1,4-bis(methoxymethyl)benzene

Endpoint summary

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 repeated-dose toxicity in males, the No-Observed-Adverse-Effect Level (NOAEL) and No-Observed-Effect Level (NOEL) was 50 mg/kg/day since bilateral focal atrophy of seminiferous tubules of the testis, bilateral germ cell debris in lumen of the epididymis and decreases in absolute and relative weights of the complex of the prostate and seminal grand were observed in males in the 200 mg/kg group. In females, the NOAEL was 200 mg/kg/day since centrilobular hypertrophy of hepatocytes, prominent nucleoli of hepatocytes and single cell necrosis of hepatocytes were observed in females in the 800 mg/kg group, and the NOEL was 50 mg/kg/day since increased levels of triglyceride and phospholipid were observed in females in the 200 mg/kg group.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

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

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:

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: 9 weeks
- Weight at study initiation: Males: 332.5-374.7 g; and 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

DETAILS OF FOOD AND WATER QUALITY:
Analytical data related to contaminants in food, bedding material, and enrichment were confirmed to meet the standards established by this laboratory based on the “Limits of Contaminants in Foods and Vehicles” (1979) established according to the US EPA Toxic Substances Control Act. Quality of drinking water was tested twice a year according to the Ministerial Ordinances Related to Water Quality Standards issued by the Ministry of Health, Labor and welfare (MHLW Ordinance Nos. 101, 135 and 174). Latest test data received before arrival of animals and after completion of experiments were confirmed to meet the standards set according to these ordinances.

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

Details on route of administration:

Dosing solutions were orally administered by gavage once a day between 9:00 and 14:00

Vehicle:

olive oil

Details on oral 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

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%).

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:

once a day

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.

- Post-exposure recovery period in satellite groups: 14 days

Observations and examinations performed and frequency:

- 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.

Sacrifice and pathology:

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

HISTOPATHOLOGY: Yes, 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%.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- 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.
- 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

Description (incidence and severity):

- Main study groups:
In males, absolute and relative weight of the prostate and seminal vesicle (prostate complex) and relative weight of the adrenals decreased significantly in the 200 mg/kg and higher dose groups, while in the 800 mg/kg group, absolute and relative weight of the liver increased significantly and absolute and relative weight of the testis and absolute weight of the adrenals decreased significantly. Absolute and relative weight of the epididymides showed a tendency to decrease although not statistically significantly. In addition, this group showed significant decreases in absolute weight of the heart. In the 200 and 50 mg/kg groups, absolute weight of the brain decreased significantly, but this change, which was not dose-dependent, was considered incidental.
In females, absolute weight of the liver increased significantly in the 50 and 200 mg/kg groups.

- Recovery groups:
In males, absolute and relative weight of the testis, epididymides and kidneys decreased significantly in the 800 mg/kg group.
In females, relative weight of the kidneys increased significantly in the 800 mg/kg group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Main study groups:
In males, the 800 mg/kg group showed enlargement of the liver in 5/7 animals, small testes in 5, softened testes in 6, partial whitening of the epididymides in one (No. 37), and nodules in the epididymides in another (No. 41). Other test substance groups and the control group showed no abnormality.
In females, the 50 mg/kg group showed edematous changes of the right submandibular gland in 1/12 animals (No. 128), small right lobe of the thyroid in one (No. 123) and small left lobe of the thyroid in another (No. 127), while the 200 mg/kg group showed pyelectasis of the left kidney in 1/11 animals (No. 132). These changes were considered spontaneous or incidental because they were also seen in the control group and were not dose-dependent.
In the control group, vaginal cysts were observed in 1/9 animals (No. 112) and miniaturization of the left thyroid lobe in 2 (Nos. 103 and 111).

- Recovery groups:
In males, the 800 mg/kg group showed miniaturization of the testes in all 5 animals, while the control group showed no abnormality.
In females, mo abnormality was seen in either the 800 mg/kg or control group.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- 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.

- 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.

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:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

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:

body weight and weight gain

clinical signs

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:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

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

Conclusions:

Regarding the repeated-dose toxicity in males, the No-Observed-Adverse-Effect Level (NOAEL) and No-Observed-Effect Level (NOEL) was 50 mg/kg/day since bilateral focal atrophy of seminiferous tubules of the testis, bilateral germ cell debris in lumen of the epididymis and decreases in absolute and relative weights of the complex of the prostate and seminal grand were observed in males in the 200 mg/kg group. In females, the NOAEL was 200 mg/kg/day since centrilobular hypertrophy of hepatocytes, prominent nucleoli of hepatocytes and single cell necrosis of hepatocytes were observed in females in the 800 mg/kg group, and the NOEL was 50 mg/kg/day since increased levels of triglyceride and phospholipid were observed in females in the 200 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.

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

Slight or moderate bilateral focal atrophy of seminiferous tubules of the testis and slight bilateral germ cell debris in lumen of the epididymis in males in the 200 mg/kg group were observed at the end of the administration period. Macroscopically-observed softening of1the testis in five of seven animals and small of the testis in six animals and histologically-corresponding moderate or severe bilateral degeneration of seminiferous tubules, moderate bilateral diffuse atrophy of seminiferous tubules of the testis, moderate or severe bilateral decreased spermatozoa in lumen and slight or moderate bilateral germ cell derbis in lumen of the epididymis in all seven animals, slight diffuse Leydig cell hyperplasia of the testis in five animals, moderate bilateral or unilateral spermatic granuloma of the epididymis in one animal each in the 800 mg/kg group were observed. In relation to these changes, decreases in absolute and relative weights of the testis and tendencies to decreases in absolute and relative weights of the epididymis in the 800 mg/kg group were noted. Decreases in absolute and relative weights of the complex of the prostate and seminal grand included a part of the urinary tract were also noted in the groups of 200 mg/kg or more. The effects of the male reproductive organs showed no clear reversibility.

As effects on the liver, slight or moderate centrilobular eosinophilic granular change of hepatocytes in all seven animals and increases in absolute and relative weights in males, slight centrilobular hypertrophy of hepatocytes in six of 12 animals, slight single cell necrosis of hepatocytes in one female and slight prominent nucleoli of hepatocytes in three males and two females in the 800 mg/kg group were observedat the end of the administration period. In relation to these changes, increased levels of alanine aminotransferase, alkaline phosphatase and total bile acids were noted in males in the 800 mg/kg group.Although increased levels of triglyceride andphospholipid were observed in females in the 200 mg/kg group, these changes were not found in the 800 mg/kg group in which all animals were not pregnant. The effects on the livershowedreversibility.

Slight degeneration of squamous epithelium in limiting ridge of the forestomach was observed in males in the 800 mg/kg group at the end of the administration period. Although anemia was not shown, slight hemosiderin deposition in the spleen in nine of 12 animals and slight congestion of the spleen in one animal in females in the same dose group were noted. The effect on the forestomach recovered. However, the effects on the spleen showed no reversibility.

In general condition during the administration period, toxic effects like decreased spontaneous activity, decreased respiratory rate and incomplete eyelid opening were observed in males and females in the 800 mg/kg group. These changes showed reversibility.

Regarding the repeated-dose toxicity in males, the No-Observed-Adverse-Effect Level (NOAEL) and No-Observed-Effect Level (NOEL) was 50 mg/kg/day since bilateral focal atrophy of seminiferous tubules of the testis, bilateral germ cell debris in lumen of the epididymis and decreases in absolute and relative weights of the complex of the prostate and seminal grand were observed in males in the 200 mg/kg group. In females, the NOAEL was 200 mg/kg/day since centrilobular hypertrophy of hepatocytes, prominent nucleoli of hepatocytes and single cell necrosis of hepatocytes were observed in females in the 800 mg/kg group, and the NOEL was 50 mg/kg/day since increased levels of triglyceride and phospholipid were observed in females in the 200 mg/kg group.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

1 (reliable without restriction)

System:

other: gastrointestinal tract and male reproductive system

Organ:

liver

testes

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test:

28 -days NOAEL: 50 mg/kg/day (male); 200 mg/kg/day (female)

Toxic effects:

Male: bilateral focal atrophy of seminiferous tubules of the testis, bilateral germ cell debris in lumen of the epididymis and decreases in absolute and relative weights of the complex of the prostate and seminal grand were observed in males in the 200 mg/kg group

Female: cell necrosis of hepatocytes were observed in females in the 800 mg/kg group

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.9.1 and 3.9.3, this substance should classified as Category 2 for specific target organ toxicity-repeated exposure.