenicity v2.26.3. The model predicts that the test substance is not a mutagen.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The study was conducted prior to the current OECD 476 guideline. The study design does not include a continuous exposure.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

The study design does not include a continuous exposure.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RMPI 1640, supplemented with 0.1% Synperonic F68, 0.011% sodium pyruvate, 2 mM L-glutamine, 50 µg/mL gentamicin and buffered with 2 mg/mL sodium bicarbonate, is referred to as R0p. A variation buffered with HEPES called R0 (HEPES) was also used. A third media used was R0p supplemented with 10% HiDHS and referred to as R10p. R10p from which growing L5178Y cells had been removed was used as conditioned medium. R0 (HEPES) containing 5% HiDHS, designated R5 (HEPES), was used as the treatment medium. R0p in which the amount of Synperonic F68 had been reduced to 0.02% and supplemented with 30% HiDHS, referred to as R30p, formed the basis of the cloning medium. This was semi-solidified by the addition of Noble agar to a final concentration of approximately 0.4%. Selective medium consisted of cloning medium containing 4 µg/mL TFT.

- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced Sprague-Dawley rat liver homogenate (S-9)

Test concentrations with justification for top dose:

With and without activation, Test 1: 0, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, and 25 µg/m
With and without activation, Test 2: 0, 5, 10, 15, 20, 30, 40, 50, 60, and 80 µg/m

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide
- Justification for choice of solvent/vehicle: The maximum solubility of the test substance was 393.8 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

200 µL added to each culture

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Ethyl methane sulphonate was used without activation, and 20-methylcholanthrene was used with activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 48 hours after the 3 hour treatment period

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The criteria for a positive response were:
At least a 2-fold increase in mutant frequency in treated cultures relative to the control
Demonstration of a statistically significant increase in mutant frequency following treatment with the test substance
Evidence of a dose relationship over at least 2 dose levels, in any increase in mutant frequency
Demonstration of reproducibility in any increase in mutant frequency
The observed increases in mutant frequency must lie outside the upper limit of the historical control range, 150 mutants per 10E6 survivors.

Statistics:

The statistical significance of the data was analysed by weighted analysis of variance following the methods described by Arlett et al. (1989)

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: In the range-finding study, treatment with 1-200 µg/mL in the absence and presence of S-9 mix resulted in relative growth in suspension of 70-2% and 89-2% respectively compared to the solvent controls. Concentrations used in the main test were based upon this data.

COMPARISON WITH HISTORICAL CONTROL DATA: There were no increases in mutant frequency outside the upper limit of the historical control range, 150 mutants per 10E6 survivors.

Conclusions:

The test substance did not demonstrate mutagenic potential in this in vitro gene mutation assay.

Executive summary:

The test substance was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in a subline of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-). Two independent tests in the absence of exogenous metabolic activation (S-9 mix) and two independent tests in the presence of S-9 mix were carried out. Toxicity was observed after treatment with the test substance in all the tests both in the absence and the presence of S-9 mix. No biologically significant increases in mutant frequency, according to the criteria used for a positive response, were observed after treatment with the test substance either in the absence or the presence of S-9 mix. Decreases in mutant colony size in comparison with the controls were not observed in any of the tests. It was concluded that the test substance did not demonstrate mutagenic potential in this in vitro mammalian cell mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In Vivo (Cytogenic effects - mammalian): equivalent to OECD 474; Mouse micronucleus. Negative. Reliability = 2.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The study was conducted prior to the current OECD 474 guideline. Only 1000 celss were scored for MN (a total of 5000 for each group) instead of 2000 (or a total of 10,000 for each group). However, the dose setting and data set is robust.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 35 days
- Weight at study initiation: 22-24 g
- Fasting period before study: not reported
- Housing: plastic disposable cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): not reported
- Air changes (per hr): 30
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: aqueous 1% methylcellulose
- Justification for choice of solvent/vehicle: not reported
- Concentration of test material in vehicle:
Preliminary toxicity test:
Phase I: 25, 50, 100, and 200 mg/mL
Phase II: 27, 45, 75, and 125 mg/mL
Micronucleus test: 6.25, 12.50, 25.00, and 50.00 mg/mL

Details on exposure:

PREPARATION OF TEST SUBSTANCE FORMULATION:
-Solvent used: aqueous 1% methylcellulose
-Preparation frequency: daily
-Preparation details: not reported
-Adjusted for purity: not reported

Duration of treatment / exposure:

5 days

Frequency of treatment:

daily intraperitoneal injection

Post exposure period:

24 hours

Dose / conc.:

125 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5/sex/concentration, plus 5 additional animals per sex at the 1000 mg/kg dose

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive Control: 5-fluorouracil
- Justification for choice of positive control(s): not reported
- Route of administration: intraperitoneal
- Doses / concentrations: a concentration of 1.25 mg/mL in sterile 0.9% saline was used; the dose was 4 mg/kg

Tissues and cell types examined:

PCEs and NCEs from mouse bone marrow taken from the femur

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The highest dose, 1000 mg/kg, was estimated to be the maximum tolerable dose based on the preliminary toxicity study.

DETAILS OF SLIDE PREPARATION:
A direct bone marrow smear was made onto a slide containing a drop of calf serum. One smear was made from each femur. The prepared smears were air-dried and fixed in methanol (>10 minutes). The smears were air-dried and stained for 10 minutes in 10% Giemsa. After rinsing in distilled water and differentiation in buffered distilled water (pH 6.8), the smears were air-dried and mounted with coverslips using DPX.

METHOD OF ANALYSIS:
One stained smear per animal was examined (under code) by light microscopy to determine the incidence of micronucleated cells per 1000 polychromatic erythrocytes.

Evaluation criteria:

A positive response is normally indicated by a substantial, dose-related and statistically significant increase in the incidence of micronucleated polychromatic erythrocytes compared with the incidence for the concurrent vehicle control group. In borderline cases e.g. where the response is not dose-related or where individual group mean values do not fall outside the testing facility historical control range, further testing may be necessary.

Statistics:

Non-parametric statistical methods based on rank are chosen for analysis of results. For a comparison of an individual treated group with a concurrent control group, Wilcoxon's sum of ranks test is used. An adaptation of this method (Kruskal-Wallis test) is used for multiple group comparisons. Spearman's rank correlation test is used to measure association between values obtained and dose level of the test agent. Jonckheere's is a similar test for trend but is based on a slightly different ranking method.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: Phase I: 500, 1000, 2000, and 4000 mg/kg/day
Phase II: 540, 900, 1500, and 2500 mg/kg/day
- Clinical signs of toxicity in test animals:
Deaths/number doses
1000 mg/kg: 1/2 males
4000 mg/kg: 2/2 males and 2/2 females
1500 mg/kg: 1/2 males
2500 mg/kg: 1/2 males and 1/2 females
Clinical signs included piloerection, ptosis, lethargy, hunched posture, wounds from fighting, feels cold to touch, and pallor of extremities
- Rationale for exposure: The dosages in Phase II were based on the outcome of Phase I and were used to confirm the results obtained
- Harvest times: Mice were sacrificed 24 hours after exposure

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no statistically significant increase was observed
- Ratio of PCE/NCE (for Micronucleus assay): no statistically significant decrease in the ratio was observed
- Appropriateness of dose levels and route: Deaths occurred in the 500 and 1000 mg/kg dosage groups, indicating that absorption of the test substance occurred.
- Statistical evaluation: No increase in the number of micronucleated polychromatic or normochromatic erythrocytes or decreases in the ratio of polychromatic to normochromatic erythrocytes was found at any dose level [p>0.05 using Kruskal-Wallis test, Jonckheere's test for trend and Spearman's correlation test]. 5-Fluorouracil caused highly significant increases in the micronucleated polychromatic erhthrocyte frequency [p>0.05 using Wilcoxon's sum of ranks test] and a moderate increase in the incidence of micronucleated normochromatic erythrocytes. It did not cause any signifcant decreases in the ratio of polychromatic to normochromatic erythrocytes [p>0.05 using Kruskal-Wallis test, Jonckheere's test for trend and Spearman's correlation test].

Conclusions:

The test substance does not show any evidence of mutagenic activity or bone marrow cell toxicity when administered as 5 daily intraperitoneal injections, in this in vivo test procedure.

Executive summary:

A micronucleus study was conducted in mice to determine whether the test substance induces an increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow. In this study, groups of male and female CD-1 mice were given single daily intraperitoneal injections for 5 consecutive days at doses of 250, 500, and 1000 mg/kg. Bone marrow smears were prepared approximately 24 hours after the final treatment and 1000 polychromatic erythrocytes per animal were evaluated for the presence of micronuclei. Treated mice did not show any increase in the frequency of micronucleated polychromatic erythrocytes or decrease in the PCE to NCE ratio. The positive control compound, 5 -fluorouracil, produced large, highly significant increases in the frequency of micronucleated polychromatic erythrocytes. The test substance has not shown any evidence of mutagenic activity or bone marrow cell toxicity/depression when administered as 5 daily intraperitoneal doses in this in vivo test procedure.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Qualitative structure activity relationships (QSAR), documented in QMRF/QPRF, predict that the test substance is not inherently electrophilic thus presents no alerts to indicate likely mutagenic potential; the test substance is expected to be non-mutagenic in Ames with or without metabolic activation. In addition, the test substance did not produce a positive response for mutagenicity when evaluated in vitro (mammalian cell lines) or in vivo (mouse micronucleus assay).

Justification for classification or non-classification

The test substance is predicted to be non-mutagenic in bacterial cells, and did not produce mutagenicity in mammalian cell culture or laboratory animals. The substance does not need to be classified for mutagenicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.