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

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study in accordance with relevant guideline; selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Data source

Reference

Reference Type:

other: reference to body responsible for the test

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Remarks:

Conducted according to guideline in effect at time of study conduct. Additional histopathology was completed on stored tissues in 2005 to bring the study closer to the 1995 edition of OECD Guideline 407.

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

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:

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

Details on test material:

- Purity: 99%

Test animals

Species:

rat

Strain:

other: Crl:CD® (SD) BR VAF Plus

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 28 ± 1 days
- Weight at study initiation: 60-87 g
- Fasting period before study: Not reported.
- Housing: Groups of 5 according to sex; cages distributed in batteries to minimize environmental influences from spatial distribution.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8-21.1, mean minimum and maximum temperatures.
- Humidity (%): 55.0-59.9%, mean minimum and maximum humidities.
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1 % aqueous methylcellulose

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
-Preparation frequency: Daily.
-Preparation details: The test substance was homogenized with the vehicle to obtain a suspension.
-Adjusted for purity: No.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not reported.
- Concentration in vehicle: Test substance concentration was 10.0, 1.0 and 0.1% w/v.
- Amount of vehicle (if gavage): Total dosage volume was 10 mL/kg/day.
- Lot/batch no. (if required): Not reported.
- Purity: Not reported.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Method of Analysis: HPLC

Concentration verification (% of Target)
Conducted on all dose levels: yes
Results:
Table 1: Mean concentration of test substance in dose formulations
Test Concentration Time Frame Result
1 mg/L Day 1, Replicate 1 0.97533 mg/mL
1 mg/L Day 1, Replicate 2 1.0398 mg/mL
10 mg/L Day 1, Replicate 1 10.919 mg/mL
10 mg/L Day 1, Replicate 2 10.167 mgm/L
100 mg/L Day 1, Replicate 1 108.17 mgmL
100 mg/L Day 1, Replicate 2 103.8 mg/mL
Control Day 1, Replicate 1 ND
Control Day 1, Replicate 2 ND

1 mg/L Day 22, Replicate 1 0.97465 mg/mL
1 mg/L Day 22, Replicate 2 1.0134 mg/mL
10 mg/L Day 22, Replicate 1 10.809 mg/mL
10 mg/L Day 22, Replicate 2 10.194 mg/mL
100 mg/L Day 22, Replicate 1 115.55 mg/mL
100 mg/L Day 22, Replicate 2 109.73 mg/mL
Control Day 22, Replicate 1 ND
Control Day 22, Replicate 2 ND

ND = Not Detected (limit of detection = 1.479x10e-4 mg/mL)
Homogeneity is reported in Table 2 below.
Conducted on dose levels: 1 and 100 mg/ml concentrations
Results:
Table 2: Physical stability of test substance in 1% methylcellulose formulations
Test Concentration Time Frame Result
1 mg/mL Top, Storage 0 hours 0.93169 mg/mL
1 mg/mL Middle, Storage 0 hours 1.0201 mg/mL
1 mg/mL Bottom, Storage 0 hours 0.95525 mg/mL
1 mg/mL Mean, Storage 0 hours 0.96901 mg/mL
1 mg/mL Top, Storage 0.5 hours 0.95106 mg/mL
1 mg/mL Middle, Storage 0.5 hours 0.94207 mg/mL
1 mg/mL Bottom, Storage 0.5 hours 0.93246 mg/mL
1 mg/mL Mean, Storage 0.5 hours 0.94186 mg/mL
1 mg/mL Top, Storage 1 hours 0.97522 mg/mL
1 mg/mL Middle, Storage 1 hours 0.98487 mg/mL
1 mg/mL Bottom, Storage 1 hours 0.84032 mg/mL
1 mg/mL Mean, Storage 1 hours 0.93347 mg/mL
1 mg/mL Top, Storage 4 hours 0.89724 mg/mL
1 mg/mL Middle, Storage 4 hours 0.88917 mg/mL
1 mg/mL Bottom, Storage 4 hours 0.93672 mg/mL
1 mg/mL Mean, Storage 4 hours 0.90771 mg/mL

100 mg/mL Top, Storage 0 hours 106.26 mg/mL
100 mg/mL Middle, Storage 0 hours 104.73 mg/mL
100 mg/mL Bottom, Storage 0 hours 104.49 mg/mL
100 mg/mL Mean, Storage 0 hours 105.16 mg/mL
100 mg/mL Top, Storage 0.5 hours 108.44 mg/mL
100 mg/mL Middle, Storage 0.5 hours 99.325 mg/mL
100 mg/mL Bottom, Storage 0.5 hours 103-57 mg/mL
100 mg/mL Mean, Storage 0.5 hours 103-78 mg/mL
100 mg/mL Top, Storage 1 hours 106.86 mg/mL
100 mg/mL Middle, Storage 1 hours 104.44mg/mL
100 mg/mL Bottom, Storage 1 hours 104.81 mg/mL
100 mg/mL Mean, Storage 1 hours 105.37 mg/mL
100 mg/mL Top, Storage 4 hours 107.88 mg/mL
100 mg/mL Middle, Storage 4 hours 108.94 mg/mL
100 mg/mL Bottom, Storage 4 hours 103.17 mg/mL
100 mg/mL Mean, Storage 4 hours 106.66 mg/mL

Stability (% of Time Zero)
Conducted on dose levels: 1 and 100 mg/mL concentrations
Storage was at room temperature in the dark
Results:
Table 3: Chemical stability of test substance in 1% methylcellulose formulations
Test Concentration Time Frame Result
1 mg/mL Storage 0 hours, Replicate 1 1.1313 mg/mL
1 mg/mL Storage 0 hours, Replicate 2 0.81800 mg/mL
1 mg/mL Storage 0 hours, Mean 0.97465 mg/mL
1 mg/mL Storage 4 hours, Replicate 1 0.93275 mg/mL
1 mg/mL Storage 4 hours, Replicate 2 0.90313 mg/mL
1 mg/mL Storage 4 hours, Mean 0.91794 mg/mL
1 mg/mL Storage 24 hours, Replicate 1 0.93222 mg/mL
1 mg/mL Storage 24 hours, Replicate 2 0.86380 mg/mL
1 mg/mL Storage 24 hours, Mean 0.89801 mg/mL

100 mg/mL Storage 0 hours, Replicate 1 102.50 mg/mL
100 mg/mL Storage 0 hours, Replicate 2 104.39 mg/mL
100 mg/mL Storage 0 hours, Mean 103.45 mg/mL
100 mg/mL Storage 4 hours, Replicate 1 102.71 mg/mL
100 mg/mL Storage 4 hours, Replicate 2 106.77 mg/mL
100 mg/mL Storage 4 hours, Mean 104.74 mg/mL
100 mg/mL Storage 24 hours, Replicate 1 106.20 mg/mL
100 mg/mL Storage 24 hours, Replicate 2 118.79 mg/mL
100 mg/mL Storage 24 hours, Mean 112.50 mg/mL

Duration of treatment / exposure:

Test duration: 28 days.

Frequency of treatment:

Daily (7 days/week).

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
10
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
100
Basis:
nominal in diet

Doses / concentrations 4

Remarks:

Doses / Concentrations:
1000
Basis:
nominal in diet

No. of animals per sex per dose:

Male: 5 animals at 0 mg/Kg bw/day
Male: 5 animals at 10 mg/Kg bw/day
Male: 5 animals at 100 mg/Kg bw/day
Male: 5 animals at 1000 mg/Kg bw/day
Female: 5 animals at 0 mg/Kg bw/day
Female: 5 animals at 10 mg/Kg bw/day
Female: 5 animals at 100 mg/Kg bw/day
Female: 5 animals at 1000 mg/Kg bw/day

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: Doses were selected based on an acute oral toxicity study (HRC Report no. 90505D/HST 327/AC) and a preliminary toxicity study (HRC Report no. 90654D/HST 336/ST). In the preliminary study, rats were given seven daily gavage doses of 1000 mg/kg of the test substance suspended in 1% aqueous methylcellulose. No treatment-related effects were found.

- Rationale for animal assignment (if not random): Test organism assignment was random to minimize body weight variation between groups
- Rationale for selecting satellite groups: Based on test organism numbers.
- Post-exposure recovery period in satellite groups: 2 weeks.

Positive control:

None reported.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: Twice daily for mortality and moribundity; daily for signs of ill health, behavioural changes or toxicosis.

BODY WEIGHT: Yes
- Time schedule for examinations: before dosing and subsequently at weekly intervals.

FOOD CONSUMPTION :
- Food consumption for each cage determined weekly: Yes.


WATER CONSUMPTION : Yes.
- Time schedule for examinations: Daily monitoring by visual appraisal of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: No.

HAEMATOLOGY: Yes.
- Time schedule for collection of blood: Day 30 for main study groups; Day 44 for recovery groups.
- Anaesthetic used for blood collection: Yes (ether).
- Animals fasted: Yes.
- How many animals: All surviving animals.
- Parameters checked in Table 4 below were examined.

CLINICAL CHEMISTRY: Yes.
- Time schedule for collection of blood: Day 30 for main study groups; Day 44 for recovery groups.
- Animals fasted: Yes .
- How many animals: All surviving test organisms.
- Parameters checked in Table 4 below were examined.

URINALYSIS: Yes.
- Time schedule for collection of urine: Day 30 for main study groups; Day 44 for recovery groups.
- Metabolism cages used for collection of urine: No data.
- Animals fasted: Yes.
- Parameters checked in Table 4 below were examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 5).
HISTOPATHOLOGY: Yes (see Table 5).

A complete necropsy was conducted on all test organisms. Animals were euthanized by carbon dioxide inhalation and exsanguinated. Microscopic examination was performed on all tissues from all test organisms in the control and high-dose groups euthanized at the scheduled primary necropsy. Gross lesions were examined from animals in the low- and mid-dose groups euthanized at the primary necropsy and animals in the control and high-dose groups euthanized at the recovery necropsy. Additionally, the liver was evaluated from animals in the low- and mid-dose groups euthanized at the primary necropsy, as well as animals in the control and high-dose group at the recovery necropsy.

Other examinations:

Food consumption data were analysed on a cage basis using cumulative cage totals. The individual test organism was the basic unit for all other parameters. Body weight data were analysed using weight gains. The following sequence of statistical tests were used for body weight, food consumption, organ weight and clinical pathology data:

If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%), the proportion of values different from the mode were analysed by appropriate methods. Otherwise, Bartlett's test was applied to test for heterogeneity of variance between treatments. Where significant (at the 1% level) heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained. If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out.

If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal-Wallis analysis of ranks was used. Analyses of variance were followed by Student's 't' test and Williams' test for a dose-related response, although only the one thought more appropriate for the response pattern observed was reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the 't' test and Williams' test (Shirley's test). Organ weight analysis initially involved a correlation analysis between organ weights and final body weight. For organs where a correlation at the 10% level of significance was established, analysis of organ weight data was performed using adjusted organ weights by analysis of covariance with final body weight as covariate.

Where a correlation between organ weight and body weight was not established the organ weight analysis was carried out using routine analysis of variance on unadjusted organ weights.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

Clinical observations:
There were no deaths. Piloerection, increased salvation and slight diarrhoea were observed among animals in groups 3, 4 and 6. Bodyweight gain was slightly reduced in males ingroups 3, 4 and 6 during the dosing period (statistically significant only at the top dose) but had returned to control values by the end of the recovery period in group 6.

Laboratory findings:
Clinical chemistry: Triglyceride levels were significantly increased, to about double those of the controls, in males from groups 3 and 4. Males in group 4 also showed significantly increased total protein and globulin levels. Aspartate aminotransferase levels were significantly decreased in males from groups 3 and 4. No clinical chemistry changes were evident in animals from group 6.

Haematology: The only significant effect observed was an increase in platelets in males of groups 4 and 6 at Week 5 and at Week 7.

Urinalysis: The only effect observed was a significant increase in urine protein concentration in both sexes from group 4. In group 6 males the urine protein level was still higher than controls, although the difference was not statistically significant.

Effect in organs:
Absolute and bodyweight-adjusted liver weight was significantly increased in both sexes from group 4. Adjusted liver weight was also significantly increased in females from group 3 and in males from group 4. The only macroscopic finding of note was distended caecum in both sexes from groups 3, 4 and 6. The only microscopic change of note was minimal hyperplasia of the caecum mucosal epithelium in three males from group 4.

Histopathological findings of 28d oral gavage study

- Males

Controls - all showed slight signs of lung inflammation, and a single animal had synovitis of a femural joint

10 mg/Kg/d - increased cellularity of mandibular lymph nodes (5/5), slight focal capsular fibrosis of the spleen (1/5).

100 mg/Kg/d - increased cellularity of mandibular lymph nodes (5/5), slight focal extramedullary haemopoiesis of the liver (1/5).

1000 mg/Kg/d - slight peribronchial inflammatory cells (2/5), urinary bladder had slight transitional cell hyperplasia and slight subepithelial inflammatory cells; slight interstitial inflammatory cells of the prostrate; prominent microfollicles of the thyroid (1/5)

- Females

Control females - slight signs of lung inflammation (1/5), slight epithelia hyperplasia of the cervix & vagina and slight luminal dilation of the uterus is observed variously across control animals.

10 mg/Kg/d - increased cellularity of mandibular lymph nodes (5/5), slight luminal dilation of the uterus (2/5)

100 mg/Kg/d - increased cellularity of mandibular lymph nodes (5/5), slight luminal dilation of the uterus (2/5) and ectopic non-gradular epithelium was observed in the glandular mucosa of the stomach of a single animal.

1000 mg/Kg/d - (1/5) Slight epithelia hyperplasia (3/5) and mucification (1/5) of the vaginas and cervix. Ectopic thymus was observed in a single animal on examination of the thyroid.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

10 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: No treatment related effects observed. Original NCD unit is mg/Kg/day

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: Original NCD unit is mg/Kg/day

3

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability). The test substance was determined to show NOEL (male and female) = 10 mg/kg/day , LOEL (male and female) = 105.2 mg/kg/day.

Executive summary:

The test substance was administered daily as a suspension in 1% methylcellulose to rats by oral gavage 1000, 100, and 10 mg/kg/day doses, for a minimum of 28 consecutive days. Control rats received 1% methylcellulose. Test organisms were euthanatized following the 28-day period, for additional animals in the control and high dosage groups, following further 2 -week post-treatment recovery period with gross necropsy examination.The effects observed at 100 and 1000 mg/Kg/day were considered not to be of appreciable significance and attributed to the laxative effect of the substance and nutritional impairment. The dosage of 10 mg/kg/day was a clear no effect level.