Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

The NOAEL for reproductive and developmental toxicity was judged to be 250 mg/kg bw/day due to decreased body weight of F1 offspring at 1000 mg/kg bw/day on PND 13.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 December 2018 to 12 April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same 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:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Testing Methods Concerning New Chemical Substances
Version / remarks:
Notification No. 0331-7, PFSB, MHLW; No. 5 of March 29, 2011, MIB, METI; No.
110331009, EPB, MOE; dated March 31, 2011
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on species / strain selection:
This strain is widely used in toxicity studies using rodents, there is abundant historical data and a large number of animals are available.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7 weeks old at receipt, 9 weeks old at the start of administration
- Weight at receipt: 224.8 to 247.6 g (males), 162.3 to 188.7 g (females)
- Fasting period before study: No, but animals were fasted before scheduled necropsy for about 17 to 24.5 hours.
- Housing: Animals were housed 2 or 3 animals per cage before grouping. After gouping, animals were housed 1 male and 1 female per cage during the mating period, one dam and its litter per cage during the lactation period and one animal per cage for the other periods. For males and females except for copulated females and dams, animals were housed in hanging type stainless wire mesh cages (W × D × H: 226 × 346 × 198 mm). Copulated females were housed in polymethylpentene cages (W × D × H: 220 × 380 × 195 mm). Both types of cages were furnished with two types of nesting material (Paper Clean, Diamond Twists) and one kind of gnawing material (Diamond Twists) for the improvement of animal welfare.
- Diet: Pelleted diet, ad libitum (except during fresh urine collection and measurement of motor activity)
- Water: Well water admixed with NaClO, ad libitum (except during the measurement of motor activity)
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY:
Data for each lot of the diet were obtained from the supplier and contaminants in the diet were confirmed to be within the acceptable limits established by the test facility.
Water was analysed twice a year. The results were confirmed to be within the acceptable limits established by the test facility.

ENVIRONMENTAL CONDITIONS
- Temperature: 22.1 to 23.7 °C
- Humidity: 51.4 to 72.6 %
- Air changes: 10 to 20 air changes per hour
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Following correction for substance purity, a prescribed amount of the test material was was weighed into a beaker and an appropriate amount of the vehicle was added and stirred with a magnetic stirrer.
After confirming of dissolution, it was transferred to a measuring cylinder. The final volume was adjusted by adding a proper quantity of the vehicle to provide the required concentration of dosing solution. Further dilution provided lower concentration dosing solutions.
Dosing formulations were prepared once within 12 days (3- to 10-day interval). The dosing formulations after preparation were divided into glass vials for each dosing day and stored at room temperature (17.9°C to 22.4°C).

DOSE VOLUME:
Individual volume was calculated on the basis of the most recently measured body weights.
Details on mating procedure:
- M/F ratio per cage: The test males and females of each dose level were cohabited at one-on-one basis for 24 hours from Day 15 to the end of the mating.
- Proof of pregnancy: The vaginal smears were collected in the morning from the day after the initiation of mating, and copulation was confirmed by the presence of the vaginal plug or sperm in the smear sample. The day of successful mating was regarded as Day 0 of gestation (GD 0).
Analytical verification of doses or concentrations:
yes
Remarks:
HPLC
Details on analytical verification of doses or concentrations:
At the first preparation, 10 mL each of the analytical samples was taken from the whole dosing formulation at each concentration and the test material concentration analysed by HPLC according to a previously validated method. Nominal test material concentrations of 12.5, 50 and 200 mg/mL provided mean measured concentrations of 12.68, 50.11 and 203.1 mg/mL, respectively and were within 101.4, 100.2, and 101.6% of nominal, respectively.
Test material formulations at 1 and 200 mg/mL were confirmed to be stable after storage for 12 days at room temperature.

High Performance Liquid Chromatography (HPLC) conditions:
Instrumentation: Prominence UFLC (Shimadzu Corporation)
Data processing: LabSolutions (Shimadzu Corporation)
Column: Ascentis Express RP-Amide (2.7 μm, 3.0 mm I.D. × 50 mm, Sigma-Aldrich Co.)
Column temperature: A constant temperature of about 40°C (set at 40°C)
Mobile phase:
Mobile phase A; water*
Mobile phase B; acetonitrile*
Linear gradient conditions and flow rate:
0.00 and 3.00 mins (55% A : 45% B) at a flow rate of 0.9 mL/min
3.01 and 12.00 mins (0% A : 100% B) at a flow rate of 1.5 mL/min
21.01 and 22.00 min (55% A : 45% B) at a flow rate of 0.9 mL/min
Analysis time: 22 minutes
Wavelength for detection: UV 200 nm
Injection volume: 10 μL
Auto-sampler set temperature: Room temperature (Not setting)
Needle wash solvent: Acetonitrile*
* sonicated under vacuum and degassed.
Duration of treatment / exposure:
Males: From 14 days before mating until the day before necropsy through the mating period (42 days in total)
Females: From 14 days before mating until Day 13 of lactation (day of delivery was Day 0 of lactation) through the mating and gestation periods and delivery.
Recovery females (Satellite females), for 42 days without mating (as for males)
Frequency of treatment:
daily
Details on study schedule:
n/a
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
0 (corn oil control) and 1000 mg/kg bw: 7 males and 12 females (main dosing group) and an additional 5 males and 5 females (recovery group)
62.5 and 250 mg/kg bw: 12 males and 12 females (main dosing group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Doses were selected following consideration of the effects observed in a 14-day study, during which 5 animals/sex/dose received oral gavage doses of test material at 0 (corn oil control), 110, 330, and 1000 mg/kg bw, for 14 consecutive days.
Following administration of test material, a lowering tendency of body weight was noted in males of the 1000 mg/kg group throughout the dosing period; however, a low value of body weight gain was transient. Haematology revealed anaemia in females of the 330 and 1000 mg/kg groups, and prolonged PT and APTT in males of the 1000 mg/kg group as an effect on the coagulation system.
At necropsy, enlargement of the liver was noted in both sexes of the 1000 mg/kg group and a high value of liver weight was noted in both sexes of the 330 and 1000 mg/kg groups. In the blood chemistry, a high value of ALAT was noted in both sexes of the 1000 mg/kg group, suggesting hepatic injury. The effects on protein/lipid metabolism were noted in males of the 1000 mg/kg group and females of the 110 mg/kg group and above. Moreover, whitish patch of mucosa in the forestomach was noted in 1 female of the 1000 mg/kg group, suggesting that the test material is irritating. Although various treatment-related changes were observed, none of the changes was severe. Therefore, the high dose in this study set at 1000 mg/kg, at which clear toxicity effects are expected to be seen. Lower doses were set at 250 and 62.5 mg/kg, with a common ratio of 4. A control group (0 mg/kg group) dosed with the vehicle (corn oil) alone was also established.
- Rationale for animal assignment: All animals except for the animals with abnormal estrous cycle (as determined during the quarantine and acclimation period) were used for grouping. Animals were assigned to groups by the stratified randomisation on the basis of body weight measured on the day before the first dosing. Animals weighing within the mean body weights ± 20% (calculated for each sex) were used.
- Post-exposure recovery period in satellite groups:
For 5 males and 5 females (non-mating satellite females) each in the control and 1000 mg/kg groups, a 14-day recovery period was set after the end of the dosing period.
Positive control:
n/a
Parental animals: Observations and examinations:
The initial day of dosing was designated as Day 1, Day 1 to Day 7 as Week 1, period after Day 43 as the recovery period. For the test females, the day of successful copulation was designated as Day 0 of gestation (GD 0) and the day of delivery as Day 0 of lactation (LD 0).

CLINICAL OBSERVATIONS: Yes
- Time schedule:
All animals were observed twice a day (before dosing and after dosing) during the dosing period and once a day in the other periods.

DETAILED CLINICAL OBSERVATIONS: Yes (as part of functional observation battery)
- Time schedule: For all animals, detailed clinical observations were performed once before the start of dosing and once a week in the afternoon during the dosing and recovery periods (day of gestation or parturition was prescinded).
Detailed clinical observations included:
- Hand-held observation, during which animals were removed from the cage for the observation by grasping their trunk gently from behind. Their reactivity to handling, trauma, colour of skin, soiled fur, piloerection, secretion, salivation, lacrimation, exophthalmos, palpebral closure, colour of conjunctiva and pupil size were observed.
- Observation on open field, during which animals were placed in the center of the open field and observed quietly for one minute. Parameters observed included arousal, posture/body position, gait, tremor, convulsion, respiration, stereotypy, bizarre behaviour, urination, defecation and number of rearings.

OBSERVATIONS DURING DELIVERY AND LACTATION PERIODS
All copulated females were allowed natural delivery. The observation of delivery was conducted once daily (a.m. 9:00) from GD 21 to GD24. Females that delivered their litter completely by 9:00 a.m. were judged as “delivered” on the corresponding day (the delivery day was regarded as Day 0 of lactation [LD 0]). When delivery was completed at 9:00 or later, the following day was defined as LD 0. The delivered animals (dams) were allowed to nurse offspring until LD 13, and postpartum behaviour such as lactation, nesting and presence or absence of cannibalism was observed every day. One female (No. 691) not delivered even after 24 days after the copulation confirmation was determined as non-delivered females.

BODY WEIGHT: Yes
- Time schedule for examinations:
The test and recovery males were weighed on Days 1, 8, 15, 22, 29, 36 and 42. The recovery males were also weighed on Days 43, 50 and 56. The satellite females were weighed at the same frequency as the recovery males. The test females were weighed on Days 1, 8 and 15, once every 7 days after the initiation of cohabitation, on GDs 0, 7, 14 and 20, and on LDs 0, 4, 7 and 13.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption was measured between Days 1 and 8, 8 and 15, 22 and 29, 29 and 36 and 36 and 41 for the test and recovery males, and between Days 43 and 50 and 50 and
55 for the recovery males. For the satellite females, it was measured between Days 1 and 8, 8 and 15, 15 and 22, 22 and 29, 29 and 36, 36 and 41, 43 and 50 and 50 and 55. Food consumption of the test females was measured at the same frequency as body weight measurement. However, food consumption was not measured for either sex during the mating period. After the completion of copulation, the measurement for males was started from the nearest measurement day. Gross weight of each feeder was weighed, and the mean daily food consumption for each period was expressed as the start day of each measurement.

WATER CONSUMPTION: No

HORMONE MEASUREMENT
Hormone (total T4) concentrations in rat plasma samples (parental animals [F0 male and F0 female], offspring [F1 offspring on PND 13], and Satellite female) were measured. A total of 80 samples (30 samples of F0 male, 20 samples of F1 offspring on PND 13, 20 samples of F0 female, and 10 samples of Satellite female) were assayed.
The plasma samples were stored frozen until analysis. The frozen plasma samples were thawed immediately before use, and then centrifuged (1710 × g for 10 minutes at set 4°C) to remove any aggregates in the plasma. The supernatant was used for assay.
An aliquot (60 μL) of the test sample or “standard solution (20 μL) (0, 25.5, 51.0, 102, 204, and 408 nmol/L) and PBS (phosphate buffered saline, 40 μL)” was pipetted into a tube coated with anti-T4 antibody. An aliquot (500 μL) of the tracer (125I-labelled T4) was added to the tube and the solution was mixed and shaken for 1 hour at room temperature. After the solution was aspirated, the remaining 125I radioactivity (cpm) in the tube was measured for 1 minute using the gamma counter.
A calibration curve was prepared with the mean cpm values of the duplicate assay for the standard solutions. The concentrations in the test samples were calculated using the calibration curve and the cpm value of the single assay for the test samples and corrected with the volume factor at the time of measurement. Hormone concentrations were calculated using the software (RiaSmart, PerkinElmer, Inc.) and Excel 2010 (Microsoft Corporation). The lower limit of quantification (LLOQ) was set at the lowest concentration of the standard solution (except for the zero concentration).

OTHERS:
The following were included as part of the repeated dose toxicity assessment: haematology, clinical chemistry, urinalysis, functional observational battery (including sensory activity, grip strength, motor activity).
Oestrous cyclicity (parental animals):
Vaginal smears were collected with swabs from all test females in the morning (same time every day) from the initial dosing day to the day of successful copulation to confirm the oestrous cycle. The obtained smears were collected on a plate for each animal, and stained with Giemsa. The oestrous cycle was classified into diestrus (D), proestrus (P), oestrus (E) and metestrus (M). The mean oestrous cycle (number of days from the estrous period to the next estrous period) and the number of the oestrous period during the test period were calculated.
Litter observations:
The day of birth was defined as “Postnatal day 0 (PND 0)” for offspring.

BODY WEIGHT
All live offspring were weighed individually on PNDs 0, 4, 7 and 13.

EXTERNAL EXAMINATION
All live offspring on PND 13, stillborn and dead offspring were observed for external anomaly. Based on the results, the following parameters were calculated.
- External anomaly index (%): (Number of offspring with external anomaly / Number of observed offspring) × 100
- External anomaly typing index (%): (Number of offspring with external anomaly by each type / Number of observed offspring) × 100

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
On PND 4, the litter size was standardised to 8 (4/sex/litter) by random removal of offspring. Even when the number of either males or females per litter was less than 4, the litter size was adjusted to 8 regardless of the sex ratio. Litter with less than 8 offspring was maintained as is. The offspring culled at the litter size adjustment (offspring excluded from blood collection) were euthanised by overdose with pentobarbital sodium (undiluted solution, 0.1 to 0.5 mL/body) via an intraperitoneal injection and discarded.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
- AGD (Anogenital distance)
Anogenital distance (AGD: distance between the anus and the genital node) was determined on PND 4 following adjustment of the number of offspring with a micrometer caliper. In order to correct variations in measured values due to weight differences among individuals, the AGI (anogenital index) divided by the cubic root of the body weight on the measurement day was also calculated.
- Nipple development
All live male offspring were observed for the appearance of nipples (or areolae) on PND 12. Based on the results, the following parameter was calculated.
Nipple development anomaly index (%): (Number of offspring with nipple development anomaly / Number of observed offspring) × 100
Postmortem examinations (parental animals):
TERMINAL PROCEDURES
All surviving animals were euthanised by exsanguination under anaesthesia and subjected to necropsy. In addition, the vaginal smears were collected from the females on LD 14 (necropsy day) in the morning and the stage of the oestrous cycle was examined under a microscope. At necropsy on LD 14, the ovaries and uterus were excised to examine for the numbers of implantation sites.
Non-delivered female (No. 691) was necropsied on GD 26. This animal was euthanised and necropsied in the same manner as the surviving animals.

GROSS PATHOLOGY: Yes
The following organs/tissues were collected and examined: Heart, thymus, spleen, mandibular lymph node, mesenteric lymph node, femur/bone marrow (femur), sternum/bone marrow (sternum), trachea, lung (including bronchus), stomach, duodenum, jejunum, ileum (including Payer’s patch), cecum, colon, rectum, liver, pancreas, submandibular gland, kidney, urinary bladder, testis, epididymis, prostate (ventral lobe), seminal vesicle (including dorsolateral lobe and coagulating gland), cowper’s gland, glans penis, ovary, uterus, vagina, pituitary, thyroid/parathyroid, adrenal, spinal cord (cervical), brain (cerebrum, cerebellum and medulla oblongata/pons), eyeball, femoral muscle/Sciatic nerve (femoral region), levator ani/bulbocavernosus muscle (LABC), and any other organs/tissues indicating gross lesion.

ORGAN WEIGHTS: Yes
After the scheduled necropsy, the organs listed above were weighed (absolute weight) and the ratios of the organ weights to body weight (relative weight) were calculated on the basis of the body weight measured on the day of necropsy. The measurement was conducted in 5 animals per group with the smallest animal numbers of the test males; all recovery males; all satellite females; 5 animals per group from the earlier parturition date with the smallest animal numbers in the test females. However, the testis and epididymis weights were measured in all males. Paired organs were weighed together.

HISTOPATHOLOGY: Yes
After necropsy, the organs/tissues listed above were fixed in 10 vol% phosphate buffered formalin solution; however, the testes and epididymides were fixed in Bouin’s solution and the eyes were fixed in Davidson’s solution, and they were preserved in 10 vol% phosphate buffered formalin solution. As for the dead animal, all of the organs/tissues were preserved in 10 vol% phosphate buffered formalin solution. The organs/tissues of 5 animals per group with the smallest animal numbers of the test males and 5 animals per group from the earlier parturition date with the smallest animal numbers for the test females in the control and 1000 mg/kg groups collected at the end of the dosing period, one dead animal (No. 693) and gross lesion were embedded in paraffin, sectioned and stained with hematoxylin and eosin, and then examined by microscopy.
As for the dead animal, the following organs could not be examined due to autolysis: mandibular lymph node, bone marrow (sternum and femur), trachea, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, submandibular gland, urinary bladder, ovary, uterus, vagina, pituitary, thyroid, adrenal, spinal cord and sciatic nerve.
According to these results, a change suspected to be attributable to the test material treatment was observed in the 1000 mg/kg group as follows: liver, pituitary, thyroid, adrenal, femur, spleen, stomach, cecum and kidney of both sexes and heart, sternum, thymus and lung of females. Therefore, the relevant organs/tissues of the relevant sex in the low and middle dose groups and all groups in the recovery study were additionally examined, according to the above criteria. Vacuolation was noted in the adrenal and kidney and foam cells were noted in the lung. To confirm whether the vacuole and foam cells are derived from lipid, these organs from one representative each in the 1000 mg/kg group (adrenal: No. 637, kidney: No. 693 and lung: No. 688) was subjected to Oil red O staining for microscopic examination.
Postmortem examinations (offspring):
The necropsy was performed on PND 13. The offspring subjected to blood sampling were necropsied after blood sampling by decapitation after anaesthetising by inhalation of isoflurane. Other animals were euthanised by exsanguination from the lateral iliac artery under anaesthesia with pentobarbital sodium (undiluted solution, 0.1 to 0.5 mL/body) via the intraperitoneal injection and necropsied. The thoracoabdominal organs/tissues were examined macroscopically.
As the results, an abnormal finding was noted in the liver of one offspring in one dam (offspring No. F04 in dam No. 698). Therefore, the organ with macroscopic lesion was collected and fixed and preserved in 10 vol% phosphate buffered formalin along with the same organs and tissues from one dam (No. 653) of the control group for comparison.
Furthermore, the thyroid glands of 1 male and 1 female (animals subjected to blood sampling for hormone level measurement) per litter were collected and fixed and preserved in 10 vol% phosphate buffered formalin. As for one dam (No. 667), the thyroid gland was collected from 2 females because there were no males in this litter.

BLOOD SAMPLING
The blood sampling was performed on PNDs 4 and 13 and conducted for the offspring from 5 dams per group from the earlier parturition date with the smallest animal numbers in the test females. On PND 4, the blood was collected from at least 1 culled animal of each sex in each litter. On PND 13, the blood sampling was performed on 1 animal of each sex in each litter. When a blood sample could not be obtained from either sex, it was obtained from 2 animals of the same sex in the litter.
The blood sampling was performed by decapitation after anesthetizing by inhalation of isoflurane. Approximately 0.4 mL of blood (including humor liquid, pooled blood per litter) was collected into a polypropylene tube containing heparin sodium. The blood was immediately cooled on ice and then promptly centrifuged (approximately 1870 × g, 10 min, 4°C) to obtain plasma (120 μL or more). The collected plasma was stored frozen.
Statistics:
Statistical analysis was performed with a computer system (tsPharma LabSite, Fujitsu Limited). However, a statistical analysis system EXSUS Version 8.1.0 (CAC Croit Corporation, statistical analysis software: SAS 9.4 [SAS Institute Japan Inc.]) was used for sex ratio. In all cases, levels of p<0.01 (1%) and p<0.05 (5%) were considered to be significant and two-tailed test was used. Analysis for concentration of total T4 was conducted according to Work Plan. The data of offspring were handled on a litter-basis.
Multiple comparison test:
For the following numeral data, mean values and standard deviations were calculated in each group. Bartlett’s test was performed to compare variances among groups (significance level: 5%). When variance of data was homogeneous, Dunnett’s multiple comparison test was performed to compare with the control group. When variance of data was heterogeneous, Steel’s multiple comparison test was performed to compare with the control group.
Relevant parameters included: oestrous cycle, number of estrus, days until copulation, gestation length, number of implantations, number of delivered offspring, number of live offspring, body weight of offspring and AGD.
The following items were tested with the chi-square test for comparison between the control and each test material group: copulation index, fertility index, gestation index and sex ratio
The following items were tested with the Wilcoxon’s rank sum test for comparison between the control and each test material group: delivery index, stillborn index, external anomaly index, external anomaly typing index, birth index, viability index on Days 4 and 13 and nipple development anomaly index.
Reproductive indices:
Based on the results of the vaginal smears, the following parameters were calculated:
- Days until copulation: Number of days from the start of mating to the detection of copulation
- Copulation index (%): (Number of animals with successful copulation / Number of animals cohabited) × 100
- Fertility index (%): (Number of pregnant animals / Number of females with successful copulation) × 100

Based on the findings following excisation of the ovaries and uterus at necropsy (on LD 14), the following parameters were calculated.
(As for non-delivered one female (No. 691), the presence of implantation was examined for the uterus at necropsy on GD 26. As a result, the implantation was confirmed macroscopically. Thus, this female was judged to be pregnant.)
- Gestation length: Days until completion of delivery from GD 0
- Gestation index (%): (Number of pregnant animals delivered live offspring / Number of pregnant animals) × 100
- Delivery index (%): (Number of delivered offspring / Number of implantations) × 100
Offspring viability indices:
The number of litter (numbers of live newborn and stillborn), sex and the presence or absence of external anomalies were examined on PND 0. Thereafter, mortality was observed daily. Based on the results, the following parameters were calculated. The stillborn and dead offspring before culling were discarded.
- Birth index (%): (Number of live offspring at birth / Number of implantations) × 100
- Stillborn index (%): (Number of stillborns / Total number of delivered offspring) × 100
- Viability index on Day 4 (%): (Number of live offspring on PND 4 / Number of live offspring at birth) × 100
- Viability index on Day 13 (%): (Number of live offspring on PND 13 / Number of live offspring after culling) × 100
- Sex ratio: (Number of male live offspring / Number of all live offspring)
- External anomaly index (%): (Number of offspring with external anomaly / Number of observed offspring) × 100
- External anomaly typing index (%): (Number of offspring with external anomaly by each type / Number of observed offspring) × 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related change was noted. Mass was observed in the axilla of one female (No. 698) treated with 1000 mg/kg on LD 4 and thereafter. However, it was judged that this change was not related to treatment with the test material.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
One female (No. 693) treated with 1000 mg/kg was found dead on LD 2. In this animal, reduced locomotor activity was observed the day before, but no other remarkable change was observed. A suppressed body weight gain was noted in this animal until the death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A low value of body weight was noted in males treated with 1000 mg/kg throughout the dosing and recovery periods (statistically significant compared to vehicle control).
A low value of body weight (statistically significant compared to vehicle control) was noted in females treated with 1000 mg/kg on GD 20.
In satellite females, no significant difference was noted between the control and 1000 mg/kg group throughout the dosing or recovery period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A low value of food consumption (statistically significant compared to vehicle control) was noted in males treated with 1000 mg/kg during Day 1-8. Thereafter, food consumption in males treated with 1000 mg/kg was similar to or higher (during Day 29-36) than that of Controls throughout the dosing and recovery periods.
Prior to pairing, food consumption was low in females treated with 1000 mg/kg during Day 1-8 (statistically significant compared to vehicle control). Thereafter, food consumption was similar to Controls for females at all dose levels during the gestation and lactation periods.
In satellite females (after Day 8), food consumption was similar to or higher than (statistically significant compared to vehicle control) that of Controls throughout the dosing and recovery periods.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the dosing period, some erythroid parameters at 1000 mg/kg were marginally and not statistically significantly lower compared to the concurrent vehicle control as follows: a low value of RBC count in females (93.9% of Controls), a lower value of haemoglobin concentration in males (94.9%) and females (93.6%), a lower value of haematocrit in males (93.3%) and females (94.3%).
At the end of the recovery period, lower values of RBC count, haemoglobin concentration and haematocrit and a higher value of reticulocyte ratio were noted in males treated with 1000 mg/kg (statistically significant compared to vehicle control).
Besides, at the end of the dosing period, shortening of PT and APTT was noted in females treated with 1000 mg/kg, the differences from the control attained the statistical significance. However, these were judged not to be treatment-related as the changes were slight compared to those of Controls and opposite to the toxicity effect. A statistically significant prolongation of PT was noted in males treated with 250 mg/kg, however, the change was not accompanied by a dose-dependency and considered not to be treatment-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the dosing period, treatment-related changes were noted as follows: higher values of albumin and A/G ratio in males treated with 1000 mg/kg; a higher value of ALAT in males treated with 250 mg/kg and above and females treated with 1000 mg/kg; a high value or higher tendency of total cholesterol and Ca in both sexes treated with 1000 mg/kg and a lower value of Na in males treated with 1000 mg/kg.
At the end of the recovery period, none of the above-mentioned changes was noted.
Besides, at the end of the dosing period, lower values of ALP and total bile acid were noted in females treated with 1000 mg/kg, attaining statistical significance. However, these were judged not to be treatment-related as the changes were opposite to the toxicity effect.
At the end of the recovery period, a lower value of glucose was noted in males treated with 1000 mg/kg as well as K in females treated with 1000 mg/kg. However, these were judged not to be treatment-related as the similar changes were not noted at the end of the dosing period and no related change was noted in any examination. Lower values of γGT, ALP and total bile acid were noted in males treated with 1000 mg/kg. However, these were judged not to be treatment-related as the changes were opposite to the toxicity effect.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
During the dosing period, pH was biased toward the acidic side in males treated with 1000 mg/kg and significant difference was noted when compared with Controls. An increase of urine volume was noted in males treated with 1000 mg/kg.
During the recovery period, none of the above-mentioned changes was noted.
Besides, high values of Na, K and Cl were noted in males treated with 1000 mg/kg during the recovery period. However, these were judged not to be treatment-related as the changes, although statistically different, were slight compared to those of the control group, the similar changes were not noted during the dosing period or no related change was noted in the histopathology.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
- Detailed clinical observations: No treatment-related change was noted in any animal in the hand held observation or observation on the open field.
The following changes were noted in the number of rearing: a low value in males treated with 1000 mg/kg on Day 35, a high value in females treated with 250 mg/kg and above on Day 7 and a low value in females treated with 1000 mg/kg on Days 35 and 42, with the differences from the control attained the statistical significance. These were transient changes and no related changes were noted in the other parameters. Therefore, it was judged that the changes were not related to the test material treatment.
- Sensory reactivity to stimuli: Reactivity to stimuli was comparable in males and females at all dose levels and no abnormality was observed.
- Grip strength: No significant difference was noted in males or females between the control and test material groups.
- Motor activity: No treatment-related change was noted.
A low value of motor activity was noted between 20 and 30 minutes in females treated with 1000 mg/kg (statistically significant compared to vehicle control). However, this was judged not to be treatment-related as the moving pattern was normal and no related change was noted in the detailed clinical observations. As a change without dose-dependency and considered as not treatment-related, a high value of motor activity was noted between 40 and 50 minutes in females treated with 62.5 mg/kg (statistically significant compared to vehicle control).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver:
- Hypertrophy of centrilobular hepatocytes was noted in 3/5 males and 1/5 females treated with 250 mg/kg (grade: minimal) and 5/5 males and 5/5 females treated with 1000 mg/kg (minimal or mild). After recovery, this finding was noted in only 1/5 satellite females treated with 1000 mg/kg (minimal).
Pituitary:
- Minimal hypertrophy of basophilic cells in the anterior lobe was noted in 3/5 males treated with 250 mg/kg and 5/5 males and 1/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
Thyroid gland:
- Minimal hypertrophy of follicular cells was noted in 2/5 males treated with 250 mg/kg and 5/5 males and 3/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
Adrenal gland:
- Minimal vacuolation of cortical cells in the fascicular zone was noted in 1/5 females treated with 250 mg/kg and 4/5 males and 5/5 females treated with 1000 mg/kg. After recovery, this finding was noted in only 1/5 satellite females treated with 1000 mg/kg (minimal).
- Minimal vacuolation of cortical cells in the glomerular zone was noted in 4/5 males treated with 250 mg/kg and 3/5 males and 5/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
- These vacuoles were positive for Oil red O staining.
Sternum:
- Minimal increase of trabecular bone was noted in 1/5 females treated with 250 mg/kg and 5/5 females treated with 1000 mg/kg. After recovery, this finding was noted in all satellite females treated with 1000 mg/kg (minimal).
Femur:
- Minimal increase of trabecular bone was noted in 1/5 males and 1/5 females treated with 250 mg/kg and 4/5 males and 5/5 females treated with 1000 mg/kg. After recovery, this finding was noted in 2/5 males and 5/5 satellite females treated with 1000 mg/kg (minimal).
Spleen:
- Minimal erythrocytic extramedullary hematopoiesis was noted in 1/5 males and 1/5 females treated with 250 mg/kg and 1/5 males and 4/5 females treated with 1000 mg/kg. After recovery, this finding was noted in 1/5 males and 1/5 satellite females treated with 1000 mg/kg (minimal).
Cecum:
- Minimal thickening of mucosa was noted in 1/5 males and 3/5 females treated with 250 mg/kg and 2/5 males and 4/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
Kidney:
- Mild basophilic tubule was noted in 1/5 males treated with 1000 mg/kg. After recovery, this finding was not noted.
Stomach:
- Minimal mineralisation of mucosa in the glandular stomach was noted in 1/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
- Minimal erosion in the limiting ridge was noted in 1/5 males treated with 250 mg/kg and 2/5 males treated with 1000 mg/kg. After recovery, this finding was not noted.
- Minimal hyperkeratosis in the limiting ridge was noted in 3/5 males treated with 1000 mg/kg. After recovery, this finding was not noted.
- Minimal parakeratosis in the limiting ridge was noted in 3/5 males treated with 1000 mg/kg. After recovery, this finding was not noted.
- Minimal inflammatory cell infiltration in the submucosa of limiting ridge was noted in 2/5 males treated with 1000 mg/kg. After recovery, this finding was not noted.
- Minimal vacuolation of squamous epithelium in the limiting ridge was noted in 1/5 males treated with 1000 mg/kg. After recovery, this finding was not noted.
Lung:
- Minimal accumulation of foam cells in the alveolus was noted in 1/5 females treated with 250 mg/kg and 3/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
- Fine vacuoles of the foam cells were positive for Oil red O staining.
Heart:
- Minimal mineralisation of aortic wall was noted in 2/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.
Thymus:
- Minimal atrophy was noted in 1/5 females treated with 1000 mg/kg. After recovery, this finding was not noted.

> [Dead animal (No. 693) treated with 1000 mg/kg]
Thymus: Moderate atrophy was noted.
Spleen: Mild atrophy was noted.
Liver: Minimal hypertrophy of centrilobular hepatocytes was noted.
Kidney: Mild vacuolation of proximal tubular epithelium was noted. The vacuoles were positive for Oil red O staining.
Sternum: Minimal increase of trabecular bone was noted.
Femur: Minimal increase of trabecular bone was noted.

> Others:
Various histopathological changes were noted in both sexes of the control and test material groups. However, these changes were judged not to be treatment-related as they are observed occasionally in normal rats and there was no clear dose-dependency in the incidence.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Mass in the axilla noted in 1 female (No. 698) treated with 1000 mg/kg was adenocarcinoma of the mammary gland in the histopathology. Adenocarcinoma of mammary gland is known to occur infrequently in young mature rats and no gross abnormalities were noted in the mammary gland of any other animals, therefore, it was judged not to be treatment-related.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
- Hormone measurement:
In parental animals (F0 male), plasma total T4 concentrations of the test material-treated group (Group 1000 mg/kg) tended to be lower compared to those in the control group after the dosing period. In parental animals (F0 female), plasma total T4 concentrations of the test material-treated groups (Groups 62.5, 250, and 1000 mg/kg) were equivalent to those of the control groups. The plasma total T4 concentration of recovery groups (Group 1000 mg/kg) were equivalent to those of the control groups in F0 male and Satellite female.
All values in the quality control sample were within the range of 100 ± 25% of the nominal value, and variations in duplicate cpm of the standard solutions were within the range of acceptability. There were no abnormalities in the procedures for the determination or in the values of the test samples. The results were therefore considered acceptable.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No significant difference was noted in the mean oestrous cycle or count of oestrus, indicating no effect of the test material on prolongation or shortening of the oestrous cycle between the control and test material groups.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
- Mating: No significant difference was noted in the copulation index, duration of mating or fertility index between the control and test material groups.
- Observation during delivery and lactation periods: No abnormality was noted in the delivery or nursing. No significant difference was noted in the gestation length, number of implantations and litter, delivery index or gestation index between the control and test substance groups.
One female (No. 691) treated with 1000 mg/kg, which had only one implantation, did not deliver. However, this was judged not to be treatment-related due to its low incidence.
Dose descriptor:
NOAEL
Remarks:
(systemic toxicity)
Effect level:
62.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: See 'Remarks'
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (nominal)
System:
other: effects on calcium metabolism and injury of the kidney, hepatic effects, effects on bone/lipid metabolism, and reaction to anaemia.
Organ:
kidney
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
no effects observed
Description (incidence and severity):
No abnormality was observed in any offspring following external examination.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
No significant difference was noted in viability index on PNDs 4 or 13 between the control and test material groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant low value of body weight was noted in both sexes of the 1000 mg/kg group on PND 13.
Besides, a high value of body weight was noted in females of the 62.5 mg/kg group on PNDs 7 and 13. However, this was judged not to be treatment-related as there was no dose-dependency.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
No significant difference was noted in males or females between the control and test material groups.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No significant difference was noted in the anomaly index between the control and test material groups.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related change was noted in any offspring.
Whitish patch of the liver was noted in one female of the 1000 mg/kg group (offspring No. F04 in dam No. 698). However, this was judged not to be treatment-related due to its low incidence.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No significant difference was noted in the number of litter, live newborns and stillborn, birth index, sex ratio or viability index on PNDs 4 or 13 between the control and test material groups.

- Hormone concentration (total T4) analysis [Offspring on PND 13]
No significant difference was noted between the control and test material groups.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Generation:
F1
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Remarks:
(developmental toxicity)
Generation:
F1
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Critical effects observed:
no
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
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:
not specified

- PARENTAL GENERATION

Summary of bodyweights, g (mean ± SD)

Day

Male

Female

Dose (mg/kg bw/day) (no. of animals)

0 (12)

62.5 (12)

250 (12)

1000 (12)

0 (17)

62.5 (12)

250 (12)

1000 (17)

1

370.18 ± 11.31

368.34 ± 10.91

368.61 ± 13.93

367.61 ± 11.12

230.74 ± 10.20

231.73 ± 9.27

231.80 ± 12.70

233.05 ± 11.49

8

411.53 ± 17.39

403.28 ± 17.29

404.06 ± 15.94

386.56 ± 15.33**

248.94 ± 9.37

246.68 ± 11.24

247.23 ± 14.02

244.68 ± 10.47

15

440.40 ± 20.25

426.15 ± 26.46

426.96 ± 20.61

408.28 ± 17.71**

258.92 ± 13.60

256.73 ± 11.96

258.48 ± 15.67

255.48 ± 10.28

22

465.89 ± 20.21

453.23 ± 32.50

453.22 ± 26.16

425.06 ± 18.46**

263.62 ± 9.64

256.60 ± 17.43

29

495.29 ± 22.27

476.41 ± 38.61

476.37 ± 30.82

451.31 ± 20.10**

267.86 ± 7.71

269.38 ± 11.31

36

519.59 ± 22.65

497.98 ± 42.47

498.53 ± 33.35

469.81 ± 22.99**

280.20 ± 9.14

278.80 ± 17.93

42

536.88 ± 21.91

514.63 ± 39.88

511.93 ± 35.54

484.18 ± 25.06**

286.10 ± 9.73

284.80 ± 21.75

43

537.84 ± 24.80

479.94 ± 31.80*

284.56 ± 13.69

283.80 ± 17.71

50

553.80 ± 24.88

494.18 ± 34.65*

292.34 ± 9.20

291.18 ± 17.98

56

562.62 ± 24.74

511.18 ± 32.89*

296.32 ± 10.78

301.28 ± 19.97

Significantly different from the control group ; * p < 0.05, ** p < 0.01 (Dunnett test)

Results in itallics relate to 5 animals

Day 1 to 42; Treatment period, Day 43 to 56; Recovery period

Summary of female bodyweights, g (mean ± SD)

Day

Dose (mg/kg bw/day) (no. of animals)

0 (12)

62.5 (12)

250 (12)

1000 (12)

Gestation period

0

266.18 ± 16.28

266.75 ± 13.71

266.59 ± 18.10

263.90 ± 13.36

7

301.64 ± 20.12

300.63 ± 16.86

298.92 ± 18.85

284.27 ± 16.47

14

336.97 ± 25.50

340.59 ± 21.19

342.07 ± 19.36

316.99 ± 19.42

20

419.53 ± 33.02

419.03 ± 32.66

425.38 ± 24.23

379.78 ± 38.42*

Lactation period

0

325.03 ± 30.56

329.48 ± 22.57

335.88 ± 19.11

303.72 ± 23.02

4

340.01 ± 31.55

343.58 ± 21.36

351.46 ± 20.97

322.94 ± 25.21

7

346.39 ± 31.44

348.66 ± 19.08

354.37 ± 24.07

336.30 ± 23.07

13

354.93 ± 33.29

361.38 ± 15.85

368.46 ± 19.88

359.01 ± 21.42

- OFFSPRING

Summary of bodyweights in offspring, g (mean ± SD)

Parameter

Sex

Dose (mg/kg bw/day) (no. of animals)

0 (12)

62.5 (12)

250 (12)

1000 (11)

Day 0

No of pups

Male

87

70

82

61

Female

80

86

84

76

Mean pup weight

Male

7.5 ± 0.5

7.8 ± 0.6 (11)

7.4 ± 0.7

6.8 ± 0.7

Female

7.0 ± 0.4

7.4 ± 0.8

7.1 ± 0.7

6.6 ± 0.7

Day 4

No of pups

Male

87

69

82

58

Female

80

85

84

70

Mean pup weight

Male

12.0 ± 1.0

12.9 ± 1.3 (11)

12.5 ± 1.6

11.2 ± 1.7 (10)

Female

11.2 ± 0.9

12.3 ± 1.8

11.8 ± 1.4

10.7 ± 1.4 (10)

Day 7

No of pups

Male

49

44

48

38

Female

47

46

48

42

Mean pup weight

Male

20.2 ± 1.8

21.9 ± 1.8 (11)

20.9 ± 2.0

18.9 ± 2.3 (10)

Female

18.6 ± 1.5

20.9 ± 2.2*

19.8 ± 2.0

17.5 ± 1.8 (10)

Day 13

No of pups

Male

49

44

48

38

Female

47

46

48

42

Mean pup weight

Male

37.3 ± 2.8

40.0 ± 2.7 (11)

38.0 ± 2.5

33.8 ± 2.5** (10)

Female

35.3 ± 2.2

38.4 ± 2.9**

37.2 ± 1.8

31.6 ± 2.5** (10)

Significantly different from the control group ; * p < 0.05, ** p < 0.01 (Dunnett test)

Number in parentheses indicates the number of litters.

Conclusions:
The NOAEL for reproductive and developmental toxicity was judged to be 250 mg/kg due to decreased body weight of F1 offspring at 1000 mg/kg on PND 13.
Executive summary:

The reproductive toxicity of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 422 and the Japanese Testing Methods Concerning New Chemical Substances, and under GLP conditions.

During the study, the test material was repeatedly administered by oral gavage at 0 (control group), 62.5, 250 and 1000 mg/kg from 14 days before mating through mating for 42 days in males and satellite females, and from 14 days before mating through gestation and parturition until Day 13 of lactation in females to assess the repeated dose toxicity and reproductive and developmental toxicity. In addition, a 14-day recovery period was set for the control and 1000 mg/kg groups and the reversibility of the toxicity effect was assessed.

One dam treated with 1000 mg/kg was found dead on LD 2. In the dead animal, no other remarkable change was observed in the clinical observation. In the histopathology, the following changes were noted in this animal: moderate atrophy of the thymus, mild atrophy of the spleen, mild vacuolation of proximal tubular epithelium (positive for Oil red O staining, indicating that the vacuoles were lipid), minimal hypertrophy of centrilobular hepatocytes and minimal increase of trabecular bone in the sternum and femur. The cause of death remains unclear; however, it was possible that the death occurred due to overlapping effects of the test material and postpartum stress.

In the surviving animals, a lower body weight was noted in males treated with 1000 mg/kg throughout the dosing period, relative to the concurrent vehicle control. A lower body weight was noted in females treated with 1000 mg/kg on GD 20. Food consumption in both sexes treated with 1000 mg/kg was lower than the control during the first week of treatment.

Adaptive physiological responses attributable to the test material treatment were noted at 250 mg/kg and above and this is not considered to be adverse in nature. The related changes were noted as follows: enlargement of the liver in males treated with 1000 mg/kg; a higher liver weight in males treated with 250 mg/kg and above and females treated with 1000 mg/kg; a higher thyroid weight in females treated with 1000 mg/kg; minimal or mild hypertrophy of centrilobular hepatocytes with a tendency of increasing incidence and severity, and minimal hypertrophy of basophilic cells in the anterior lobe of the pituitary in both sexes treated with 250 mg/kg and above; minimal hypertrophy of follicular cells of the thyroid in males treated with 250 mg/kg and above and females treated with 1000 mg/kg and a low total T4 level in males treated with 1000 mg/kg. However, the above-mentioned pathological changes in males treated with 250 mg/kg and above and females treated with 1000 mg/kg were judged to be toxic effects as the blood chemistry revealed the effects on protein/lipid metabolism in both sexes treated with 1000 mg/kg and increased ALAT in males treated with 250 mg/kg above and females treated with 1000 mg/kg. Increased calcium level was noted in both sexes treated with 1000 mg/kg, suggesting an association with hyperthyroidism.

Minimal increase of trabecular bone in the sternum and femur was noted in females treated with 250 mg/kg and above and both sexes treated with 250 mg/kg and above, respectively. The cause remains unclear.

Lipid accumulation in the cells or dysbolism of lipid was noted in the adrenal and alveolus of the lung; however, the cause remains unclear. Related changes were noted as follows. A high total cholesterol level was noted in both sexes treated with 1000 mg/kg. A higher adrenal weight was noted in females treated with 1000 mg/kg.

Histopathological examination revealed the following: minimal vacuolation of cortical cells in the fascicular zone of the adrenal in males treated with 1000 mg/kg and females treated with 250 mg/kg and above; minimal vacuolation of cortical cells in the glomerular zone of the adrenal in males treated with 250 mg/kg and above and females treated with 1000 mg/kg and minimal accumulation of foam cells in the alveolus of the lung in females treated with 250 mg/kg and above. The vacuoles in the adrenal and fine vacuoles in the alveolus of the lung were positive for Oil red O staining. The changes suggesting the irritation of the test material were noted in the stomach and the cecum. In the stomach, the following changes were noted in the limiting ridge (all findings were of minimal severity): erosion in males treated with 250 mg/kg and above; hyperkeratosis, parakeratosis, inflammatory cell infiltration of submucosa and vacuolation of squamous epithelium in males treated with 1000 mg/kg. In the cecum, minimal thickening of mucosa was noted in both sexes treated with 250 mg/kg and above.

The damage to the tubular epithelium in the kidney was noted. In the urinalysis, pH was biased toward the acidic side and urine volume increased in males treated with 1000 mg/kg. In the blood chemistry, a low Na level was noted, but slightly, in males treated with 1000 mg/kg. A higher kidney weight was noted in both sexes treated with 1000 mg/kg. In the histopathology, mild basophilic tubule was noted in only one male treated with 1000 mg/kg.

Dysbolism of calcium was noted: a high calcium level was noted in both sexes treated with 1000 mg/kg in the blood chemistry and minimal mineralisation of aortic wall and mucosa in the glandular stomach was noted in females treated with 1000 mg/kg. Slight anaemia was noted in both sexes treated with 1000 mg/kg. In the spleen, minimal erythrocytic extramedullary hematopoiesis was noted in both sexes treated with 250 mg/kg and above, indicating a reaction to anaemia.

A lower thymus weight was noted in females treated with 1000 mg/kg. In the histopathology, minimal atrophy of the thymus was noted in one female treated with 1000 mg/kg. It was judged to be a secondary reaction associated with stress as this animal had a suppressed body weight gain.

In the recovery test, among the changes noted at the end of the dosing period, except for the changes noted in the sternum and femur, reversibility or a tendency to recover was observed.

In the sternum, minimal increase of trabecular bone was noted in satellite females treated with 1000 mg/kg and the incidence was similar to that of females treated with 1000 mg/kg at termination. In the femur, minimal increase of trabecular bone was noted in males for recovery test and satellite females treated with 1000 mg/kg. The incidence in males for recovery test was lower than that of males treated with 1000 mg/kg at termination, while the incidence in satellite females was similar to that of females treated with 1000 mg/kg at termination. These changes may be reversible if the recovery period is extended.

A lower value of body weight was noted in males for recovery test treated with 1000 mg/kg throughout the recovery period. However, body weight gains during the recovery period were similar to or higher than that of Controls, indicating reversibility.

A higher value of the liver weight was noted in satellite females treated with 1000 mg/kg. Minimal hypertrophy of centrilobular hepatocytes was noted in one satellite female treated with 1000 mg/kg and the incidence and severity were lower than those of females treated with 1000 mg/kg at termination. This change was not noted in males for recovery test, indicating reversibility.

In the adrenal gland, minimal vacuolation of cortical cells in the fascicular zone was noted in one satellite female treated with 1000 mg/kg. The incidence was lower than that of females treated with 1000 mg/kg at termination. This change was not noted in males for recovery test, indicating reversibility. Minimal vacuolation of cortical cells in the glomerular zone was not noted in any animal after recovery, indicating reversibility.

A higher value of the spleen weight was noted in males for recovery test treated with 1000 mg/kg. Minimal erythrocytic extramedullary haematopoiesis was noted in one male for recovery test and one satellite female treated with 1000 mg/kg. The incidence in satellite females was lower than that of females treated with 1000 mg/kg at termination. On the other hand, the incidence in males for recovery test was similar to that of males treated with 1000 mg/kg at termination and haematological examination also revealed anemia in males for recovery test. However, the severity was slight and increased hematopoiesis was noted, indicating a tendency to recover.

A higher kidney weight was noted in both sexes treated with 1000 mg/kg. However, this was judged not to be toxicologically significant as no related change was noted in any examination.

There were no effects on parental reproductive parameters.

In offspring, a low value of body weight was noted in both sexes at 1000 mg/kg on PND 13. However, since the viability was not affected, the toxic effect on offspring was judged not to be severe.

Under the conditions of the study, the No-Observed-Adverse-Effect Level (NOAEL) for repeated dose toxicity was estimated to be 62.5 mg/kg bw/day.

The NOAEL for reproductive and developmental toxicity was judged to be 250 mg/kg bw/day due to decreased body weight of F1 offspring at 1000 mg/kg bw/day on PND 13.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A guideline study is available to address the REACH endpoint. The study was conducted under GLP conditions and is assigned a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997). The quality of the database is therefore good.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The reproductive toxicity of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 422 and the Japanese Testing Methods Concerning New Chemical Substances, and under GLP conditions.

During the study, the test material was repeatedly administered by oral gavage at 0 (control group), 62.5, 250 and 1000 mg/kg from 14 days before mating through mating for 42 days in males and satellite females, and from 14 days before mating through gestation and parturition until Day 13 of lactation in females to assess the repeated dose toxicity and reproductive and developmental toxicity. In addition, a 14-day recovery period was set for the control and 1000 mg/kg groups and the reversibility of the toxicity effect was assessed.

One dam treated with 1000 mg/kg was found dead on LD 2. In the dead animal, no other remarkable change was observed in the clinical observation. In the histopathology, the following changes were noted in this animal: moderate atrophy of the thymus, mild atrophy of the spleen, mild vacuolation of proximal tubular epithelium (positive for Oil red O staining, indicating that the vacuoles were lipid), minimal hypertrophy of centrilobular hepatocytes and minimal increase of trabecular bone in the sternum and femur. The cause of death remains unclear; however, it was possible that the death occurred due to overlapping effects of the test material and postpartum stress.

In the surviving animals, a lower body weight was noted in males treated with 1000 mg/kg throughout the dosing period, relative to the concurrent vehicle control. A lower body weight was noted in females treated with 1000 mg/kg on GD 20. Food consumption in both sexes treated with 1000 mg/kg was lower than the control during the first week of treatment.

Adaptive physiological responses attributable to the test material treatment were noted at 250 mg/kg and above and this is not considered to be adverse in nature. The related changes were noted as follows: enlargement of the liver in males treated with 1000 mg/kg; a higher liver weight in males treated with 250 mg/kg and above and females treated with 1000 mg/kg; a higher thyroid weight in females treated with 1000 mg/kg; minimal or mild hypertrophy of centrilobular hepatocytes with a tendency of increasing incidence and severity, and minimal hypertrophy of basophilic cells in the anterior lobe of the pituitary in both sexes treated with 250 mg/kg and above; minimal hypertrophy of follicular cells of the thyroid in males treated with 250 mg/kg and above and females treated with 1000 mg/kg and a low total T4 level in males treated with 1000 mg/kg. However, the above-mentioned pathological changes in males treated with 250 mg/kg and above and females treated with 1000 mg/kg were judged to be toxic effects as the blood chemistry revealed the effects on protein/lipid metabolism in both sexes treated with 1000 mg/kg and increased ALAT in males treated with 250 mg/kg above and females treated with 1000 mg/kg. Increased calcium level was noted in both sexes treated with 1000 mg/kg, suggesting an association with hyperthyroidism.

Minimal increase of trabecular bone in the sternum and femur was noted in females treated with 250 mg/kg and above and both sexes treated with 250 mg/kg and above, respectively. The cause remains unclear.

Lipid accumulation in the cells or dysbolism of lipid was noted in the adrenal and alveolus of the lung; however, the cause remains unclear. Related changes were noted as follows. A high total cholesterol level was noted in both sexes treated with 1000 mg/kg. A higher adrenal weight was noted in females treated with 1000 mg/kg.

Histopathological examination revealed the following: minimal vacuolation of cortical cells in the fascicular zone of the adrenal in males treated with 1000 mg/kg and females treated with 250 mg/kg and above; minimal vacuolation of cortical cells in the glomerular zone of the adrenal in males treated with 250 mg/kg and above and females treated with 1000 mg/kg and minimal accumulation of foam cells in the alveolus of the lung in females treated with 250 mg/kg and above. The vacuoles in the adrenal and fine vacuoles in the alveolus of the lung were positive for Oil red O staining. The changes suggesting the irritation of the test material were noted in the stomach and the cecum. In the stomach, the following changes were noted in the limiting ridge (all findings were of minimal severity): erosion in males treated with 250 mg/kg and above; hyperkeratosis, parakeratosis, inflammatory cell infiltration of submucosa and vacuolation of squamous epithelium in males treated with 1000 mg/kg. In the cecum, minimal thickening of mucosa was noted in both sexes treated with 250 mg/kg and above.

The damage to the tubular epithelium in the kidney was noted. In the urinalysis, pH was biased toward the acidic side and urine volume increased in males treated with 1000 mg/kg. In the blood chemistry, a low Na level was noted, but slightly, in males treated with 1000 mg/kg. A higher kidney weight was noted in both sexes treated with 1000 mg/kg. In the histopathology, mild basophilic tubule was noted in only one male treated with 1000 mg/kg.

Dysbolism of calcium was noted: a high calcium level was noted in both sexes treated with 1000 mg/kg in the blood chemistry and minimal mineralisation of aortic wall and mucosa in the glandular stomach was noted in females treated with 1000 mg/kg. Slight anaemia was noted in both sexes treated with 1000 mg/kg. In the spleen, minimal erythrocytic extramedullary hematopoiesis was noted in both sexes treated with 250 mg/kg and above, indicating a reaction to anaemia.

A lower thymus weight was noted in females treated with 1000 mg/kg. In the histopathology, minimal atrophy of the thymus was noted in one female treated with 1000 mg/kg. It was judged to be a secondary reaction associated with stress as this animal had a suppressed body weight gain.

In the recovery test, among the changes noted at the end of the dosing period, except for the changes noted in the sternum and femur, reversibility or a tendency to recover was observed.

In the sternum, minimal increase of trabecular bone was noted in satellite females treated with 1000 mg/kg and the incidence was similar to that of females treated with 1000 mg/kg at termination. In the femur, minimal increase of trabecular bone was noted in males for recovery test and satellite females treated with 1000 mg/kg. The incidence in males for recovery test was lower than that of males treated with 1000 mg/kg at termination, while the incidence in satellite females was similar to that of females treated with 1000 mg/kg at termination. These changes may be reversible if the recovery period is extended.

A lower value of body weight was noted in males for recovery test treated with 1000 mg/kg throughout the recovery period. However, body weight gains during the recovery period were similar to or higher than that of Controls, indicating reversibility.

A higher value of the liver weight was noted in satellite females treated with 1000 mg/kg. Minimal hypertrophy of centrilobular hepatocytes was noted in one satellite female treated with 1000 mg/kg and the incidence and severity were lower than those of females treated with 1000 mg/kg at termination. This change was not noted in males for recovery test, indicating reversibility.

In the adrenal gland, minimal vacuolation of cortical cells in the fascicular zone was noted in one satellite female treated with 1000 mg/kg. The incidence was lower than that of females treated with 1000 mg/kg at termination. This change was not noted in males for recovery test, indicating reversibility. Minimal vacuolation of cortical cells in the glomerular zone was not noted in any animal after recovery, indicating reversibility.

A higher value of the spleen weight was noted in males for recovery test treated with 1000 mg/kg. Minimal erythrocytic extramedullary haematopoiesis was noted in one male for recovery test and one satellite female treated with 1000 mg/kg. The incidence in satellite females was lower than that of females treated with 1000 mg/kg at termination. On the other hand, the incidence in males for recovery test was similar to that of males treated with 1000 mg/kg at termination and haematological examination also revealed anemia in males for recovery test. However, the severity was slight and increased hematopoiesis was noted, indicating a tendency to recover.

A higher kidney weight was noted in both sexes treated with 1000 mg/kg. However, this was judged not to be toxicologically significant as no related change was noted in any examination.

There were no effects on parental reproductive parameters.

In offspring, a low value of body weight was noted in both sexes at 1000 mg/kg on PND 13. However, since the viability was not affected, the toxic effect on offspring was judged not to be severe.

Under the conditions of the study, the No-Observed-Adverse-Effect Level (NOAEL) for repeated dose toxicity was estimated to be 62.5 mg/kg based on occurrence of death, decreased body weight, effects on calcium metabolism and injury of the kidney occurring at 1000 mg/kg and hepatic effects, effects on bone/lipid metabolism, injury of stomach and cecum and reaction to anaemia occurring at 250 mg/kg and above.

The NOAEL for reproductive and developmental toxicity was judged to be 250 mg/kg due to decreased body weight of F1 offspring at 1000 mg/kg on PND 13.

Effects on developmental toxicity

Description of key information

The NOAEL for reproductive and developmental toxicity was judged to be 250 mg/kg bw/day due to decreased body weight of F1 offspring at 1000 mg/kg bw/day on PND 13.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A guideline study is available to address the REACH endpoint. The study was conducted under GLP conditions and is assigned a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997). The quality of the database is therefore good.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The developmental toxicity of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 422 and the Japanese Testing Methods Concerning New Chemical Substances, and under GLP conditions.

During the study, the test material was repeatedly administered by oral gavage at 0 (control group), 62.5, 250 and 1000 mg/kg from 14 days before mating through mating for 42 days in males and satellite females, and from 14 days before mating through gestation and parturition until Day 13 of lactation in females to assess the repeated dose toxicity and reproductive and developmental toxicity. In addition, a 14-day recovery period was set for the control and 1000 mg/kg groups and the reversibility of the toxicity effect was assessed.

One dam treated with 1000 mg/kg was found dead on LD 2. In the dead animal, no other remarkable change was observed in the clinical observation. In the histopathology, the following changes were noted in this animal: moderate atrophy of the thymus, mild atrophy of the spleen, mild vacuolation of proximal tubular epithelium (positive for Oil red O staining, indicating that the vacuoles were lipid), minimal hypertrophy of centrilobular hepatocytes and minimal increase of trabecular bone in the sternum and femur. The cause of death remains unclear; however, it was possible that the death occurred due to overlapping effects of the test material and postpartum stress.

In the surviving animals, a lower body weight was noted in females treated with 1000 mg/kg on GD 20. Food consumption in females treated with 1000 mg/kg was lower than the control during the first week of treatment.

Adaptive physiological responses attributable to the test material treatment were noted at 250 mg/kg and above and this is not considered to be adverse in nature. The related changes were noted as follows: a higher liver weight in females treated with 1000 mg/kg; a higher thyroid weight in females treated with 1000 mg/kg; minimal or mild hypertrophy of centrilobular hepatocytes with a tendency of increasing incidence and severity, and minimal hypertrophy of basophilic cells in the anterior lobe of the pituitary in animals treated with 250 mg/kg and above; minimal hypertrophy of follicular cells of the thyroid in females treated with 1000 mg/kg. However, the above-mentioned pathological changes in females treated with 1000 mg/kg were judged to be toxic effects as the blood chemistry revealed the effects on protein/lipid metabolism in animals treated with 1000 mg/kg and increased ALAT in females treated with 1000 mg/kg. Increased calcium level was noted in females treated with 1000 mg/kg, suggesting an association with hyperthyroidism.

Minimal increase of trabecular bone in the sternum and femur was noted in females treated with 250 mg/kg and above. The cause remains unclear.

Lipid accumulation in the cells or dysbolism of lipid was noted in the adrenal and alveolus of the lung; however, the cause remains unclear. Related changes were noted as follows. A high total cholesterol level was noted in animals treated with 1000 mg/kg. A higher adrenal weight was noted in females treated with 1000 mg/kg.

Histopathological examination revealed the following: minimal vacuolation of cortical cells in the fascicular zone of the adrenal in females treated with 250 mg/kg and above; minimal vacuolation of cortical cells in the glomerular zone of the adrenal in females treated with 1000 mg/kg and minimal accumulation of foam cells in the alveolus of the lung in females treated with 250 mg/kg and above. The vacuoles in the adrenal and fine vacuoles in the alveolus of the lung were positive for Oil red O staining. The changes suggesting the irritation of the test material were noted in the stomach and the cecum. In the cecum, minimal thickening of mucosa was noted in animals treated with 250 mg/kg and above.

The damage to the tubular epithelium in the kidney was noted. A higher kidney weight was noted in animals treated with 1000 mg/kg.

Dysbolism of calcium was noted: a high calcium level was noted in animals treated with 1000 mg/kg in the blood chemistry and minimal mineralisation of aortic wall and mucosa in the glandular stomach was noted in females treated with 1000 mg/kg. Slight anaemia was noted in animals treated with 1000 mg/kg. In the spleen, minimal erythrocytic extramedullary hematopoiesis was noted in animals treated with 250 mg/kg and above, indicating a reaction to anaemia.

A lower thymus weight was noted in females treated with 1000 mg/kg. In the histopathology, minimal atrophy of the thymus was noted in one female treated with 1000 mg/kg. It was judged to be a secondary reaction associated with stress as this animal had a suppressed body weight gain.

In the recovery test, among the changes noted at the end of the dosing period, except for the changes noted in the sternum and femur, reversibility or a tendency to recover was observed.

There were no effects on parental reproductive parameters.

In offspring, a low value of body weight was noted in both sexes at 1000 mg/kg on PND 13. However, since the viability was not affected, the toxic effect on offspring was judged not to be severe.

No abnormality was noted in the delivery or nursing. No significant difference was noted in the gestation length, number of implantations and litter, delivery index or gestation index between the control and test substance groups. No significant difference was noted in the number of litter, live newborns and stillborn, birth index, sex ratio or viability index on PNDs 4 or 13 between the control and test material groups. No abnormality was observed in any offspring and no significant difference was noted in the anomaly index (concerning nipple development) between the control and test material groups.

Under the conditions of the study, the No-Observed-Adverse-Effect Level (NOAEL) for repeated dose toxicity was estimated to be 62.5 mg/kg bw/day.

The NOAEL for reproductive and developmental toxicity was judged to be 250 mg/kg bw/day due to decreased body weight of F1 offspring at 1000 mg/kg bw/day on PND 13.

Justification for classification or non-classification

Effects seen in the study are not of concern for reprotoxicity. The offspring are born slightly lighter at the high dose, but the maternal weight gain is also lower in gestation for this group, suggesting effects are secondary to the maternal toxicity. The effect in the offspring persists until PND13 (weight and gain remaining around 10 % less than controls in both sexes) but there are no adverse consequences to development, with no impact on survival or any other parameters evaluated.

Overall, in accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification for reproductive or developmental toxicity.

Additional information