Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4',4''-(ethan-1,1,1-triyl)triphenol
EC Number:
405-800-7
EC Name:
4,4',4''-(ethan-1,1,1-triyl)triphenol
Cas Number:
27955-94-8
Molecular formula:
C20H18O3
IUPAC Name:
4-[1,1-bis(4-hydroxyphenyl)ethyl]phenol
Details on test material:
- Purity: 99%

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Test results
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].

Applicant's summary and conclusion

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.