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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
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

Constituent 1
Chemical structure
Reference substance name:
Bumetrizole
EC Number:
223-445-4
EC Name:
Bumetrizole
Cas Number:
3896-11-5
Molecular formula:
C17 H18 Cl N3 O
IUPAC Name:
2-tert-butyl-6-(5-chloro-2H-1,2,3-benzotriazol-2-yl)-4-methylphenol
Test material form:
solid
Specific details on test material used for the study:
- Name of test material (as cited in study report): bumetrizole and 2-(2'-Hydroxy-3-tert -butyl-5’-methyphenyl) -5-chlorobenzotriazole
- Appearance/physical state: light yellow/powder
- Analytical purity: 99.9% w/w
- Lot/batch No.: 01721IW4
- Stability under test conditions: Stable
- Storage condition of test material: Stored sealed in a cabinet at 20.0 - 25.3° C
- Supplier: Ciba Specialty Chemicals, Osaka, Japan

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles river Laboratories Japan (Hino Breeding Center)
- Age at study initiation/start of treatment: 7 weeks/10 weeks
- Weight at study initiation: Test group males 230 - 249 g; Test group females 176 - 202 g; Recovery group females 179 - 198 g
- Fasting period before study: Day before administration
- Housing: Individually
- Diet: CRF-1 (Oriental Yeast Co.), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 5 days quarantine, 16 days acclimation (17 days for recovery group)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 25
- Humidity (%): 40 - 56 (on 5 occasions, deviations below 40% humdity were recorded. As humidity deviation was slight (36 - 39.8%) and humidity for other period was within allowable limits, it is considered that this deviations had no influence to the test results.
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From May 13th, 2005 to July 13-15th (main group) and July 27-28th, 2005 (recovery group)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5 % w/v methyl cellulose solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
After the necessary quantity of bumetrizole was weighed (Electronic balance: AT250 and PM2500, METTLER-TOLEDO), the specified concentration was prepared by suspension in 0.5% (w/v) methylcellulose solution using a conditioning mixer (AR-250, THINKY). Besides, for preparation, the test material was not adjusted according to the content, and administration dose was indicated as bulk powder weight.

VEHICLE
- Lot/batch no: Lot No. 110654
- Concentration in vehicle: 1.25, 5 and 20%
- Amount of vehicle: 5 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): After day 18 of pregnancy, females were housed individually in a plastic cage until day 4 of lactation, then in a stainless steel cage.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of test material in the dosing material used for administration was determined by HPLC on the first and last days of administration.
Duration of treatment / exposure:
Males: Total of 42 days (from 14 days before mating to 28 days after mating)
Females: Total of 44 - 56 days (from 14 days before mating, during pregnancy and up to lactation day 6)
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Test group (male): 12/dose (6 were assigned to the recovery group)
Test group (female): 12/dose
Recovery group females: 6/group at control, 250 or 1000 mg/kg bw/day.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses selected on the basis of a preliminary repeated dose study (no adverse influence on mortality, clinical signs, food consumption or necropsy findings at 1000 mg/kg bw/day).
- Rationale for animal assignment: random
- Post-exposure recovery period: Males - half the animals in each dose group were allowed a recovery period of 14 days after the administration period; Recovery group females - 14 day recovery period after a 42 day administration period.

Examinations

Parental animals: Observations and examinations:
Refer to Section 7.5.1 Repeated dose toxicity: oral for details
Oestrous cyclicity (parental animals):
The estrus cycle of the test group females was observed once a day from the start of the administration period until the day before copulation verification. When the estrous cycle was observed for two consecutive days, it was counted as one.
Sperm parameters (parental animals):
Parameters examined in male parental generations: testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
- number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain (birthday and on day 4 after birth)

GROSS EXAMINATION OF DEAD PUPS: Yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
Refer to Section 7.5.1 Repeated dose toxicity: oral for details
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.
Statistics:
The test of significance was performed between control groups and each test group and recorded separately at p<0.05 and p<0.01. Unimpregnated females after copulation were excluded from the counting of general signs, body weight, food consumption and FOB. For body weights of pups, the average of one litter defined as one unit, the average and total value of one litter were calculated.

Averages and standard deviations in each group were calculated for body weights (parent animals, pups), food consumption, frequency of rearing and grooming for Functional Observational Battery (FOB), grip strength, spontaneous motor activity, urinary volume, urinary specific gravity, records of hematological/blood chemical examination, absolute and relative weight of organs, estrus frequency, copulation required days, pregnancy period, number of corpora lutea, number of implantations, implantation index, total number of pups born, number of live pups, number of stillborns, delivery index, rate of pups delivery, live birth index, live pups on lactation day 4, viability index, occurrence rate of abnormal appearance. Subsequently, the test of distribution uniformity by Bartlett's test performed, and the Dunett Test was done when the distribution is uniform. In contrast, the Dunett Test using rank was done when the homoscedastic was not recognized.

For the Functional Observational Battery (FOB)(however, excluded frequency of rearing and grooming) and sensory response, the average and range in each group were calculated. Then, the Dunett Test using rank was done.

The copulation index, fertility index, and gestation index were calculated by chi-square test.
Reproductive indices:
Copulation required days
Copulation index [(number of pairs with successful copulation/number of pairs mated) x 100]
Fertility index [(number of pregnant females/number of pairs with successful copulation) x 100]
Gestation index [(number of females with live pups/number of pregnant females) x 100]
Pregnancy period [delivery date (lactation day 0 – copulation verification day)]
Number of corpora lutea
Number of implantations
Implantation index [(number of implantations/number of corpora lutea) x 100]
Offspring viability indices:
Total number of pups born (number of live pups + number of stillborns)
Number of live pups
Number of stillborns
Delivery index [(number of pups born /number of implantations) x 100]
Rate of pups delivery [(number of live pups on day 0/number of implantations) x 100]
Live birth index [(number of live pups on day 0/ number of pups born) x 100]
Survival pups on lactation day 4
Viability index [(number of live pups on lactation day 4/number of live pups on lactation day 0) × 100]
Occurrence rate of abnormal appearance [(number of pups with abnormal appearance/number of neonates) x 100]

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

Refer to Section 7.5.1 Repeated dose toxicity: oral for details of general toxicity effects

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No significant difference was observed in the estrus frequency during the administration period (14 days) before the start of mating in administered groups compared to controls.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Copulation required days, copulation index, number of pregnant females, fertility index: All animals copulated in every group. The copulation index was 100 %. No significant difference was observed in the copulation required days between administered groups and controls. There was one case of an unimpregnated female in both the controls and the 100 mg/kg bw/day group. No significant difference was observed in fertility index between the administered groups and the controls.

Pregnancy period: No significant differences between administered groups and controls.

Number of corpora lutea, number of implantations and implantation index: No significant difference was observed in the number of corpora lutea, number of implantations and implantation index in 1000 and 250 mg/kg bw/day groups compared to the control group. Significant high value of implantation index was seen in 62.5 mg/kg bw/day group compared to the control group, however because this effect was not dose related, it was considered to not be due to administration of the test material.

Gestation index, delivery conditions, nursing conditions: Gestation index was 100 % in all groups. No abnormality was observed in the delivery conditions and in the lactation conditions in any group.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Highest dose tested. No effects seen in the reprodcutive parameters up to the highest dose tested.

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
There was no siginificant difference in viability index between any dose group and the controls.

CLINICAL SIGNS (OFFSPRING)
There was no significant difference in clinical signs between any dose group and controls. There was one acaudal pup in the 1000 mg/kg bw/day group, but this was considered to be a natural occurrence and not related to administration of the test material.

BODY WEIGHT (OFFSPRING)
No significant difference was observed in dose groups compared to controls in the average body weight by sex on lactation day 0 or 4, the average body weights per litter on lactation day 0 or 4, and the total body weights per litter on lactation day 0 or 4.

SEXUAL MATURATION (OFFSPRING)
No data

ORGAN WEIGHTS (OFFSPRING)
No data

GROSS PATHOLOGY (OFFSPRING)
In pups born dead, no significant difference was observed in the 1000 and 62.6 mg/kg bw/day groups as well as in the controls. Though an absence of intraabdominal organ was observed in one case in the 250 mg/kg bw/day group, as it was not relevant to the administration dose, it was not considered to be due to administration of the test material.

In live pups examined at the end of the study, no abnormalities were observed in any group.

HISTOPATHOLOGY (OFFSPRING)
No data

OTHER FINDINGS (OFFSPRING)
Total number of pups born, number of dead pups, number of live pups on day 0, sex ratio on day 0, delivery index, rate of pups delivery, live birth index: No significant difference between controls and 1000 or 62.5 mg/kg bw/day dose groups in any of these parameters. Delivery index and rate of pups delivery were significantly lower than controls at 250 mg/kg bw/day, however as the effect was not dose related, it was considered to not be due to administration of the test material.

Sex ratio on day 4: No significant difference between controls and any dose group.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Highest dose tested. No effects seen in developmental parameters examined at the highest dose tested

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Please refer to Section 7.5.1 for tables related to repeat dose toxicity in parental animals

2)  Reproductive Developmental Toxicity

Administration dose

Control group

Low Dose Group

Medium Dose group

High Dose Group

(mg/kg)

0

62.5

250

1000

Parental animal

Estrus frequency

3.1

3.5

3.4

3.3

Copulation rate (%)

100.0

100.0

100.0

100.0

Conception rate (%)

91.7

100.0

100.0

91.7

Number of corpus luteum of   

pregnancy

16.4

15.6

17.3

16.5

Number of implantations

14.4

15.0

15.7

15.4

Implantation rate (%)

88.0

96.3↑↑a)

90.3

93.7

Number of pregnant dams

11

12

12

11

Number of dams with live births

11

12

12

11

Birth rate (%)

100.0

100.0

100.0

100.0

Pregnancy period

22.3

22.5

22.5

22.3

Neonate 

Number of total births

13.8

13.8

14.0

14.7

Delivery rate (%)

95.2

89.7

85.5↓↓a)

93.5

Rate of pups delivery (%)

95.3

89.7

85.5↓↓a)

93.5

Number of survival pups   (lactation day 0 )

13.6

13.4

13.4

14.4

Survival birth rate (%)

98.9

97.0

95.8

97.5

Sex ratio (♂♀, lactation day 0)

1.06

1.34

2.24

1.21

Number of survival pups  (lactation day 4)

13.5

13.1

13.3

14.2

Survival rate (%)

98.6

97.5

98.9

98.7

Sex ratio(♂♀, lactation day 4)

1.06

1.43

2.25

1.21

Body weights (lactation day 0)

-/-

-/-

-/-

Body weights (lactation day 4)

-/-

-/-

-/-

Abnormality of appearance (N)

-

-

-

Acaudal (1)b)

Necropsy findings (N)

-

-

Interperitoneal fracture (1)b)

-

NOAEL

Parental animal: = 1000 mg/kg/day for both male and female rats.

Pups: = 1000 mg/kg/day.

Change for estimated grounds of NOAEL

Because no effect was observed in any parameter at 1000 mg/kg administration for male rats.

Because no effect was observed in any parameter at 1000 mg/kg administration for female rats.

Because no effect was observed in any parameter at 1000 mg/kg administration for pups.

NOAEL: No Observed Adverse Effect Level

-/- :/

Copulation rate (%) = (Number of copulation verified animals/Number of hybridization animals) x100

Conception rate (%) = (Number of pregnant animals/Number of copulation verified animals) x100

Implantation Rate (%) = (Number of implantations/Number of corpus luteum of pregnancy) x100

Birth rate (%) = (Number of dams with live births/Number of pregnant dams) x100

Pregnancy period = Nursing day 0 [Delivery verified day] (date) - Pregnancy day 0 (date)

Delivery rate (%) = (Number of total births/Number of implantations) x100

Pups birth rate (%) = (Number of neonatal pups on lactation 0 day/Number of implantations) x100

Survival birth rate (%) = (Number of survivals on lactation day 0/ Number of total births) x100

Survival rate (%) = (Number of survivals on lactation day 4/Number of survivals on lactation day 0) x 100

-: No change, or no significant difference with control group.

↑↑ ↓↓: 5% significant difference

a): Judged as Not Toxicological Effect.

b): Judged as contingent case.

Applicant's summary and conclusion