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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
Study period:
2006-2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Japan, Inc. (Atsugi Breeding Center)
- Age at study initiation: 10 -11 weeks
- Weight at study initiation: 370 to 422 g in males, 225 to 263 g in females
- Fasting period before study: none
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2°C
- Humidity (%): 55 ± 15%
- Air changes (per hr): 15 to 17
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing samples were prepared once a week based on the results of the 8-day stability study conducted at the Japan Bioassay Research Center and stored in a dark place at room temperature and were used within 7 days after preparation.


VEHICLE
- Justification for use and choice of vehicle (if other than water): The substance is not soluble in water, but suspendable in olive oil
- Concentration in vehicle: adjusted to volume of 5 ml/kg bw
- Amount of vehicle (if gavage): 5 ml/kg bw
Details on mating procedure:
The males and females of the same group were mated by cohabitation in 1:1 sex ratio every day for 14 days at the longest starting from Day 15 of treatment. Copulation was confirmed by the presence of vaginal plug in the vagina or presence of sperms in the vaginal smear and the day when these were found was defined as Day 0 of gestation. The females with establishment of copulation were promptly separated from males and housed individually.
The time to copulation (number of days taken from the start of mating to establishment of copulation) and the copulation rate [(number of pairs that established copulation / number of pairs mated) x 100] were calculated based on the results of mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance was checked by comparing the chromatograms determined using high-performance liquid chromatography (Hewlett Packard 1090) before and after use. The concentration and uniformity of the test substance in the dosing samples were checked by the measurement using high-performance liquid chromatography (Hewlett Packard 1090) at the time of the initial preparation.
Duration of treatment / exposure:
Males, 42 days
Females, from 14 days before mating to day 4 of lactation
Females (satellite), 42 days

Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Males, 12 (5 for recovery)
Females, 12; satellite females, 5

Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose selection rationale: The doses in this study were determined based on the results of the preliminary study. The dose levels in the preliminary study were determined by setting 1000 mg/kg as the highest dose level as specified in the OECD Guideline for Testing of Chemicals, followed by 300 mg/kg, 100 mg/kg, and 30 mg/kg.

Five each of male and female Crl:CD(SD) rats aged 10 weeks per group received 2-(2’-hydroxy-5’-methylphenyl) benzotriazole orally for 14 consecutive days and were autopsied on the day after completion of administration. As a result, the test substance had no effect on the body weight or general health condition of the males and females in all treatment groups. Both the males and females given 100 mg/kg and higher showed an increase in liver weight and the females also showed an increase in total cholesterol. Increased phospholipid was also noted in the females given 300 mg/kg and higher. Based on these results, 300 mg/kg was selected as the high dose level in this study followed by 100 mg/kg as the intermediate dose level and 30 mg/kg as the low dose level.
Positive control:
not required

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week
In the observation of females in Week 6 of treatment, however, only the satellite animals were observed to reduce the burden on the animals due to parturition and nursing.
The animals were observed for contact reaction, vocality and easiness of taking out when they were taken out from cages, easiness for handling, body temperature, fur condition, skin color, eye condition and salivation after taking out from cages, posture, activity, alert/searching behavior, gait condition, stereotypical behavior, respiration, and tremor/spasm as well as numbers of events of urination and defecation, grooming and face washing per minute on the work table.

BODY WEIGHT: Yes
- Time schedule for examinations: The males were weighed on Days 1, 8, 15, 22, 29, 36 and 42 of treatment, and the recovery animals were weighed on Days 1, 8 and 14 of recovery.
All the females were weighed on Days 1, 8 and 15 of treatment before mating. After the start of mating, they were weighed on Days 0, 7, 14 and 20 of gestation, on the parturition day and Day 4 after parturition (day 0 and 4 of nursing). The satellite females were weighed on Days 1, 8, 15, 22, 29 and 36 of treatment and Days 1, 8 and 14 of recovery.
On the autopsy day, the body weight was measured in all animals after fasting.

Food consumption: Yes
For the males, food consumption was measured between Days 1 and 8, 8 and 15, 29 and 36, and 36 and 42 in the treatment period and between Days 1 and 8, and 8 and 14 in the recovery period.
For the females, food consumption was measured for all animals before mating between Days 1 and 8, and 8 and 15. After the start of mating, food consumption was measured between Days 0 and 7, 7 and 14, 14 and 20 of gestation and between Days 0 and 4 of nursing. For the satellite females, food consumption was measured between Days 1 and 8, 8 and 15, 29 and 36, and 36 and 43 of treatment and between Days 1 and 8, and 8 and 14 of recovery.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at autopsy
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No data
- How many animals: all
- Parameters checked: Red blood cell count, platelet count, reticulocyte ratio, white blood cell count, mean corpuscular volume (the light scattering method, hereinbefore), hemoglobin concentration (cyanmethemoglobin method), hematocrit, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration (by calculation, hereinbefore) were determined by the general hematology system (ADVIA 120: Bayer), differential leukocyte classification (by Wright’s stain) using the automatic blood cell analyzer (MICROXHEG-120NA: Omron), and prothrombin time (Quick one-step method) and activated partial thromboplastin time (ellagic acid activation method) using the full automatic blood coagulation tester (Sysmex CA-5000: Sysmex).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at autopsy
- How many animals: all
- Parameters checked: for total protein (biuret method), albumin (BCG method), A/G ratio (calculation), total bilirubin (azobilirubin method), glucose (GlcK・G-6-PDH method), total cholesterol (CE・COD・POD method), triglyceride (MGLP・GK・GPO・POD method), phospholipid (PLD・ChOD・POD method), AST, ALT, γ-GTP, CK (JSCC method, hereinbefore), LDH (SFBC method), ALP (GSCC method), urea nitrogen (urease GLDH method), creatinine (Jaffé method), sodium, potassium, chlorine (ion-selective electrode method, hereinbefore), calcium (OCPC method) and inorganic phosphorus (PNP・XOD・POD method) using an automatic analyzer (Hitachi 7080: Hitachi, Ltd.).

URINALYSIS: Yes
Fresh urine was collected before administration from all males and satellite females in Week 6 and pH, protein, glucose, ketone bodies, bilirubin, occult blood and urobilinogen were tested using a urine test paper (Multistix, Bayer).
Urinalysis was also performed for all male and female recovery animals in Week 2 of recovery as changes suspected of the effect of the test substance administration were found in the males given 100 mg/kg and higher.


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: All the males were tested in Week 6 of treatment. As for the females, 5 animals that underwent parturition on the same day or closer days were selected in each group and the tests were performed on the day before autopsy in the combined repeated-dose oral toxicity and reproductive and developmental toxicity study (day 4 after parturition).
- Battery of functions tested: sensory activity, grip strength and motor activity
In the reactivity test, vision, hearing, pain sensation, pupil reflex and aerial righting reflex were examined.
Grip strength was measured using a grip strength meter (MK-380CM, Muromachi Kikai Co., Ltd.). Both the forelimbs and hindlimbs were measured twice and the means were defined as the grip strengths of the individuals.
Locomotor activity was measured for 60 minutes per animal using a locomotor measuring instrument (SCANET MV-10, Melquest).
Oestrous cyclicity (parental animals):
Vaginal smears were collected from all the females (excluding satellite females) every morning from the start of treatment to the first day of mating (day 15 of treatment), stained with Giemsa stain, and observed for sex cycle under a light microscope.
Sperm parameters (parental animals):
none
Litter observations:
The body weight of pups was measured per dam (litter) on Days 0 and 4 of nursing by the units of males and females and the mean per pup was calculated.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

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):
The ovaries were excised from copulated females at the time of autopsy and the number of corpus luteum verum was counted under a stereomicroscope. In addition, the uterus was excised, stained with 10% ammonium sulfate solution, and implantation sites were counted on the following day.
The implantation rate [(number of implantation sites / number of corpus luteum verum) x 100] was calculated based on these results.


GROSS PATHOLOGY: Yes
Autopsy was performed for the males excluding recovery animals on the day after Day 42 of treatment and for the recovery animals on the day after Day 14 of recovery. The females that underwent parturition were autopsied on Day 5 after parturition and those that did not give parturition were autopsied on the day corresponding to Day 26 of gestation to check presence or absence of conception. The satellite females were autopsied on the day after Day 14 of recovery.

HISTOPATHOLOGY: Yes
The testes, epididymides, seminal vesicles/coagulating gland, prostate (ventral lobe) and ovaries of all animals and trachea, lungs, bone marrow (femur), lymph nodes (axillary, peritoneal, etc.), thymus, spleen, heart, stomach, small intestines (including duodenum), large intestines, liver, kidneys, bladder, pituitary body, thyroid gland, parathyroid gland, adrenal glands, uterus (only in the satellite females), vagina, mammary glands, brain, spinal cord, peripheral nerve (sciatic nerve), muscle, and bone (femur) of 5 animals in each group (in the ascending order of animal numbers, excluding infertile females) and recovery animals were excised, embedded in paraffin, sectioned, and stained in hematoxylin and eosin, and observed under a light microscope.
Postmortem examinations (offspring):
GROSS NECROPSY
All the pups that survived on Day 4 of nursing were laparotomied under ether anesthesia, exsanguinated by cutting the abdominal aorta, and autopsied. The pups that died in the course were also autopsied.

Statistics:
The chi-square test was performed for the copulation rate, conception rate, birth rate, urinalysis and histopathology results between the control group and each of the treated groups.
For the numeric data obtained in other tests, equal variation was examined preliminary by the Bartlett method using the control group as the basis. The one-way analysis of variance was performed if equal variation was shown and the Dunnett’s multiple comparison was performed for testing the means when a significant difference was detected between the groups. If the variation was not equal, the measured values were ranked across the groups and the Kruskal-Wallis rank test was performed followed by the Dunnett-type multiple comparison if a significant difference was detected between the groups. In the statistical analysis of the numeric data in the recovery animals, the F-test was performed first followed by the Student’s t-test if there was no difference in distribution and by the Aspin-Welch’s t-test if the distribution showed a difference.
The significance level was set at 5% and 1% on both sides and 5% significance level was set as the judgment criteria for the effect of the test substance
Reproductive indices:
Copulation index = (No. of copulated pairs / No. of mated pairs) × 100.
Fertility index = (No. of pregnant females / No. of copulated pairs) × 100.
Gestation index = (No. of pregnant females with live pups / No. of pregnant females) × 100.
Implantation index = (No. of implantations / No. of corpora lutea) × 100.
Delivery index = (No. of pups born / No. of implantations) × 100.
Offspring viability indices:
Viability index = (No. of live pups on day 4 / No. of live pups on day 0) × 100.
Birth index = (No. of live pups on day 0 / No. of implantations) × 100.
Live birth index = (No. of live pups on day 0 / No. of pups born) × 100.
Sex ratio on day 0 = No. of male live pups on day 0 / No. of live pups on day 0.
Sex ratio on day 4 = No. of male live pups on day 4 / No. of live pups on day 4.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
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:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

A NOAEL of 30 mg/kg bw was defined based on the increase in relative liver weight for males. In the histological examination, liver and kidney lesions occurred in the dosed group of both sexes. The hepatic lesions were characterized by increased incidences of hypertrophy of the centrilobular hepatocytes in the males given 300 mg/kg and in the females given 100 mg/kg and above. Theses changes were not observed at the end of the recovery period. The kidney lesions exhibited a gender difference, as characterized by increased incidences of an eosinophilic body of proximal tubules in the males given 300 mg/kg, and hydropic change and regeneration of proximal tubules in the females given 100 mg/kg and above. The eosinophilic body of proximal tubules remained affected in the recovery group of males given 300 mg/kg, whereas female kidney lesions were not observed in the satellite females given 300 mg/kg.



No adverse effects on reproductive performance was observed for parental animals at the highest tested dose of 300 mg/kg bw.

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
>= 30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: effects on liver at clincial chemistry observed in dose-range finding study at 1000 mg/kg bw

Target system / organ toxicity (P0)

Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
kidney
liver
Treatment related:
yes
Dose response relationship:
yes

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:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

No effect of the compound was observed on the reproductive performances, such as the estrous cycle, copulation, fertility, delivery and lactation. No effect of the compound was observed on the implantation, the number of pups, sex ratio of pups, viability of the pups during 4 days of lactation and weights of the pups. No external abnormalities or macroscopic findings were detected in any pup.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: as far as F1 can be assessed in a screening study.

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Table 1: Estrous cycle and reproductive performance

Dose (mg/kg) 0 30 100 300
Estrous cycle        
No. of animals examined 12 12 12 12
Type of cycle
4-day 10 9 9 12
5-day 2 2 1 0
Others
Length (day)a)
0
4.2±0.4
1
4.2±0.4
2
4.2±0.4
0
4.0±0.0
Reproductive performance
No. of mated pairs 12 12 12 12
No. of copulated pairs 12 12 12 12
Copulation index (%) 100 100 100 100
No. of pregnant females 12 12 12 12
Fertility index (%)
Pairing days until copulationa)
100
2.3±1.1
100
4.0±3.1
100
2.3±1.2
100
2.4±1.2
Parturition and lactation
Non-remarkable 12 12 12 11
Abnormal lactation 0 0 0 1

a) Data represent mean ± S.D.

Copulation index = (No. of copulated pairs / No. of mated pairs) × 100.

Fertility index = (No. of pregnant females / No. of copulated pairs) × 100.

Table 2 Summary of development of pups from dams treated orally with 2-(2'-hydroxy-5'-methylphenyl)benzotriazole in the combined repeated dose and reproductive/developmental toxicity screening test

Dose (mg/kg) 0   30   100   300  
No. of pregnant females 12   12   12   12
No. of pregnant females with live pups 12 12 12 12
Gestation index (%) 100 100 100 100
Gestation length (days) 21.9±0.5 (12) 21.9±0.3 (12) 22.1±0.3 (12) 21.9±0.3 (12)
No. of corpora lutea 16.0±1.3 (12) 16.2±1.2 (12) 16.0±2.0 (12) 16.3±1.7 (12)
No. of implantations 15.3±1.5 (12) 14.7±1.8 (12) 15.0±1.3 (12) 15.4±1.4 (12)
Implantation index (%) 95.2±2.9 (12) 90.8±9.5 (12) 94.5±9.3 (12) 95.1±5.5 (12)
Day 0 of lactation           
No. of pups born 14.4±1.4 (12) 13.6±2.0 (12) 13.5±2.8 (12) 14.0±1.4 (12)
Delivery index (%) 94.7±6.5 (12) 92.8±9.5 (12) 90.0±17.0 (12) 91.0±6.8 (12)
No. of live pups 14.3±1.5 (12) 13.5±2.0 (12) 13.4±3.1 (12) 13.8±1.5 (12)
Birth index (%) 93.6±8.0 (12) 92.2±9.3 (12) 89.4±18.8 (12) 89.5±9.0 (12)
Live birth index (%) 98.7±3.0 (12) 99.4±2.0 (12) 98.6±4.8 (12) 98.2±3.3 (12)
Pups weight (g)
Male 6.6±0.4 (12) 6.5±0.5 (12) 6.8±0.5 (12) 6.8±0.5 (12)
Female 6.3±0.5 (12) 6.1±0.4 (12) 6.5±0.4 (12) 6.4±0.4 (12)
Sex ratio on day 0 0.56±0.12 (12) 0.48±0.10 (12) 0.50±0.16 (12) 0.50±0.10 (12)
Day 4 of lactation             
No. of live pups 14.2±1.6 (12) 13.3±2.2 (12) 13.2±2.9 (12) 12.6±3.9 (12)
Viability index (%) 99.4±2.2 (12) 98.0±4.9 (12) 98.4±3.9 (12) 91.7±26.7 (12)
Pups weight (g) Male 10.3±1.0 (12) 10.5±1.2 (12) 10.8±1.6 (12) 10.5±0.8 (11)
Female 9.9±1.1 (12) 9.9±1.2 (12) 10.4±1.5 (12) 9.5±1.8 (12)
Sex ratio on day 4 0.56±0.12 (12) 0.48±0.11 (12) 0.50±0.16 (12) 0.46±0.18 (12)

Data represent mean ± S.D.

Parentheses represent the number of litters examined.

Gestation index = (No. of pregnant females with live pups / No. of pregnant females) × 100.

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

Delivery index = (No. of pups born / No. of implantations) × 100.

Birth index = (No. of live pups on day 0 / No. of implantations) × 100.

Live birth index = (No. of live pups on day 0 / No. of pups born) × 100.

Sex ratio on day 0 = No. of male live pups on day 0 / No. of live pups on day 0.

Viability index = (No. of live pups on day 4 / No. of live pups on day 0) × 100.

Sex ratio on day 4 = No. of male live pups on day 4 / No. of live pups on day 4.

Applicant's summary and conclusion

Conclusions:
No indication of reproductive toxicity by the oral route was identified in this screening study.
Executive summary:

No effect of the compound was observed on the reproductive performances, such as the estrous cycle, copulation, fertility, delivery and lactation. No effect of the compound was observed on the implantation, the number of pups, sex ratio of pups, viability of the pups during 4 days of lactation and weights of the pups. No external abnormalities or macroscopic findings were detected in any pup.