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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study under GLP condition

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007
Reference Type:
publication
Title:
Reproductive and developmental toxicity screening study of 4-aminophenol in rats.
Author:
Harada T, Kimura E, Hirata-Koizumi M, Hirose A, Kamata E, Ema M.
Year:
2008
Bibliographic source:
Drug Chem Toxicol.2008;31(4):473-86.

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 4-aminophenol
- Physical state: white or pale-yellow crystalline powder
- Analytical purity: 99.00%
- Lot/batch No.: Lot No.044K0101 (Tokyo Chemical Industry Co., Ltd.)
- Storage condition of test material: The test material was sealed up, and it was saved in the cool and dark space
- Other: The purity and stability of the chemical were verified by analysis before the study.

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc., Atsugi
- Age at study initiation: (P) 10 weeks old
- Housing:Rats were housed individually, except during the acclimation, mating, and nursing periods. From Day 0 of pregnancy to the day of sacrifice, individual dams and litters were reared by using wooden chips (White Flake; Charles River Japan, Inc.) as bedding.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 ± 2 ℃
- Humidity (%): 55 ± 5 %
- Air changes (per hr): 10 - 20 times/hr
- Photoperiod (hrs dark / hrs light): 12 hrs dark /12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% carboxymethylcellulose-Na solution
Details on exposure:
Dosing volume: 10 mL/kg
Stability (test solutions): The stability of formulations in the dark at room temperature has been confirmed for up to 6 h.
Frequency of preparation: The formulations were prepared just before use and were used within 6 h.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): From Day 0 of pregnancy to the day of sacrifice, individual dams and litters were reared by using wooden chips (White Flake; Charles River Japan, Inc.) as bedding.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
(P) Male: 49 days (from 14 days before mating to the day before sacrifice through the mating period)
(P) Female: 40 - 60 days (from 14 days before mating to 3 days after delivery through the mating and gestation periods)
Frequency of treatment:
once-daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 20, 100, 500 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
12 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dosage levels were determined based on the results of a previous 28-day repeated-dose toxicity study in rats given 4-aminophenol by gavage at 0 (vehicle), 4, 20, 100, or 500 mg/kg/day (See repeated dose toxicity_001). At 500 mg/kg/day, the death in 1 male with renal necrosis, decreases in body-weight gain, food consumption, erythrocyte count, hematocrit value (Ht) and hemoglobin content (Hb), increased relative weight of the liver and spleen, basophilic tubules in the kidney, and brown urine were observed. At 100 mg/kg/day, brown urine and basophilic tubules in the kidney were also observed. No toxicological effects were detected at 4 and 20 mg/kg/day.

- Rationale for animal assignment (if not random): Body weight-balanced randomization

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations:
Male: Twice a week
Female:
Premating period: Twice a week
Mating period: Twice a week
Pregnancy period: Days 0, 7, 14 and 20
Lactation period: Days 1 and 4

FOOD CONSUMPTION: Yes
Male:
Premating period: Twice a week
Female:
Premating period: Twice a week
Pregnancy: Days 1, 7, 14 and 20
Lactation: Days 1 and 4"

WATER CONSUMPTION: No
Oestrous cyclicity (parental animals):
Daily vaginal lavage samples of each female were evaluated for estrus cycle throughout the premating period.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
Testis weight, epididymis weight
Litter observations:
PARAMETERS EXAMINED:
The following parameters were examined in F1 offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE:
Male animals: Rats were euthanized by exsanguination under anesthesia on the day after the last administration.
Maternal animals: Rats were euthanized by exsanguination under anesthesia on Day 4 of lactation.
GROSS NECROPSY:
Yes, Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
HISTOPATHOLOGY / ORGAN WEIGHTS:
- Histopathological examined organ:
Control and 500 mg/kg/day groups: Epididymides, Testes, Ovaries
100 mg/kg/day group: Epididymides, Testes
- Weighted organ:
All groups of the male: Epididymides, Testes
Postmortem examinations (offspring):
SACRIFICE
The F1 offspring were sacrificed on PND 4.

GROSS NECROPSY
On PND 4, the pups were euthanized by exsanguination under anesthesia and gross internal examinations were performed.
Statistics:
Statistical analyses were conducted by Mann-Whitney U-test, Fischer’s exact probability test, Dunnett’s test, and Kruskal-Wallis rank sum test.
Reproductive indices:
Number of pregnant females
Number of corpora lutea
Number of implantation sites
Pre-coital interval (days)
Gestation length
Number of pups delivered
Number of live pups delivered
Number of stillborn pups
Number of dams delivererd
Number of dams with total litter loss
Copulation index (%)=(No. of pairs with successful copulation / No. of pairs mated) x 100
Fertility index (%)=(No. of pregnant animals / No. of pairs with successful copulation) x 100
Gestation index (%)=(No. of females with live pups delivered / No. of pregnant females) x 100
Implantation index = (No. of implantations/no. of corpora lutea) x 100
Delivery index = (No. of pups born/no. of implantation sites) x 100
Rate of stillborn pups(%)=(No. of stillborns pups/total no. of pups delivered) x 100
Sex ratio = (No. of live male pups/no. of live female pups) x 100
Offspring viability indices:
Offsprings viability indices:
Viability index on day0 of lactation (%)=(No. of live pups delivered / total no. of pups delivered) x 100
Viability index on day4 of lactation (%)Z=(No. of live pups on day 4 / No. of live pups delivered) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- Mortality:
Male:
- 500 mg/kg group: 4 males died.
Female:
- 500 mg/kg group: 2 females died.
- Clinical signs:
Male:
- 100 mg/kg group: Brown urine was observed.
- 500 mg/kg group: Brown urine, prone position, decrease in locomotor activity, bradypnea, and salivation was observed.
Female:
- 100 mg/kg group: Brown urine was observed.
- 500 mg/kg group: Brown urine, decrease in locomotor activity, bradypnea, pale skin, pale eye, loss of hair, hypothermia, lateral position, and salivation was observed.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
[Body weight] (See Table 1)
Male:
- 500 mg/kg group: A low value of body weight was recognized.
Female:
- 500 mg/kg group: A low value of body weight was recognized.

[Food consumption]
Male:
- 500 mg/kg group: A low value of food consumption was recognized.
Female:
- 100 mg/kg group: A low value of food consumption was recognized.
- 500 mg/kg group: A low value of food consumption was recognized.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS) (See Table 3)
Female:
- 500 mg/kg group: 4 females terminated their estrus cycles and showed extended diestrous vaginal smears.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS) (See Table 3)
One pair did not copulate at 500 mg/kg/day.
No significant effects of 4-aminophenol were observed on precoital interval or copulation index.
One female did not become impregnated in each of the control, 20-, and 500-mg/kg/day groups.
No significant differences were noted in fertility index or gestation index between the control and 4-aminophenol-treated groups.
Gestation length was significantly prolonged at 500 mg/kg/day.
At 500 mg/kg/day, the delivery index was significantly reduced and the rate of stillborn pups was increased significantly. At this dose, almost all dams neglected their pups, some dams showed cannibalism, and all pups of 6 dams died.
Longer gestation period, decreased delivery index, and lower body weight of pups on postnatal day (PND) 0 and increased number of stillborns at 500 mg/kg/day were also observed. At this dose, the viability of pups on PND 4 was decreased markedly.

ORGAN WEIGHTS (PARENTAL ANIMALS) (See Table 2)
Male:
Male:
- 500 mg/kg group: Absolute and relative weights of the testes and epididymides were decreased.
Female: No effect

GROSS PATHOLOGY (PARENTAL ANIMALS)
[Dead animal]
Male:
- 500 mg/kg group: Discoloration and enlargement of the kidney (4/4) was observed.
Female:
- 500 mg/kg group: Small of thymus (1/2), black-brownish colored kidney (2/2), reddish urine retention in urinary bladder (1/2) were observed.
[Surviving animals]
Male:
- 500 mg/kg group: Black-brownish colored spleen (8/8), discoloration of kidney (4/8), small testis (1/8) were observed.
Female:
- 500 mg/kg group: Black-brownish colored spleen (2/8) was observed.

HISTOPATHOLOGY (PARENTAL ANIMALS)
[Dead animal]
Male:
- 500 mg/kg group: Tubular necrosis (4/4), basophilic tubules (4/4), protein cast (4/4), brown granular deposit (4/4) on epithelial cells of the proximal tubule in the kidney, vacuolation of Sertoli cells (1/4), degeneration/necrosis of spermatocytes (1/4) in the testis were observed.
Female:
- 500 mg/kg group: Atrophy of thymus (1/2), Basophilic tubules (2/2), protein cast (2/2), tubular necrosis (1/2), hyaline and/or brown granular deposit on epithelial cells of the proximal tubule (2/2) in the kidney were observed.
[Surviving animals]
Male:
- 500 mg/kg group:
Basophilic tubules (4/8), protein cast (4/8), and granular cast (4/8) in the kidney and deposits of hemosiderin in the red pulp (8/8) and extramedullary hematopoiesis (3/8) in the spleen, decreased spermatocyte (8/8) and spermatid levels (8/8) , vacuolation of Sertoli cells (8/8), degeneration/necrosis of spermatocytes (3/8) in the testis, and decreased sperm counts (4/8) and debris of germ cells (8/8) in the epididymis lumen were observed.
Female:
- 500 mg/kg group:
Deposits of hemosiderin in the red pulp (2/8) and extramedullary hematopoiesis (1/8) in the spleen were observed.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
for general toxicity
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Decreased food consumption in males at 500 mg/kg/day and females at 100 and 500 mg/kg/day, and brown urine in both sexes at 100 and 500 mg/kg/day were also observed.
Dose descriptor:
NOAEL
Remarks:
for reproductive or developmental toxicity
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Longer gestation period, decreased delivery index, and lower body weight of pups on postnatal day (PND) 0 and increased number of stillborns at 500 mg/kg/day were also observed. At this dose, the viability of pups on PND 4 was decreased markedly.
Remarks on result:
other: Generation not specified (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING) (See Table 4)
Although no significant difference was observed in viability index at PND 0 between control and 4-aminophenol-treated groups, the index was significantly decreased at PND 4 at 500 mg/kg/day.

BODY WEIGHT (OFFSPRING) (See Table 4)
At 500 mg/kg/day, the body weight of live male and female pups were significantly lowered on PND 0 and were decreased on PND 4.

GROSS PATHOLOGY (OFFSPRING) (See Table 4)
At 500 mg/kg/day, pups with external malformations were found in 2 pups; 1 showed a vestigial tail and the other showed an open auricle, short tail, and kinky tail. No significant difference was observed in the incidence of pups with malformations between control and 500-mg/kg/day groups. No pups with external malformations were observed in the control or groups given 4-aminophenol at 20 and 100 mg/kg/day. No pups with internal malformations were found in any groups.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
gross pathology

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1. Body-weight gain in male and female rats given 4-aminophenol

Dose (mg/kg/day) 0 20 100 500
Male
Number of animals 12 12 12 12
 Number of deaths 0 0 0 4
 Initial body weight (g)  373 ± 18 372 ± 20 371 ± 19 368 ± 18
Body weight gain (g) 
 Days 1-8 29 ± 7 33 ± 7 22 ± 15 -7 ± 10**
 Days 8-15 29 ± 8 28 ± 8 26 ± 10 19 ± 5*
 Days 15-22 32 ± 5 30 ± 11 34 ± 8 26 ± 8
 Days 22-29 23 ± 5 24 ± 5 24 ± 7 21 ± 7
 Days 29-36 28 ± 8 22 ± 5 23 ± 5 21 ± 10
 Days 36-43 21 ± 8 22 ± 7 25 ± 6 26 ± 10
 Days 43-50 20 ± 7 22 ± 6 21 ± 5 17 ± 8
Female        
Number of animals 12 12 12 12
 Number of deaths 0 0 0 2
 Initial body weight (g)  225 ± 13 224 ± 12 223 ± 8 224 ± 9
Body weight gain (g) 
 Days 1-8 16 ± 8 14 ± 7 9 ± 5 -13 ± 11**
 Days 8-15 9 ± 6 7 ± 5 9 ± 6 11 ± 16
 Days 0-7 of pregnancy 36 ± 6 36 ± 7 32 ± 8 26 ± 9*
 Days 7-14 of pregnancy 30 ± 4 31 ± 7 33 ± 6 30 ± 7
 Days 14-20 of pregnancy 74 ± 12 74 ± 14 69 ± 15 50 ± 10
 Days 0-4 of lactation 23 ± 15 19 ± 14 26 ± 7 6
         

Significantly different from 0 mg/kg group, *; at P<0.05, **; P<0.01.

Table 2. Reproductive organ weights in rats given 4-aminophenol

Dose (mg/kg/day) 0 20 100 500
Male
Number of animals 12 12 12 8
 Weight of testes (g) 3.53 ± 0.143 3.50 ± 0.33 3.34 ± 0.24 2.40 ± 0.29*
 Relative weight of testes (g) 0.64 ± 0.05 0.63 ± 0.04 0.62 ± 0.05 0.49 ± 0.05*
 Weight of epididymides (g) 1.27 ± 0.07 1.27 ± 0.08 1.23 ± 0.09 0.92 ± 0.05*
 Relative weight of epididymides (g) 0.23 ± 0.02 0.23 ± 0.01 0.23 ± 0.02 0.19 ± 0.02*
Female        
Number of animals 11 11 12 2
 Weight of ovaries (g) 90.7 ± 9.9 95.8 ± 11.0 96.7 ± 8.2 81.7
 Relative weight of ovaries (g) 28.1 ± 1.4 29.9 ± 3.1 30.7 ± 2.9 29.2
         

Significantly different from 0 mg/kg group, *; at P<0.05, **; P<0.01.

Table 3. Reproductive findings in rats given 4-aminophenol.

Dose (mg/kg/day) 0 20 100 500
 Number of females examined 12 12 12 11
 Count of estrus  3.8 ± 0.5 3.8 ± 0.6 3.9 ± 0.9 2.6 ± 1.6
 Females showing abnormal estrous cycles (%)  0 8.3 0 45.5*
 Number of mated (male/female) 12/12 12/12 12/12 7/10a
 Precoital interval (day)  2.5 ± 1.2 2.6 ± 1.2 2.9 ± 3.3 4.6 ± 4.0
 Copulation index (%, male/female)  100/100 100/100 100/100 85.7/90.0
 Fertility index (%, male/female) 91.7/91.7 91.7/91.7 100/100 100/88.9
 Gestation index (%) 100 100 100 100
 Gestation length (day) 22.2 ± 0.4 22.2 ± 0.4 22.6 ± 0.7 23.3 ± 0.50**

Abnormal estrous cycle (%)= (No. of females showing abnormal estrous cycles /No. of females) X 100

Copulation index (%)= (No. of rats copulated / No. of pairs) X 100

Fertility index (%)= (No. of pregnant / No. of copulated) X 100

Gestation index (%)= (No. of females with live pups born / No. of pregnant females) X 100

a: One female was not used for mating because this female showed severely toxicological sign.

Significantly different from 0 mg/kg group, *; at P<0.05, **; P<0.01.

Table 4. Developmental findings in rats given 4-aminophenol.

Dose (mg/kg/day) 0 20 100 500
Number of pregnant females 11 11 12 8
 Number of corpora lutea  15.4 ± 1.6 14.1 ± 2.0 15.3 ± 1.8 15.6 ± 1.5
 Number of implantations 14.4 ± 1.0 13.6 ± 2.0 14.2 ± 2.8 14.8 ± 0.9
 Implantation index (%) 93.5 96.8 92.9 94.4
 Number of pups delivered 12.8 ± 3.1 13 .0± 2.0 13.3 ± 2.8 11.1 ± 3.5
 Number of live pups delivered 12.7 ± 3.2 12.9 ± 2.0 13.1 ± 2.6 10.1 ± 4.4
 Number of stillborn pups 0.1 ± 0.3 0.1 ± 0.3 0.3 ± 0.5 1.0 ± 1.2
 Delivery index (%) 88.6 94.7 92.4 68.6**
 Rate of stillborn pups (%) 0.7 0.7 1.9 9*
 Sex ratio of live pups (males/females) 74/66 66/76 69/88 50/31
 Number of dams delivered 11 11 12 8
 Number of dams with total litter loss 0 0 0 6
 Viability index (%)
   Day 0 of lactation 99.3 99.3 98.1 91
   Day 4 of lactation 99.3 99.3 98.7 24.7**
 Body weight of pups (g)
   Male PND 0 6.9 ± 6.0 6.9 ± 0.3 6.7 ± 0.9 4.9 ± 0.6**
   Male PND 4 10.9 ± 1.6 11.0 ± 0.94 10.7 ± 2.2 6.1
   Female PND 0 6.5 ± 0.7 6.5 ± 0.4 6.4 ± 0.8 4.5 ± 0.6**
   Female PND 4 10.4 ± 1.6 10.5 ± 0.9 10.2 ± 2.0 6.9
Number of pups (litters) examined externally on PND 0 141 (11) 143 (11) 160 (12) 89 (8)
 Number of pups (litters) with malformations 0 0 0 2 (2)
   Open auricle 0 0 0 1 (1)
   Vestigial tail 0 0 0 1 (1)
  Short tail 0 0 0 1 (1)
  Kinky tail 0 0 0 1 (1)
Number of pups (litters) examined internally on PND 4 139 (11) 141 (11) 155(12) 20 (2)
 Number of pups (litters) with malformations 0 0 0 0

Implantation index (%)= (No. of implantations / No. of corpora lutea) X 100

Delivery index (%)= ( No. of live pups delivered / No. of implantations) X 100

Rate of stillborn pups (%)= (No. of stillborn pups / total No. of pups delivered) X 100

Viability index on Day 0 of lactation (%)= (No. of live pups delivered / total No. of pups delivered) X 100

Viability index on Day 4 of lactation (%)= (No. of live pups on Day 4 of lactation / No. of live pups delivered) X 100

Significantly different from 0 mg/kg group, *; at P<0.05, **; P<0.01.

Applicant's summary and conclusion