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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Japanese Ministry of Health Study - English translation - well documented

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2001

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
not specified
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Reference substance name:
o-sec butyl phenol
IUPAC Name:
o-sec butyl phenol
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): o-sec butyl phenol
- Analytical purity: 99.15%
- Lot/batch No.: Lot Number: 970922
- supplied by Honshu Chemical Industry Co., Ltd. (Wakayama, Japan)
- Stability under test conditions: The stability of the test article during the study period was confirmed by the supplier by re-analysis of the test article remaining after use in the study
- Storage condition of test material: preserved in light-resistant containers at room temperature after receipt

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc
- Age at study initiation: 8 wks
- Mean Body Weight at study initiation: Males: 300+/- 6 g; Females: 220 +/-7.5 g
- Housing: individually in metallic cages. From day 14 of gestation (the day sperm was confirmed = Day 0 of gestation), dams were housed in breeding cages for rats and paper pulp chips (ALPHA-dri, Kasho Co., Ltd.) supplied ad libitum as bedding.
- Diet (ad libitum): pelleted diet (CE-2, CLEA Japan, Inc.)
- Water (ad libitum): tap water (supplied from Waterworks Department, Hadano-shi)
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1°C
- Humidity (%): 50-65%
- Air changes (per hr): 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hours a day (from 7:00 to 19:00)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The test article was dissolved in corn oil
Amount administered (if gavage): 2ml/kg

Details on mating procedure:
Males and females in the same group were housed together on 1:1 basis from the evening of day 15 of administration for 14 days at maximum
Copulation was confirmed by examination for the presence of vaginal plug and sperms in vaginal smears, and the females for which copulation was confirmed were separated from males and housed individually thereafter.
Sperm in vaginal smear referred to as day 0 of pregnancy
From day 14 of gestation (the day sperm was confirmed = Day 0 of gestation), dams were housed in breeding cages for rats and paper pulp chips
(ALPHA-dri, Kasho Co., Ltd.) supplied ad libitum as bedding.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of the test article in the dose formulations were confirmed by Hatano Research Institute. No detailed information.
Duration of treatment / exposure:
Single daily gavage exposures, for 42 days males.
28 days plus gestation period up to day 3 lactation in females
Frequency of treatment:
Once daily
Details on study schedule:
Test article was administered once daily to animals 2 weeks before mating, 2 weeks during the mating period.
Administration was repeated once daily for 2 weeks after the end of the mating period for males and throughout the gestation period up to day 3 of
lactation after delivery for females.
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (vehicle control), 12, 60 and 300 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
n= 13 males, n= 13 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels of this study were selected based on the preliminary test in which o-sec-butylphenol was administered
orally by gavage repeatedly for 14 days at dose levels of 0 (corn oil), 30, 100 or 300 mg/kg to groups of rats of the same strain as this study each
group consisting of 5 males and 5 females. The high dose level in the main study was set at 300 mg/kg, at which effects were observed in general condition, body weight and liver weight in males and females.
Positive control:
None

Examinations

Parental animals: Observations and examinations:
All males and females were observed at least once a day

BODY WEIGHT: All males and females were weighed once a week during the study period (Days 1, 8, 15, 22, 29, 36 and 42 of administration for
males and days 1, 8 and 15 of administration for females). The females that were housed together with males were weighed also on day 22 of
administration. Copulated females were weighed on days 0, 7, 14 and 20 of gestation and delivered females were weighed on days 0 and 4 of
lactation.

FOOD WEIGHT AND CONSUMPTION: For all males and females, weight of the feed was measured on the same days as for body weight measurement, and the food consumption from the day of measurement to the day of next measurement calculated. During the 2-week mating period, food
consumption was not measured. Food consumption was measured from day 0 to day 7, day 7 to day 14 and day 14 to day 20 of gestation for
copulated females and from day 0 to day 4 of lactation for delivered females.

Oestrous cyclicity (parental animals):
The number of estruses during the mating period were determined.
Sperm parameters (parental animals):
The testes and epididymides were weighed
Litter observations:
On days 0 and 4 of lactation, body weight of pups was measured for each litter by sex (litter weight) and (litter weight / number of pups) was
calculated for each litter.
Postmortem examinations (parental animals):
GROSS PATHOLOGY OF MAJOR ORGANS WAS PERFORMED FOR FEMALES AND MALES
The following organs were weighed and the ratio to body weight (relative organ weight) calculated: brain, heart, thymus, liver, kidneys, spleen,
adrenals, testes and epididymides.

FEMALES: For all pregnant and non-pregnant females, the ovary and uterus were excised, the number of implantations was counted using the
uterus, and the females that showed implantations were regarded as pregnant females. For the ovary, the number of corpora lutea was counted
under a stereoscopic microscope, and the ovary was fixed in Bouin’s solution and preserved. The ovaries of the non-pregnant females were
subjected to histopathological examination. The following organs were weighed and fixed in 10 w/v% formalin and preserved: brain, heart,
thymus, liver, kidneys, spleen and adrenals.

MALES: The testes and epididymides were fixed in Bouin’s solution and preserved while the other organs, urinary bladder, lungs and stomach were
fixed and preserved in 10 w/v% formalin. For the males in the control group and the high dose group, these fixed organs were subjected to
histopathological examination. Other than these organs, histopathological examination was not done since they were judged to have no effects
from administration of the test article.
Postmortem examinations (offspring):
On day 4 of lactation, all pups were euthanized by ether inhalation and necropsied, and external and internal organs observed macroscopically.
The pups that died on delivery were autopsied and the external and internal organs observed macroscopically for the presence or absence of
abnormalities.
Statistics:
The copulation index and fertility index were analyzed by the chi-square test with Yates’ correction.
Histopathological findings: graded data were analyzed by Mann-Whitney’s U test; the sum of the positive grade analyzed by Fisher’s unilateral
exact test.Statistical analysis was not done for clinical signs, necropsy findings or urinalysis data.
For the other data, the value obtained for each animal or litter mean was regarded as one sample, and the homogeneity of variance for each group was examined first by Bartlett’s test. Based on the results, the data were subjected to one-way analysis of variance or Kruskal-Wallis rank test, and the difference between the control group and each test article administration group was tested by Dunnett’s method or Scheffé’s method, if
significance was observed between groups. The levels of statistical significance were 5% and 1%.
Reproductive indices:
Copulation index [(number of copulated pairs / number of mated pairs) × 100], fertility index [(number of pregnant animals / number of copulated pairs) × 100], the number of days from the start of mating to the day copulation was confirmed.
The length of the gestation period (the number of days from day 0 of gestation and the day of delivery) was calculated and the gestation index
[(number of pregnant females with pups alive / number of pregnant females) × 100] calculated for each group.

Offspring viability indices:
Litter size (liveborn and stillborn pups) was counted on day 0 of lactation, and the delivery index [(number of pups born / number of implantation
sites) × 100] and the birth index [(number of pups alive on day 0 / number of implantation sites) × 100] were calculated. In addition, pups were
observed for the presence or absence of external malformation and sex, and the sex ratio of liveborn pups [(number of male pups born alive /
number of female pups born alive) × 100] calculated.
The number of pups that died was counted every day and the live birth index [(number of pups alive on day 0 / number of pups born) × 100] and the viability index on day 4 after birth [(number of pups alive on day 4 of lactation / number of pups alive on day 0 of lactation) × 100] were calculated.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
For both males and females, there were neither deaths nor moribund animals in any dose group.

MALES: Two males showed decreased locomotor activity and 1 male showed transient salivation after dosing during the early administration period in the 60 mg/kg group, and all males showed transient salivation after dosing and decreased locomotor activity throughout the administration period, 9 males showed abdominal or lateral position, 2 males showed incomplete eyelid opening and 1 male showed leaning position during the early administration period in the 300 mg/kg group.

FEMALES: At the top dose of 300 mg/kg, all females showed transient salivation, decreased locomotor activity, abdominal or lateral position and 10 females showed staggering gait largely throughout the administration period, and 7 females showed incomplete eyelid opening and 3 females showed leaning position from the start to the middle of the administration period. These changes had disappeared by the time of administration of the next day.

There were no abnormalities in general condition in males or females in the control group or 12 mg/kg group or in females in the
60 mg/kg group.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): no treatment related effects observed

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): no treatment related effects observed

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): no treatment related effects observed

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): no treatment related effects observed

ORGAN WEIGHTS (PARENTAL ANIMALS): Comparison between the 300 mg/kg group and control group showed a small but significant increase
(p<0.05) in the liver weight body weight ratio. In the other organs, there were no significant differences in organ weight between the control group and any test article administration group.

GROSS PATHOLOGY (PARENTAL ANIMALS): no treatment related effects observed

HISTOPATHOLOGY (PARENTAL ANIMALS):
Males - Minimal centrilobular hypertrophy of hepatocytes was observed in the 300 mg/kg group and its incidence was significantly (p<0.01) higher than in the control group.

OTHER FINDINGS (PARENTAL ANIMALS)

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Remarks:
Reproductive effects
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: TOP DOSE tested: no effects from administration of the test article in any parameter measured.
Dose descriptor:
NOEL
Remarks:
General toxicity
Effect level:
12 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No effects seen at this dose
Dose descriptor:
NOEL
Remarks:
General toxicity
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No effects seen at this dose
Dose descriptor:
LOEL
Remarks:
General toxicity
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: 60 mg/kg - A decrease in locomotor activity was observed at this dose during the initial administration period in a small number of males. 300 mg/kg - Transient salivation, decreased activity and incomplete eyelid opening.
Dose descriptor:
LOEL
Remarks:
General toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Transient salivation, decreased activity and incomplete eyelid opening, staggering gait

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

VIABILITY (OFFSPRING): no treatment related effects observed

CLINICAL SIGNS (OFFSPRING): no treatment related effects observed

BODY WEIGHT (OFFSPRING): no treatment related effects observed

Effect levels (F1)

Dose descriptor:
NOEL
Remarks:
Pups
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were seen in pups

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
There were no adverse reproductive effects reported at any dose.
There were no effects to body weight, heamatology parametersl, blood chemistry, organ pathologies.
Clinical signs of transient salivation, decreased locomotor activity and staggering gait were observed at the top dose of 300 mg/kg (females) and at 60 mg/kg (males).
There were no adverse observations in the pups.
The NOEL for general toxicity is based on clinical signs only, as observed in parent animals.
the NOEL for reproductive is set at the top dose tested but could be higher.
Executive summary:

In a study report provided by the Japanese Ministry of Health, a combined repeat dose and reproductivescreening study on o-sec butyl phenol (OSBP) is described, as similar to the principles of an OECD guideline 422 study. Crj:CD(SD) rats (n=13 males and n=13 females per dose group) were administered with OSBP in corn oil, by oral gavage, at doses of 300, 60, 12 and 0 mg/kg. There were no changes in parent animals that were thought to be caused by administration of the test article in the results of body weight measurement, food consumption measurement, hematological examination, blood chemistry examination, urinalysis, necropsy or histopathological examination or reproductive effects. There were no adverse observations in pups.

Clinical observations that were thought to be caused by administration of the test article were observed in both males and females in the 300 mg/kg groups. These effects were transient salivation and decreased locomotor activity and (in females) staggering gait. Some clinical signs of transient salivation and decreased acitivity were observed in some males in the 60 mg/kg group. No adverse clinical effects were seen at the 12 mg/kg dose.

It is considered that the no observed effect level (NOEL) for reproductive effects of o-sec-butylphenol is >300 mg/kg/day (ie greater than the top dose tested) for both males and females under the conditions of this study. This substance is not a reproductive toxicant.

It is considered that the no observed effect level (NOEL) for general toxicity of o-sec-butylphenol is 12 mg/kg/day (males) and 60 mg/kg day (females) under the conditions of this study.

It is expected that o-tert butyl phenol with behave similarly to o-sec butyl phenol, and that OTBP would not be a reproductive toxicant.