Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
In early studies, the test substance given intravenously in dogs was found to be relatively non-toxic. Moderate toxicity after intraperitoneal injection in rats is reported in SAX’s with an LD50 of 500 mg/kg body weight. Information on oral toxicity of test substance in rodents have been found through several studies undertaken during the National Toxicology Program (NTP), including 14 day, 13 week, and 2 year feeding studies in rats and mice. These studies were published in November 1996 as NTP’s Technical Report Series, No. 465 (TR-465): Toxicology and Carcinogenesis Studies of Phenolphthalein (CAS no. 77 -09 -8) in F344/N Rats and B6C3F1 Mice (Feed Studies).1 In these studies, the dose range applied in oral feed exceeds the limit dose for acute toxicity studies according to the OECD Guideline for the Testing of Chemicals No. 423 throughout the protocols. For example, in the 13 -week study in rats groups of 10 male and 10 or 9 female F344/N rats were given 0, 3000, 6000, 12000, 25000, or 50000 ppm test item equivalent to average daily doses of approximately 200, 400, 800 , 1600, or 3500 mg/kg body weight. All study rats survived the end of the study and no mortality was observed. The final mean body weight of the 50000 ppm females and the mean body weight gains of the females were significantly lower compared to those data of the controls. The final mean body weight and mean body weight gains of all other exposed groups were similar to those of the controls. Thus, in can be concluded that the test substance does not possess any acute toxic potential below 2000 mg/kg bw. These findings suggest that further acute oral toxicity studies for the test substance are scientifically unjustified and should thus not be performed due to animal welfare reasons.
References: Repeated dose toxicity studies: (1) NTP 1996, 14-day oral feed rats (2) NTP 1996, 13-weeks oral feed rats
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
data waiving: supporting information
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 April 1987 - 29 July 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes
Remarks:
Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58)
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms (Germantown, NY)
- Age at study initiation: 6 weeks
- Weight at study initiation: 132 g (males) and 109 g (females)
- Housing: Polycarbonate suspended cages (Lab Products, Inc., Rochelle Park, NJ), changed twice per week
- Diet: NIH-07 open formula meal diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, changed weekly
- Water: Tap water (Bethesda municipal supply) via automatic watering system (Edstrom Industries, Inc., Waterford, WI), available ad libitum, changed every 2 weeks
- Acclimation period: 12 to 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 50-67
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet: The dose formulations for all studies were prepared weekly by mixing test item with feed
- Mixing appropriate amounts with feed: A test item premix was prepared by hand and was then blended with feed in a Patterson-Kelly twin-shell blender for 15 minutes using an intensifier bar for the initial 5 minutes.
- Storage temperature of food: Formulations were stored in double plastic bags at temperatures at or below -20° C for up to 3 weeks.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability was monitored during the 13-week using high-performance liquid chromatography. No degradation of the bulk chemical was detected. Homogeneity was confirmed and the stability of the dose formulation was confirmed for at least 3 weeks when stored protected from light at room temperature. All of the dose formulations analyzed during the were within 10% of the target concentrations.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
continuously via feed
Dose / conc.:
0 ppm
Dose / conc.:
3 000 ppm
Remarks:
equivalent to average daily doses of approximately 200 mg/kg bw/day
Dose / conc.:
6 000 ppm
Remarks:
equivalent to average daily doses of approximately 400 mg/kg bw/day
Dose / conc.:
12 000 ppm
Remarks:
equivalent to average daily doses of approximately 800 mg/kg bw/day
Dose / conc.:
25 000 ppm
Remarks:
males: equivalent to average daily doses of approximately 1600 mg/kg bw/day
females: equivalent to average daily doses of approximately 1700 mg/kg bw/day
Dose / conc.:
50 000 ppm
Remarks:
males: equivalent to average daily doses of approximately 3500 mg/kg bw/day
females: equivalent to average daily doses of approximately 3600 mg/kg bw/day
No. of animals per sex per dose:
10 males and 9 or 10 females
Control animals:
yes, plain diet
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed initially, weekly, and at the end of the studies

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Feed consumption was recorded weekly by cage.

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected from the retroorbital sinus of rats in the clinical pathology groups on days 5 and 21 and of core study rats at the end of the study for hematology and clinical chemistry.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: all
- Parameters checked: hematocrit, hemoglobin, erythrocyte and reticulocyte counts, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, platelet count, and total leukocyte counts and differentials. Clinical chemistry: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile acids.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected from the retroorbital sinus of rats in the clinical pathology groups on days 5 and 21 and of core study rats at the end of the study for hematology and clinical chemistry.
- Animals fasted: Not specified
- How many animals: all
- Parameters checked: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile acids.

URINALYSIS: Yes

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsy was performed on all core study rats . Organs weighed were brain, heart, right kidney, liver, lung, right testis, and thymus.

HISTOPATHOLOGY: Yes

Complete histopathology was performed on 0 and 50,000 ppm rats . In addition to gross lesions and tissue masses, the tissues examined included: adrenal gland, brain, clitoral gland (rats), esophagus, femur (including marrow), heart, gallbladder (mice), large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum, and ileum), kidney, liver, lungs (and mainstem bronchi), lymph nodes (mandibular and mesenteric), mammary gland, nose, ovaries, pancreas, parathyroid gland, pituitary gland, preputial gland (rats), prostate gland, salivary gland, skin, spleen, stomach (including forestomach and glandular stomach), testes (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus. In rats, the liver (females), lung (males), and ovaries were examined to a no effect level. In mice, the bone marrow and spleen (males) were examined to a no effect level.
Other examinations:
At the end of the study, sperm samples were collected from male rats (core study) in the 0, 12,000, 25,000, and 50,000 ppm groups for sperm morphology evaluations. The parameters evaluated included sperm density, morphology, and motility. The right cauda, right epididymis, and right testis were weighed. Vaginal fluid samples were collected for up to 7 consecutive days prior to the end of the studies from female rats (core study) in the 0, 12,000, 25,000, and 50,000 ppm groups. The parameters evaluated were relative frequency of estrous stages and estrous cycle length.
Statistics:
Williams' or Dunnett's test
Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
All rats survived to the end of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The final mean body weight of the 50,000 ppm females and the body weight gains of the 25,000 and 50,000 ppm females were significantly lower than those of the controls. The final mean body weights and body weight gains of all other exposed groups were similar to those of the controls.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Feed consumption by exposed groups was similar to that by the controls. Dietary levels of 3,000, 6,000, 12,000, 25,000, or 50,000 ppm test item resulted in average daily doses of approximately 200, 400, 800, 1,600, or 3,500 mg test item/kg body weight to males and 200, 400, 800, 1,700, or 3,600 mg/kg to females.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The few differences in the hematology parameters were sporadic and were not considered to be chemical related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The few differences in the clinical chemistry parameters were sporadic and were not considered to be chemical related.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute and relative liver weights of 25,000 and 50,000 ppm males and the relative liver weights of 12,000 ppm males and of 25,000 and 50,000 ppm females were significantly greater than those of the controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No chemical-related gross lesions were observed.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No chemical-related microscopic lesions were observed.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
The percentage of motile sperm in the 12,000 ppm males was significantly greater than that in the controls, but no other significant differences in sperm morphology or vaginal cytology were observed between exposed and control groups.
Key result
Dose descriptor:
NOEL
Effect level:
6 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Remarks on result:
other: Corresponding to 400 mg/kg bw/d.
Key result
Dose descriptor:
NOAEL
Effect level:
12 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Remarks on result:
other: Corresponding to 800 mg/kg bw/d.
Critical effects observed:
not specified
Conclusions:
Based on the results of this study, the NOAEL was 12000 ppm (800 mg/kg bw/day) for males and females.
Executive summary:

A study similar to OECD TG 408 was performed to assess the cumulative toxicity of the test item when administered daily to rats in feed for a period of 13 weeks.

Groups of 10 male and 9 or 10 female F344/N rats were given 0, 3000, 6000, 12000, 25000, or 50000 ppm test item (equivalent to average daily doses of approximately 200, 400, 800, 1600, or 3500 mg test item/kg body weight to males and 200, 400, 800, 1700, or 3600 mg/kg to females) in feed for 13 weeks. Additional groups of 10 male and 10 female rats designated for clinical pathology evaluations were also given 0, 3000, 6000, 12000, 25000, or 50000 ppm test item in feed until day 21.

All core study rats survived to the end of the study. The final mean body weight of the 50000 ppm females and the mean body weight gains of the 25000 and 50000 ppm females were significantly lower than those of the controls. The final mean body weights and mean body weight gains of all other exposed groups were similar to those of the controls. There was no cathartic action or any other clinical finding attributed to exposure to the test item. The few differences in the haematology and clinical chemistry parameters were sporadic and were not considered being chemical related. The percentage of motile sperm in the 12000 ppm males was significantly greater than that in the controls, but no other significant differences in sperm morphology or vaginal cytology between exposed and control groups were observed. Absolute and relative liver weights of 25000 and 50000 ppm males were significantly greater than those of the controls. No chemical-related gross or microscopic lesions were observed.

Based on the results of this study, the NOAEL was 12000 ppm (800 mg/kg bw/day) for males and females.

Reason / purpose for cross-reference:
data waiving: supporting information
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Jun 04,1979 - Jun 17, 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
14 days exposure/no clinical pathology
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Industries (Indianapolis, IN)
- Age at study initiation: Not available
- Weight at study initiation: 123 g (males) and 109 g (females)
- Housing: housed five per cage, Polycarbonate cages with stainless steel tops, changed twice per week
- Diet: NIH-07 open formula meal diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, changed daily
- Water: Tap water (Reading municipal supply) via water bottles changed weekly, available ad libitum
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24
- Humidity (%): 45- 55
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): The dose formulations were prepared weekly by mixing test item with feed. Dose formulations were mixed by hand for 1 minute and then blended in a Vortex feed mixer for 20 minutes using an intensifier bar for the initial 5 minutes.
- Storage temperature of food: Formulations were stored in plastic bags within metal containers at room temperature for up to 1 week.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability studies of the 6,000 ppm dose formulation were performed by the analytical chemistry laboratory using high-performance liquid chromatography. The stability of the dose formulation was confirmed for at least 2 weeks when stored at temperatures up to 25° C. Homogeneity was confirmed and the stability of the dose formulation was confirmed for at least 3 weeks when stored protected from light at room temperature.
Duration of treatment / exposure:
14 days
Frequency of treatment:
continuously via feed
Dose / conc.:
6 250 ppm
Remarks:
equivalent to average daily doses of approximately 500 mg/kg bw/day
Dose / conc.:
12 500 ppm
Remarks:
equivalent to average daily doses of approximately 1000 mg/kg bw/day
Dose / conc.:
25 000 ppm
Remarks:
equivalent to average daily doses of approximately 2000 mg/kg bw/day
Dose / conc.:
50 000 ppm
Remarks:
males: equivalent to average daily doses of approximately 4500 mg/kg bw/day
females: equivalent to average daily doses of approximately 4000 mg/kg bw/day
Dose / conc.:
100 000 ppm
Remarks:
males: equivalent to average daily doses of approximately 10500 mg/kg bw/day
females: equivalent to average daily doses of approximately 11000 mg/kg bw/day
No. of animals per sex per dose:
5/5 (m/f)
Control animals:
yes, plain diet
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: observed once daily

BODY WEIGHT: Yes
- Time schedule for examinations: initially, on day 7, at the end of the study

FOOD CONSUMPTION AND COMPOUND INTAKE:
Feed consumption was recorded by cage on days 7 and 14 and whenever the feed had to be replenished due to an inadequate feed supply.

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
At the end of the 14-day studies, a necropsy was performed on all rats.
HISTOPATHOLOGY: Yes
Complete histopathology was performed on one male rat and two female rats from the control groups and on two males and one female from the 100,000 ppm rats. In addition to gross lesions and tissue masses, the tissues examined included: adrenal glands, bone marrow, brain, ears (external and middle), esophagus, eyes, gallbladder (mice), heart, large intestine (colon and rectum), small intestine (duodenum, jejunum, and ileum) kidneys, larynx, liver, lungs (and mainstem bronchi), lymph nodes (mandibular and mesenteric), mammary gland, nose, ovaries, pancreas, parathyroid glands, pituitary gland, prostate gland, rib (and costochondral junction), salivary gland, skin, spinal cord (and sciatic nerve), spleen, stomach, testes (with seminal vesicle), thigh muscle, thymus, thyroid glands, trachea, urinary bladder, and uterus.
Statistics:
Williams' or Dunnett's test
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The feces of 100,000 ppm males and females were abnormally lighter in color than those of the other groups. This discoloration was observed on day 3 and continued throughout the study.
Mortality:
no mortality observed
Description (incidence):
All rats survived to the end of the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The final mean body weights of all exposed groups of rats were similar to those of the controls.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Feed consumption by 100,000 ppm males and females was greater than that by the controls; feed consumption by all other exposed groups of rats was similar to that of the controls. Dietary levels of 6,250, 12,500, 25,000, 50,000, or 100,000 ppm resulted in average daily doses of approximately 500, 1,000, 2,000, 4,500, or 10500 mg/kg bw/day to males and 500, 1,000, 2,000, 4,000, or 11,000 mg/kg bw/day to females.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no chemical-related gross lesions observed in rats.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no chemical-related microscopic lesions observed in rats.
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOEL
Effect level:
100 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Remarks on result:
other: Corresponding to 10500 mg/kg bw/d for males and 11000 mg/kg bw/d for females.
Key result
Critical effects observed:
no
Conclusions:
Based on the results of this study, no test item related adverse effects have been observed up to 100000 ppm (11000 mg/kg bw/day), the highest dose tested.
Executive summary:

The purpose of this oral toxicity study was to assess the cumulative toxicity of the test item when administered daily to rats in feed for a period of 14 days. Groups of five male and five female F344/N rats were given 0, 6250, 12500, 25000, 50000, or 100000 ppm test item in feed for 14 days. Dietary levels of 6250, 12500, 25000, 50000, or 100000 ppm test item resulted in average daily doses of approximately 500, 1000, 2000, 4500, or 10500 mg test item/kg body weight to males and 500, 1000, 2000, 4000, or 11000 mg/kg to females.

All rats survived to the end of the study. The final mean body weights of all exposed groups of rats were similar to those of the controls. No chemical-related gross or microscopic lesions were observed.

Based on the results of this study, no test item related adverse effects have been observed up to 11000 mg/kg bw/day (100000 ppm).

Data source

Materials and methods

Results and discussion

Applicant's summary and conclusion