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Toxicity to reproduction

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Endpoint:
toxicity to reproduction
Remarks:
other: Combined repeated dose and reproduction / developmental toxicity study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD TG 422)
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
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): p-anisaldehyde(4-methoxybenzaldehyde)
- Physical state: clear, colorless liquid
- Analytical purity: 99.9% (GC)
- Lot/batch No.: W81015 (Nippon Shokubai Co., Ltd.)
- Stability under test conditions: the remainder of the test article used for the study was subjected to a second quality test (99.34%), and thus the stability of the test article during the study period was confirmed
- Storage condition of test material: kept in an airtight container and put in the test article storage room at room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Crj: CD (SD) IGS, SPF rats from the Tsukuba Breeding Center of Charles River Laboratories Japan, Inc.
- Age at study initiation: 7 weeks
- Weight at study initiation: 243.4 – 265.0 g at arrival, 297.7 – 427.7 g after acclimatization and 403.0 – 356.5 g after grouping for males; 171.7 – 199.7 g at arrival, 205.5 – 297.8 g after acclimatization and 254.9 – 218.6 g after grouping for females
- Housing: separately housed in metal wire-mesh flooring cage (220 x 270 x 190 mm)
- Diet: pellets (CE-2 manufactured by Clea Japan, Inc.); ad libitum
- Water: tap water (supplied by Hadano City Waterworks Bureau); ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24±1
- Humidity (%): 50-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

OTHER:
Each female animal on and after day 14 of gestation (the day when copulation was confirmed = day 0 of gestation) was housed in a plastic reproduction cage for rats (350 x 400 x 180 mm), to which paper pulp chips (ALPHA-dri manufactured by Kasho Company Limited) were supplied appropriately as bedding. Moreover, the temperature and humidity of the breeding room stood at 23.5-25.5 °C and 45-70 % respectively during the breeding period, and both of them did not deviate from the reference temperature and humidity over an hour.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article was weighed on a dose basis and dissolved in corn oil and administered within 8 days after the preparation.

VEHICLE
- Concentration in vehicle: 0.4, 2 and 10%, respectively in the low, mid and high dose groups
- Amount of vehicle (if gavage): 5 ml/kg bw
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
It was also confirmed in a previous acute oral study that the prepared test samples [0.40, 2.00, 10.0 % (w/v)] contained the test substance at the predetermined concentration, respectively. Each 2 ml of the prepared samples were volume-adjusted with n-propanol and measured by high performance liquid chromatography (HPLC; analytical column: Inertsil ODS-2 [4.6 mm i.d. x 150 mm, particle size 5 μm]; mobile phase: water/acetonitrile [60 : 40]). Their concentrations were obtained by using a calibration curve constructed from the measured values (MBA: 10-50 μg/ml).
Duration of treatment / exposure:
- Males: 2 weeks premating; further 2 weeks after the completion of mating period
- Females: 2 weeks premating, throughout mating periods up to 2 weeks, then throughout pregnancy up to day 4 of lactation after parturition (females copulated without parturition were dosed until the day corresponding to day 24 of gestation).
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (vehicle), 20, 100 and 500 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
13
Control animals:
yes, concurrent vehicle
Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
Mating:
Copulation and time to copulation was checked based on the presence or absence of vaginal plug and sperms in vaginal smear every morning.


Observation of parturition and lactation state:
For animals, of which the parturition state could be monitored, their conditions were observed.
As to animals of which the parturition state could not be directly observed, the presence or absence of parturition problems,
such as parturition difficulties, delays etc., was assessed and recorded based on
clinical signs after parturition.
Parturition was checked between 9 a.m. and 11 a.m. For animals which were confirmed to have completed parturition, that day was defined as parturition day. For animals which completed parturition after 11 a.m., the following day was regarded as parturition day.
For all cases that were confirmed to have completed parturition, the gestation length (number of days from day 0 of gestation to parturition day) was calculated, and also the birth rate was obtained.
After parturition, the lactation state was observed daily.



For other endpoints assessed, see Chapter 7.5.


Oestrous cyclicity (parental animals):
For all females the estrous cycles were observed based on the vaginal smears, in order to obtain the average number of estrous days and also estrous types.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups born (stillborn + liveborn), postnatal mortality, presence of gross anomalies/external malformations, pup weights

GROSS EXAMINATION OF DEAD PUPS: all cases were subjected to autopsy on day 4 of lactation
Postmortem examinations (parental animals):
SACRIFICE
Described in Chapter 7.5.

GROSS NECROPSY
Described in Chapter 7.5.
The number of corpora lutea of pregnancy (no data derived from non-pregnant animals) and number of implantation sites were counted, respectively in ovaries and uterus under a stereoscopic microscope.

HISTOPATHOLOGY / ORGAN WEIGHTS:
Described in Chapter 7.5.
Uterus and vagina for 8 females having failed to conceive or having total embryonic resorption in the high-dose group and seminal vesicle and prostate for male mating partners of such females were additionally paraffin sectioned in the usual manner and stained with hematoxylin and eosin, in order to conduct histopathological examination.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
Statistics:
Described in Chapter 7.5.
As to the copulation and conception rates, Fisher’s exact test was conducted (level of significance: 5 %).
Reproductive indices:
Copulation index (nr. animals copulated/nr. animals mated x 100)
Fertility index (nr. animals pregnant /nr. animals copulated x 100)
Implantation index (nr. implantation sites/ nr. corpora lutea)
Gestation index (nr. females with live pups/nr. pregnant females x 100)

Offspring viability indices:
Delivery index (nr. pups born / nr. implantation sites x 100)
Birth index (nr. live pups d0 / nr. implantation sites x 100)
Live birth index (nr. live pups / nr. pups born x 100)
Viability index (nr. live pups d4 / nr. live pups d0 x 100)
Sex ratio d0 (nr. male live pups d0 / nr. live pups d0 x 100)
Sex ratio d4 (nr. male live pups d4 / nr. live pups d4 x 100)

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Reported in Chapter 7.5

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Reported in Chapter 7.5

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No significant difference in animals with normal estrous cycles and mean cycle length was observed in all groups. Mean times of vaginal estrous during mating period were not significantly different between test groups.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
500 mg/kg bw/d:
Significant decrease of number of pregnant dams (6 vs 12 in control group) and respective fertility index (46.2% vs 92.3%). No test substance related effects observed at 100 mg/kg bw/d and 20 mg/kg bw/d

All animals copulated and the copulation index and time to copulation was not significantly different in the testing groups.
There were no statistically significant differences in the number of corpora lutea, number of implantation sites, implantation index, gestation index and gestation length between the control group and each test substance-treated group.
No abnormalities were observed in the parturition and lactation state in all test groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Reported in Chapter 7.5

GROSS PATHOLOGY (PARENTAL ANIMALS)
Reported in Chapter 7.5

HISTOPATHOLOGY (PARENTAL ANIMALS)
Reported in Chapter 7.5

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Remarks:
parental toxicity
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: liver effects at 500 mg/kg bw/d, irritative GI effects at 100 mg/kg bw/d
Dose descriptor:
NOEL
Remarks:
reproductive toxicity
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: nr. of pregnant dams

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
500 mg/kg bw/d:
The number of pups born per litter was significantly decreased (9.3 vs 14 in control group), the number of liveborn pups (d0 of lactation) per litter (10.6 vs 13.8 in control group) and the number of live pups (day 4 of lactation) per litter (10.2 vs. 14.8 in control group) was significantly decreased.
The respective delivery index was significantly decreased (66.5% vs 94.7% in control group), and the birth index showed a non-significant decrease (76% vs 85.3% in control group). No significant changes in the live birth index and the viability index has been observed in all test groups.
100 and 20 mg/kg bw/d:
No test substance changes observed.

OTHER FINDINGS (OFFSPRING):
There were no statistically significant differences in the sex ratio between the control group and each test substance-treated group.

BODY WEIGHT (OFFSPRING)
No test substance related changes in pup weights on day 0 and day4 of lactation was observed.

GROSS PATHOLOGY (OFFSPRING)
No external malformations were found in all pups born; and also there were no abnormalities in internal organs at autopsy on day 4 of lactation. In addition, no abnormalities were observed at autopsy of dead pups.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: nr of liveborns/ pups born at 500 mg/kg bw/d

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Summary of pregnancy data

Parameter

Vehicle

(0 mg/kg bw)

Low dose

(20 mg/kg bw)

Mid dose

(100 mg/kg bw)

High dose

(500 mg/kg bw)

Copulation index (%)

100

100.0

100.0

100.0

Fertility index (%)

92.3

92.3

92.3

46.2*

Number of corpora lutea

17.8

18.0

16.3

13.3

Number of implantation sites

14.8

15.6

13.0

12.0

Implantation index (%)

82.3

87.0

79.1

86.4

Number of pregnant dams

12

12

12

6*

Number of dams delivering liveborns

12

12

12

5

Gestation index (%)

100.0

100.0

100.0

83.3

Gestation length

22.8

22.3

22.8

23.0

*: Significantly increased relative to control (p<0.05)

 

Table 2: Summary of litter data

Parameter

Vehicle

(0 mg/kg bw)

Low dose

(20 mg/kg bw)

Mid dose

(100 mg/kg bw)

High dose

(500 mg/kg bw)

Number of Pups Born

14.0

14.3

11.8

9.3*

Delivery Index (%)

94.7

92.2

90.9

66.5*

Number of Liveborns (Day 0 of Lactation)

13.8

14.2

11.8

10.6*

Birth Index (%)

85.3

91.6

90.9

76.0#

Live Birth Index (%)

90.5

99.3

100.0

95.4

Sex Ratio (/)

0.7

1.0

1.0

1.0

Number of Live Pups (Day 4 of Lactation)

14.8

14.2

11.8

10.2*

Viability Index of Newborns on Day 4 after

Birth (%)

98.7

100.0

100.0

96.0

Pup Weight (g) (Day 0 of Lactation)            Males

Females

7.0

7.0

7.4

6.6

6.5

6.6

7.0

6.1

Pup Weight (g) (Day 4 of Lactation)             Males

Females

11.3

11.3

12.7

11.3

10.8

10.9

12.3

11.1

External Findings

0

0

0

0

Visceral Findings

0

0

0

0

*: Significantly increased relative to control; #: tendency for decrease compared to control.

Applicant's summary and conclusion