1,4-bis(methoxymethyl)benzene

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

A Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test was performed for test substance in compliance with OECD TG422.

Regarding the reproductive/developmental toxicity, the NOAEL and NOEL were both 200 mg/kg/day since decreases in conception index and fertility index in parental animals were observed in the 800 mg/kg group.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2010-10-01 to 2011-02-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Purity: 99.80%

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

It has been established as laboratory animals and are commonly used in general toxicity studies

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hino Breeding Center of Charles River Laboratories Japan, Inc.
- Age at study initiation: (P) 9 wks
- Weight at study initiation: (P) Males: 332.5-374.7 g; Females: 206.3-245.4 g
- Housing:
Before grouping, animals were housed in stainless steel cages with a wire mesh floor (W260 x D380 x H180 mm), 1-3 animals per cage. After grouping, both males and females were individually housed in wire mesh cages of W260 x D380 x H180 mm (males) and W165 x D300 x H150 mm (females). During mating, one male was allowed to cohabit with one female in his cage. Females who had copulation were housed individually or with their pups in polycarbonate flat-floor cages (W265 x D426 x H150 mm; PC Cages) with bedding material from Day 14 of gestation until the day before autopsy. Enrichment (Wood Bite) was placed in cages from Day 0 of gestation until the day before autopsy. During the recovery period, both males and females were individually housed in W260 x D380 x H180 mm wire mesh cages. Trays were replaced at the end of the quarantine period, at the time of grouping, twice a week after grouping, and when animals were transferred from the animal room to the autopsy room. Feeders, cages and racks were replaced at the time of grouping and at the end of the treatment period (only the recovery group). PC cages were replaced on Day 17 of gestation.
- Diet: pellet food, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 10-day

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25ºC
- Humidity (%): 40-70%
- Air changes (per hr): 10-15 times/hour
- Photoperiod (hrs dark / hrs light): a light/dark cycle of 12 hours (light on 7:00 and off at 19:00)

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was precisely weighed and dissolved by adding olive oil to prepare a 20.0 w/v% solution of the test substance (dosing solution). Aliquots of the 20.0 w/v% solution of the test substance were diluted with olive oil to prepare 5.00 and 1.25 w/v% solutions of the test substance.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 20.0, 5.00 and 1.25 w/v%
- Amount of vehicle (if gavage): 4 mL/kg
- Lot/batch no. (if required): 240621

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days at the maximum
- Proof of pregnancy: vaginal plugs or sperms in vaginal smears presence referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: housed individually or with their pups in polycarbonate flat-floor cages (W265 x D426 x H150 mm; PC Cages) with bedding material (Sunflake) from Day 14 of gestation until the day before autopsy. Enrichment (Wood Bite) was placed in cages from Day 0 of gestation until the day before autopsy.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Solutions of the test substance were sampled (n=1) from the mid layer shortly after preparation. After pre-treatment, concentrations of the test substance were determined once by HPLC (n=1). The relative concentration of the test substance was 100±10% of the nominal concentration at all three concentrations (102%, 103% and 99.2%, respectively, at 20.0 w/v%, 5.00 w/v% and 1.25 w/v%). These findings indicated that all three dosing solutions were appropriately prepared for use in the study and that the preparation method was acceptable. Therefore, concentrations of dosing solutions prepared the next and subsequent time were not determined.

Duration of treatment / exposure:

The treatment period was 42 days for males from the main study groups and recovery group and females from the recovery group and from the start of treatment until Day 4 of parturition through the mating, gestation and nursing period (42-54 days) in females from the main study groups.

Frequency of treatment:

Once a day between 9:00 and 14:00 during the treatment period
Females were treated after confirming completion of parturition if they were having parturition or have just finished parturition in the designated time slot.

Details on study schedule:

- Age at mating of the mated animals in the study: 11 weeks

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Remarks:

Low dose

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Remarks:

Mid dose

Dose / conc.:

800 mg/kg bw/day (actual dose received)

Remarks:

High dose

No. of animals per sex per dose:

Male: 7 rats/group（control and high dose groups of main study) +5 rats/group (control and high dose groups of recovery); 12 rats/group (low and middle dose groups of main study).
Female: 12 rats/group (all groups of main study) + 5 rats/group (control and high dose groups of recovery).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Doses were set based on the results of a dose-setting study conducted for the combined multiple oral dose toxicity and reproduction toxicity study in rats. The test substance was orally administered at 0 (olive oil), 25, 250, 500 and 1,000 mg/kg/day for 14 days to 9-week-old Crl:CD(SD) rats, 3 males and 3 females per group, and animals were observed with respect to clinical signs and symptoms, weighed, and subjected to hematological and blood biochemical examinations, organ weighing, and autopsy. Testes were histopathologically examined in males. The test substance was found to affect mainly the testes and liver.
Thus, the test substance seemed to affect organs such as the testes and liver, but the effects on the liver were not thought to be significant toxic effects because changes such as enzyme deviations were not seen. Observation of clinical signs and symptoms showed changes which suggested effects on the nervous system such as staggering gait, but these were not thought to suggest any considerable toxic effect, either, because these were transient and were not associated with any significant changes in body weight. Toxic effects on the testes were considered to be strong at 1,000 mg/kg/day because these were moderate in two of the three animals and weak at 500 mg/kg/day because these were slight in one of the three animals. These findings suggested that a number of pregnant animals large enough to evaluate the effects on the next generation cannot be obtained due to toxic effects on the testes at 1,000 mg/kg/day and the test substance's toxicity profile cannot be appropriately evaluated due to weak toxicity at 500 mg/kg/day. Taking these findings and the scheduled treatment period in the combined study into account, 800 mg/kg/day, which was expected to give a number of pregnant animals large enough to evaluate effects on the next generation and make it possible to appropriately evaluate the test substance's toxicity profile, was thought to be appropriate as a high dose, followed by a mid dose of 200 mg/kg/day, which was expected to show slight toxic effects, and a low dose of 50 mg/kg/day, which was not expected to be toxic.

- Fasting period before blood sampling for clinical biochemistry:
Animals started being fasted on the final day of treatment in the main study groups and on the final day of the rest period in the recovery group. Animals were anesthetized with ether the following day when they were fasted for 16-20 hours.

Parental animals: Observations and examinations:

- Observation of clinical signs and symptoms:
Clinical signs and symptoms, including mortality, were observed twice a day before and during through after treatment during the treatment period and once a day during the recovery period. Parturition performance and nursing activity were also observed in dams in which gestation was confirmed. Animals were also observed shortly before their transfer from the animal room on the day of autopsy.
- Detailed observation of clinical signs and symptoms:
Detailed observation of clinical signs and symptoms was conducted once before starting treatment and once a week during the treatment period and the recovery period. However, neither males nor females were observed during the mating period. Females from the main study groups were not observed in Week 6 of treatment and were observed instead before starting fasting on Day 4 of nursing after parturition, but this observation was not conducted in any of the 12 females from the high dose group since parturition was not confirmed in them.
- Function tests:
Males with the five smallest numbers from all groups including the control group were tested on Days 36 and 37 of treatment (Week 6 of treatment). All females from the recovery group were also tested on Days 36 and 37 of treatment (Week 6 of treatment)
- Body weight:
Animals were weighed on Days mentioned below:
∙ Day of grouping
∙ During the treatment period: Males: Days 1 (1st day of treatment), 3, 7, 14, 21, 28, 35 and 42; Females: Same as males in the recovery group. In the main study groups, same as males until confirmation of copulation; Days 0, 7, 14, 17 and 20 after confirmation of copulation; and Days 0 and 4 of nursing after parturition. Non-pregnant females were weighed on Days 21 and 28 as well.
∙ During the recovery period: 1, 3, 7 and 14 days after the final administration
∙ Day animals were removed from the animal room

- Food consumption:
Food consumption was measured on the days mentioned below:
∙ During the treatment period: Males: Days 1, 3, 7, 14 and 42; Females: Days 1, 3, 7, 14, 21, 28, 35 and 42 in the recovery group. In the main study groups, same as males until confirmation of copulation; Days 0, 7, 14, 17 and 20 after confirmation of copulation; and Days 0 and 4 of nursing after parturition. Food consumption was determined on Days 21 and 28 as well in non-pregnant females.
∙ During the recovery period: 1, 3, 7 and 14 days after the final administration

- Blood tests:
Blood was sampled from animals which underwent functional tests, i.e., five animals with the smallest animal Nos. selected from all groups including the control group in the case of males and five animals which had parturition on days closest to each other selected from all groups including the control group in the case of females. In the high dose group, however, five females were selected at random for blood sampling since parturition was not confirmed in any of the 12 females. Blood was also sampled from 2 females which failed to show parturition in the control group. Blood was samples from all recovery animals in all groups.

Oestrous cyclicity (parental animals):

Vaginal smears were collected every day from Day 1 through 14 of treatment at 8:30 - 12:00 from all females including those in the recovery group and examined under an optical microscope after Giemsa staining. The number of days between an estrus and the next estrus was defined as the length (days) of sexual cycle, and the mean length (days) of sexual cycle was calculated. If multiple estruses continued, the day the first estrus was observed was defined as the first day of estrus. The percentage of individual animals which showed abnormal estrus cycle (animals which did not show regular sexual cycle) was calculated.

Sperm parameters (parental animals):

Parameters examined in all male parental generations: testis weight, epididymis weight

Litter observations:

The number of newborns (stillborn pups + live newborns) and the number of live newborns (surviving pups + dead pups) were counted and gender and external surface were observed after completion of parturition (Day 0 after birth). Pups were autopsied when they were found dead to determine whether they were live-born or stillborn. The lungs were examined after observing the body surface, orifices, and thoracic and abdominal cavities with the naked eye. Clinical signs and symptoms including mortality were observed once a day until 4 days after birth, and all pups including dead ones were sexed. Live pups were weighed by litter and sex 0 and 4 days after birth, and the body surface, orifices, and cranial, thoracic, abdominal and pelvic cavities were observed with the naked eye 4 days after birth.
The following data were determined based on findings obtained: numbers of newborns including those stillborn and those which died on day 0 after birth, by sex and both sexes combined, number of pups stillborn, numbers of live-born pups including those which died on day 0 after birth, by sex and both sexes combined, numbers of surviving pups 0 and 4 days after birth, by sex and both sexes combined, and mean body weight + SD by sex 0 and 4 days after birth. Findings obtained in the observation of clinical signs and symptoms and autopsy were recorded. Newborns and live pups delivered were sex, and the number was separately reported for males and females. Live pups with abnormalities on the external surface were observed on day 0 after birth.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were euthanized by exsanguination from the abdominal aorta under ether anesthesia the day after the final day of treatment in the main study groups and the day after the end of the recovery period in the recovery group.
- Maternal animals: All surviving animals was autopsied on Day 5 of nursing in females which had parturition, and on Day 25 of gestation in females which did not have parturition.

GROSS NECROPSY
- The body surface, orifices, subcutaneous tissues, cranial, thoracic, abdominal and pelvic cavities and their contents were examined with the naked eye.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues (Trachea and lungs, Stomach, intestine (including duodenum through rectum and Payer's patch) and liver*, Heart*, Kidneys* and urinary bladder, Testes*, epididymides*, prostate*, seminal vesicles*, ovaries*, uterus* and vagina, Brain* (including cerebrum, cerebellum and pons), spinal cord and sciatic nerves, Bone marrow (femur), lymph nodes (axillary and mesenteric), spleen* and thymus*, Pituitary*, thyroid* (including parathyroids) and adrenals*, Eye balls) were prepared for microscopic examination, and those marked by an asterisk (*) weighed, respectively.

Statistics:

Uniformity of variance in each group was tested by Bartlett's method.
Parameters which were considered to show equal variance with no significant difference at 5% by Bartlett's test were further tested by one-way layout analysis of variance. If significant differences were observed at 5%, Dunnett's test was conducted between the control group and each test substance dose group.
Parameters which were considered to show unequal variance with significant differences at 5% by Bartlett's test were further tested by Kruskal-Wallis' test. If significant differences were observed at 5%, non-parametric Dunnett's test was conducted between the control group and each test substance dose group.
Kruskal-Wallis' test was conducted with respect to the number of times defecation was made (number of fecal masses) and the number of times urination was made (number of urine pools). Non-parametric Dunnett's test was conducted between the control group and each test substance dose group with respect to parameters which showed significant differences at 5%.
Dunnett's test and non-parametric Dunnett's test were conducted at 5% and 1% on both sides.
N88-Basic statistical processing program was used for analyses of hematological and blood biochemical parameters, while the statistical tool, StatLight 2000© (Yukms), was used for all other parameters.
In the evaluation of study data, changes were considered significant if test substance groups showed statistically significant differences compared to the control group at the level of significance of 1% or 5%.

Reproductive indices:

Days needed for copulation; Copulation rate; Insemination rate; Fertilization rate; Implantation rate; Pre-implantation embryo loss rate; Post-implantation embryo loss rate; Fertility rate; Parturition rate

Offspring viability indices:

Birth rate; Sex ratio of pups delivered; Day 0 survival rate; Sex ratio of pups surviving on Day 0; Incidence of external surface abnormalities; Day 4 survival rate; Sex ratio of pups surviving on Day 4

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

1) During the treatment period
- Males:
All 12, 11, 10 and 5 animals from the 800 mg/kg group, respectively, showed salivation, decreases in spontaneous locomotion, decreases in respiration rate, and incomplete eyelid opening.
Salivation occurred shortly after treatment and disappeared within 15 min after treatment sporadically or almost continuously from Day 4 through 42 of treatment. Some animals also showed salivation shortly before treatment throughout the treatment period. Decreases in spontaneous locomotion and respiration rate were observed sporadically throughout the treatment period from Week 2 of treatment, while incomplete eyelid opening was observed sporadically throughout the treatment period from Week 4 of treatment. No abnormality was observed in any other treated group or the control group.
- Females:
The 200 mg/kg group showed no abnormality throughout the mating, gestation and nursing periods. In the 50 mg/kg and control groups, animals which became pregnant/had parturition showed the changes described below, but these changes were thought to be incidental and not related to the test substance because no abnormality was seen in the 200 mg/kg group.
Specifically, one female from the 50 mg/kg group showed torticollis before mating and throughout the gestation and nursing periods. However, this was thought to be incidental because it was seen only in this (low dose) group and was not dose-dependent. During the nursing period (on the day of parturition or Day 0 of nursing), 2/9 animals (No. 106 and No. 111) from the control group showed poor placenta processing activity and lack of retrieving behavior, while one (No. 106) showed lack of licking behavior and crouching behavior. In the 50 mg/kg group, one of the 12 animals (No. 125) showed decreases in spontaneous locomotion, soilage around the nose and mouth, poor placenta processing activity and lack of retrieving behavior on the day of parturition.
Among satellite animals, which were not mated, all 5 animals from the 800 mg/kg group showed salivation and decreases in spontaneous locomotion and respiration rate, while 4 and two animals, respectively, showed incomplete eyelid opening and a large amount of food on the tray (increases in left-over food). No abnormality was seen in the control group.
Salivation occurred shortly after treatment and disappeared within 15 min after treatment almost continuously from Day 4 of treatment. Some animals also showed salivation shortly before treatment in Weeks 2 and 3 of treatment. Decreases in spontaneous locomotion were observed sporadically or almost continuously throughout the treatment period from Week 2 of treatment, while decreases in respiration rate and incomplete eyelid opening were observed sporadically throughout the treatment period from Week 2 of treatment. A large amount of food on the tray was sporadically seen from Week 2 through 4 of treatment.
- Non-pregnant animals: Females (No. 108 from the control group and Nos. 142-150, 152 and 153 from the 800 mg/kg group)
Before mating, all 11, 10, 3, 1 and 1, respectively, from the 800 mg/kg group showed salivation, decreases in spontaneous locomotion, decreases in respiration rate, incomplete eyelid opening and a large amount of food on the tray. After mating, all 11, 9, 7 and 1, respectively, from the 800 mg/kg group showed salivation, decreases in spontaneous locomotion, decreases in respiration rate, and incomplete eyelid opening. No abnormality was seen in the control group.
Salivation occurred shortly after treatment and disappeared within 15 min after treatment sporadically or almost continuously throughout the treatment period from Day 4 of treatment. Some animals also showed salivation shortly before treatment throughout the treatment period. Decreases in spontaneous locomotion and respiration rate and incomplete eyelid opening were observed sporadically throughout the treatment period from Week 2 of treatment, while a large amount of food on the tray was observed only on Day 13 of treatment.

-Females which lost all embryos after implantation: Females (No. 109 from the control group and No. 151 from the 800 mg/kg group)
The female from the 800 mg/kg group showed salivation, decreases in spontaneous locomotion and respiration rate and incomplete eyelid opening both before mating and during the gestation period. No abnormality was seen in the control group.
Salivation occurred shortly after treatment and disappeared within 15 min after treatment almost continuously throughout the treatment period from Day 5 of treatment. Decreases in spontaneous locomotion were observed almost continuously throughout the treatment period from Day 13 of treatment, while decreases in respiration rate and incomplete eyelid opening were sporadically seen throughout the treatment period from Day 13 of treatment.

- Females which lost all pups: Females (No. 107 from the control group and No. 137 from the 200 mg/kg group)
The female from neither the control nor 200 mg/kg group showed any abnormality before mating or during the gestation period. During the nursing period, however, the female from the control group showed spoilage in the lower abdomen and a lack of retrieving and crouching behaviors on Days 2 and 3 of nursing and all their pups died by Day 4 of nursing. The female from the 200 mg/kg group showed a lack of retrieving behaviors on the day of parturition (Day 0 of nursing), and all its pups died by Day 1 of nursing.

2) During the recovery period
No abnormality was observed in either males or females from the 800 mg/kg or control group.

Mortality:

no mortality observed

Description (incidence):

No mortality observed in any group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- During the treatment period:
In males, a tendency toward decreases was observed in the 800 mg/kg group on Day 35 of treatment and onward although this change was not statistically significant. Changes observed in the 200 and 50 mg/kg groups were similar to those observed in the control group.
In females, significant decreases were observed in the 800 mg/kg group on Day 14 of treatment (before mating). One female from this group which was found to be pregnant and lost all embryos after implantation showed a tendency toward decreases from Day 14 through 20 of gestation. Satellite females from the 800 mg/kg group showed significant decreases from Day 21 through 42 of treatment. No significant change was observed in the 200 or 50 mg/kg group before mating or throughout the gestation and nursing periods.

- During the recovery period:
In males, the 800 mg/kg group showed no significant change.
In females, the 800 mg/kg group showed significant decreases throughout the recovery period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- During the treatment period:
In males, the 200 mg/kg and higher dose groups showed significant decreases on Day 3 of treatment, while the 800 mg/kg group showed significant increases on Day 42 of treatment. In addition, the 50 mg/kg group showed significant decreases on Day 14 of treatment, but this change was considered incidental because it was not dose-related.
In females, significant decreases were observed in the 800 mg/kg group on Day 3 of treatment (before mating). One female from this group which was found to be pregnant showed a tendency toward decreases from Day 14 through 20 of gestation. Satellite females from the 800 mg/kg group showed significant increases on Day 42 of treatment. No significant change was observed in the 200 or 50 mg/kg group before mating or throughout the gestation and nursing periods.

- During the recovery period:
In both males and females, the 800 mg/kg group showed no significant change.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- Main study groups:
In males, no parameter showed any significant change in any test substance group.
In females, erythrocyte counts showed significant increases in the 200 mg/kg group, but no parameter showed any significant change in the 50 mg/kg group.

- Recovery groups:
In males, the 800 mg/kg group showed significant decreases in hemoglobin concentrations and hematocrit and significant increases in platelet counts.
In females, the 800 mg/kg group showed no significant change in any parameter.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- Main study groups:
In males, the 800 mg/kg group showed significant increases in alanine transferase, alkaline phosphatase, creatinine and total bile acid. Gamma-glutamyl transpeptidase showed a tendency to increase although not statistically significantly. In the 50 mg/kg group, lactate dehydrogenase increased significantly, but this change, which was not dose-dependent, was thought to be incidental. No parameter showed any significant change in the 200 mg/kg group.
In females, the 200 mg/kg group showed significant increases in triglyceride and phospholipid and significant decreases in inorganic phosphorus. No parameter showed any significant change in the 50 mg/kg group.

- Recovery groups:
In males, the 800 mg/kg group showed significant increases in gamma-glutamyl transpeptidase and inorganic phosphorus.
In females, the 800 mg/kg group showed significant increases in alkaline phosphatase and significant decreases in triglyceride and blood glucose levels.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- During the treatment period:
Males from all test substance groups showed no significant changes in grasping power or spontaneous locomotion. Males from the test substance groups and the control group also showed no abnormality in responsiveness tests.
Satellite females from the 800 mg/kg group showed significant increases in spontaneous locomotion between 0 and 10 min of determination. However, this increase was considered incidental because it was transient and the 0-60 min statistics showed no significant change. Grasping power and spontaneous locomotion showed no significant change in the 200 or 50 mg/kg group. Responsiveness tests also showed no abnormality in any test substance group or the control group.

- During the recovery period:
No test was conducted in the recovery period because no change thought to be attributable to the test substance was observed in either males or females in Week 6 of treatment.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

1) Main study groups
In males, the 800 mg/kg group showed slight or moderate eosinophilic granular changes of centrilobular hepatocytes in the liver in all 7 animals, slight increases in the clarity of hepatocellular nucleoli in 3, moderate or severe bilateral degeneration of the seminiferous epithelium of the testes and moderate diffuse atrophy of the seminiferous tubules in all 7, slight diffuse hyperplasia of Leydig cells in 5, moderate or severe bilateral decreases in intraluminal sperms and slight or moderate residues of intraluminal sperm cells in the epididymides in all 7, and creases in medullary hematopoietic cells in 2. Two males which showed partial whitening of the epididymides (No. 37) or nodules in the epididymides (No. 41) in macroscopic examination showed bilateral or unilateral spermatic granuloma. In addition, slight degeneration of border squamous epithelium in the anterior stomach, microgranuloma in the liver, and cyst formation in the pituitary intermediate lobe were observed in one animal each. The 200 mg/kg group showed slight or moderate bilateral localized atrophy of seminiferous tubules in the testes in 2/12 animals and severe unilateral diffuse atrophy of seminiferous tubules and slight diffuse hyperplasia of Leydig cells in one. In addition, slight bilateral residue of intraluminal sperm cells was observed in the epididymides of 2 animals which showed abnormality in the testes, while moderate unilateral decreases in intraluminal sperm and slight unilateral residue of intraluminal sperm cells were observed in another. The 50 mg/kg group showed slight bilateral localized atrophy of seminiferous tubules in the testes in 1/12 animals. This animal also showed slight bilateral residue of intraluminal sperm cells in the epididymides.
The control group showed slight bilateral localized atrophy of seminiferous tubules in the testes in 1/7 animals. This animal showed slight bilateral residue of intraluminal sperm cells in the epididymides. In addition, microgranuloma was observed in the liver of two animals.
In the PAS staining conducted for the testes of all males, almost all seminiferous tubules showed atrophy and degeneration in the 800 mg/kg group and the sperm formation stage could not be determined. In the 200 and 50 mg/kg groups, the sperm formation stage could not be determined with seminiferous tubules which showed atrophy. With other seminiferous tubules, however, the sperm formation cycle and stage showed no abnormality.
In females, the 200 mg/kg group showed increases in extramedullary hematopoiesis in the spleen in one animal and unilateral pyelectasis in another (No. 132) which showed pyelectasis in the left kidney. In the 50 mg/kg group, one (No. 128) which showed edematous changes of the right submandibular gland showed unilateral submandibular hydrops, while two (Nos. 123 and 127) which showed miniaturization of the right or left thyroid lobe showed aplasia of the right lobe and isthmus or aplasia of the left lobe. Changes observed in the 200 mg/kg or lower dose groups were not dose-dependent and considered unrelated to the test substance.
In the control group, one (No. 112) which showed vaginal cysts when observed with the naked eye had solitary cysts in the vaginal muscle layer. Of the two (Nos. 103 and 111) which showed miniaturization of the left thyroid lobe, one (No. 103) had remnant branchial pouch, while the other (No. 111) showed left lobular hypoplasia. Hydrops of the submucosal layer of the anterior stomach and localized hyperplasia of the squamous epithelium were observed in one and atrophy of the thymus in two.

- Non-pregnant animals: Females (No. 108 from the control group and Nos. 142-150, 152 and 153 from the 800 mg/kg group)
In the 800 mg/kg group, slight hypertrophy of centrilobular hepatocytes was observed in the liver of 5 animals, slight increase in clarity of hepatocellular nucleolus and slight single cell necrosis in one, and increases in follicular cysts in the ovaries and increases in mucus secretion from the vaginal epithelium in two. In addition, slight deposition of hemosiderin in the spleen was observed in 8 and slight congestion in one. Two (Nos. 143 and 153) which showed blackened areas on the glandular stomach mucosa when observed with the naked eye showed fundic glandular mucosal necrosis in the glandular stomach. One showed cerebral ventricle dilatation.
In the control group, increased mucus secretion from the vaginal epithelium was observed.

- Females which lost all embryos after implantation: Females (No. 109 from the control group and No. 151 from the 800 mg/kg group)
The following were observed in the 800 mg/kg group: slight hyperplasia of centrilobular hepatocytes and slight increase in clarity of hepatocellular nucleoli in the liver, slight hemosiderin deposition in the spleen, vaginitis and atrophy of the thymus. No abnormality was seen in the control group.

-Females which lost all pups: Females (No. 107 from the control group and No. 137 from the 200 mg/kg group)
The glandular stomach showed fundic glandular mucosal necrosis in the 200 mg/kg group.
The following were observed in the control group: decreases in goblet cells in the colon, atrophy of the thymus and posterior lobe cyst formation in the pituitary.

2) Recovery groups
- In males: the 800 mg/kg group showed slight bilateral degeneration of seminiferous epithelium in the testes of 4/5 animals, slight or moderate bilateral diffuse atrophy of seminiferous tubules in two, slight bilateral localized atrophy of seminiferous tubules in 3, and slight diffuse hyperplasia of Leydig cells in 4. The epididymides showed moderate bilateral decreases in intraluminal sperms and moderate residue of intraluminal sperm cells in all 5. The anterior stomach, liver, kidneys, bone marrow and pituitary were also examined but no abnormality was found. In the control group, one animal showed slight bilateral localized atrophy of seminiferous tubules in the testes and slight bilateral residue of intraluminal sperm cells in the epididymides.
- In females: the 800 mg/kg group showed slight or moderate hemosiderin deposition in the spleen of all 5 animals. The anterior stomach, liver, kidneys, ovaries, uterus and vagina were also examined but no abnormality was found. In the control group, 3/5 animals showed slight hemosiderin deposition in the spleen.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Detailed observation of clinical signs and symptoms
- During the treatment period:
Males from all test substance groups showed no significant changes in frequency of defecation or urination. No abnormality was seen, either, with respect to any other detailed observation item in any test substance group or the control group.
Females from the 50 and 200 mg/kg groups showed significant decreases in frequency of urination on Day 4 after parturition, but frequency of defection remained normal and no other detailed observation item showed any abnormality in any test substance group or the control group. Decreases in frequency of urination, which are also reported in the background data, were thought to be an incidental change attributable to a high control value and not toxicologically significant.

- During the recovery period:
Neither males nor females in the 800 mg/kg group showed any significant changes in frequency of defection or urination. No other detailed observation item showed any abnormality in either the 800 mg/kg or control group.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The sexual cycle showed no abnormality in any animal from any group including the control group. The mean length of sexual cycle (days) showed no significant change in any test substance group.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

- Examination of reproductive ability:
All animals from all groups including the control group achieved copulation, and the copulation rate showed no abnormality attributable to the test substance.
However, the insemination rate and fertilization rate decreased significantly to 8.3% in the 800 mg/kg group with the number of pregnant females being 11, 12, 12 and 1, respectively, in the control, 50, 200 and 800 mg/kg groups. The insemination rate and fertilization rate stood at 100% in the 50 and 200 mg/kg groups but decreased to 91.7% in the control group due to one animal which failed to become pregnant.
This female which failed to become pregnant was thought to have some hormonal abnormality for the following reasons: its partner male showed no abnormal clinical sign or symptom; showed a weight gain similar to that seen in other males from the same group; showed no abnormality in the organs or tissues examined including genital organs; and the female also showed no abnormal clinical sign or symptom; showed a normal weight gain after confirmation of copulation although not as much as in other females from the same group which achieved parturition; and showed marked increases in mucus secretion from the vaginal epithelium.
In addition, one female each from the 800 mg/kg group (No. 151) and the control group (No. 109) lost all embryos after implantation although the incidence was not statically significant. The cause of total post-implantation embryo loss in the control group was unknown because this female showed no abnormal clinical sign or symptom; its weight gain was normal although smaller than in other females from the same group which achieved parturition; and its partner male also showed a normal weight gain with no abnormality in the organs or tissues examined including genital organs. However, some hormonal abnormality was suspected because of the presence of only one implantation site despite the presence of 10 corpora lutea.
The copulation rate and the number of days needed for copulation showed no significant change in any test substance group. The gestation period, numbers of corpora lutea and implantation sites, implantation rate, pre-implantation embryo loss rate, fertility rate and parturition rate showed no significant changes in the test substance groups up to 200 mg/kg.

- Parturition and nursing activity:
In the 200 mg/kg group, one female (No. 137) showed poor nursing activity due to impaired retrieving behavior on the day of parturition, and all its pups died on Day 1 of nursing. In the 50 mg/kg group, one female (No. 125) showed impaired placenta processing and retrieving behavior on the day of parturition, but no abnormality was seen from the following day. In the control group, one female (No. 107) showed poor nursing activity due to impaired retrieving and crouching behavior on Days 2 and 3 of nursing, and all its pups died on Day 4 of nursing. Two more females (Nos. 106 and 111) from the control group showed impaired placenta processing and retrieving behavior on the day of parturition, but no abnormality was seen from the following day. Poor nursing activity observed in the test substance groups was not considered attributable to the test substance because it was not clearly dose-related and was also seen in the control group.

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

body weight and weight gain

histopathology: non-neoplastic

Key result

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Key result

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical biochemistry

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

800 mg/kg bw/day (actual dose received)

System:

gastrointestinal tract

Organ:

liver

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day (actual dose received)

System:

male reproductive system

Organ:

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No breast milk was found on the day of parturition (Day 0 of nursing) in the stomach of pups from one dam (No. 137) in the 200 mg/kg group which showed poor nursing activity and lost all pups on Day 1 of nursing. No breast milk was found in the stomach and body temperature decreased from Day 2 of nursing in pups from one dam (No. 107) in the control group which showed poor nursing activity and lost all pups on Day 4 of nursing. In the 200 mg/kg group, only one dam lost all pups. This incidence was the same as in the control group. Death of all pups from the same litter was not considered related to the test substance because its incidence in the 200 mg/kg group was the same as in the control group and it is also reported in background data. Of these dams which lost all pups, the one in the control group showed no abnormal clinical sign or symptom until Day 1 of parturition and showed a body weight gain similar to that observed in other pregnant dams from the same group until Day 20 of gestation and pathological examinations showed only moderate atrophy of the thymus and slight decreases in goblet cells in the colon although they showed poor nursing activity on Days 2 and 3 of parturition and all pups died on Day 4 of parturition. The one in the 200 mg/kg group showed no abnormal clinical sign or symptom until the day before parturition and showed a body weight gain similar to that observed in other pregnant dams from the same group until Day 20 of gestation and pathological examinations showed only slight fundic glandular necrosis in the glandular stomach although it showed poor nursing activity on the day of parturition and all pups died on Day 1 of parturition. Therefore, the cause of death of all pups from a litter remained unknown in both dams.
The number of pups, birthrate, the sex ratio of pups, number of live pups on Day 0, survival rate on Day 0, sex ratio of live pups on Day 0, incidence of external surface abnormalities, the survival rate on Day 4 and sex ratio of live pups on day 4 showed no significant changes in the test substance groups up to 200 mg/kg.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Females from the 200 mg/kg group showed significant decreases in body weight on Day 0, but not on Day 4. Both males and females from the 50 mg/kg group and males from the 200 mg/kg group showed no significant changes in body weight on Day 0 or 4.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the 200 mg/kg group, the kidneys showed pyelectasis in 4/142 pups, the liver showed blackened areas in 1/142, the cerebrum showed blackened areas in 1/142, and the ureter showed dilatation in 3/142. In the 50 mg/kg group, the kidneys showed pyelectasis in 5/151 pups and the liver showed whitened areas in 1/151. The control group showed no abnormality.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no offspring delivered at 800 mg/kg/day

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no offspring delivered at 800 mg/kg/day

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

800 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

The NOAEL and NOEL for reproductive/developmental toxicity were both 200 mg/kg/day since decreases in conception index and fertility index in parental animals were observed in the 800 mg/kg group.

Executive summary:

A Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test was performed for test substance in compliance with OECD TG422.

Four groups were established, including three given the test substance (50, 200 and 800 mg/kg/day) and one vehicle control group given the vehicle. Females from the main study groups were transferred to cages of males from the same dose groups in an ascending order of animal No. in the evening of Day 15 of treatment. Males and females were allowed to cohabit overnight on a 1:1 basis, and females were returned to their cages in the morning the following day. Mating was continued in the same way for up to 14 days until copulation was confirmed.

After treatment with the test substance, toxic effects on the male reproductive organs were mainly observed.

There was only one pregnant animal out of 12 copulation confirmed females in the 800 mg/kg group. Based on the result, conception index and fertility index were decreased in this dose group. There was only one implantation site in the pregnant animal and no next generation was obtained in this dose group. Although severe toxic effects on the testis above mentioned were observed, no abnormalities attributable to the test substance were detected in the ovary, uterus or vagina in females in the 800 mg/kg group.

No abnormalities attributable to the test substance were detected in any test parameters like estrous cycle, copulation index, conception index, delivery dam index of the reproductive ability of the parental animals or the external appearance, body weight, viability index or sex ratio for development of the next generation in the 200 or 50 mg/kg groups.

Regarding the reproductive/developmental toxicity, the NOAEL and NOEL were both 200 mg/kg/day since decreases in conception index and fertility index in parental animals were observed in the 800 mg/kg group.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

1 (reliable without restriction)

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Reproductive/developmental toxicity: NOAEL and NOEL were both 200 mg/kg/day.

Adverse effects: Males received 800 mg/kg/day showed obviously testicular lesions with high frequency, and the marked decrease in fertility index was considered to be related to that.

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.7.1(a), this substance should classified as Category 2 for reproductive toxicants.