Naphthalene-2,6-dicarboxylic acid

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Test Start Date December 4, 2014 - Study End Date March 20, 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

repeated dose toxicity: oral, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Test Start Date December 4, 2014 - Study End Date March 20, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This is a study performed according to Japanese guidelines, was written in Japanese and translated for use in this submittal, some information was sanitized (i.e., kept confidential) before publication by the Ministry of Health, Labor and Welfare Pharmaceutical Food Bureau of Japan

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

"About the method of testing related ot new chemical substances" (March 31, 2011, Department of Medicine Diet 0331 No. 7 Heisei 23, 03 29 Station No. 5. Personal Protection No. 110331009). regarding the partial revision of "Regarding the method of testing related to new chemical substances" (April 2, 2012. Pharmaceutical Diet 0402. No. 1. Heisei 24, 03 28 Mfg. No. 2 Personal Protection Plan No. 120402001. Ministry of Health, Labor and Welfare Director of the Pharmaceutical Foods Division, Ministry of Economy, Trade and Industry Director of Manufacturing Industry Bureau Director of the Environmental Policy Bureau. Directorate of the Environment).

Deviations:

no

GLP compliance:

yes

Remarks:

"Regarding standards related to the test facilities for testing new chemical substances" (March 31, 2011. Pharmaceutical Diet 0331. No. 8 Heisei 23, 03 29 Mfg. No. 6 Personal Protection No. 110331010 MHLW Director, Ministry of Economy

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: J82SE
- Concentration and homogeneity of the dosing solutions: confirmed
- Physicochemical properties: white to slightly thin yellow, crystal to powder. Melting point > 300°C
- Purity test date: Test Substance received October 20, 2014 with purity of 99.7%, Initial confirmation of purity November 17, 2014 (100.3%), final confirmation of purity February 3, 2015 (100%)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (measured as 5.0 to 9.4 degrees celcius) in airtight bottles, dark
- Stability under test conditions: Stable, no measured degradation
- Solubility and stability of the test substance in the solvent/vehicle: Soluble, stability in dosing solution analytically verified


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Prepared in gavage dosing solution: (1% w/v) methyl cellulose aqueous solution
- Preliminary purification step (if any): none

FORM AS APPLIED IN THE TEST: prepared and administered in gavage dosing solution

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Japan Charles River Company, Ltd.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks
- Weight at study initiation: males 240 to 330 g, females 160 to 230 g
- Housing: In general, suspended wire mesh cages. With respect to mating female animals, from the 17th day of pregnancy to 4th day of nursing, the wire mesh floor of the same cage was replaced with a small saucer as a floor covering for experimental animals (white Flake, Japan Charles River Co., Ltd.) was used.

Number of housed animals per cage: two animals during the quarantine and acclimatization period, one mouse after group assignments, one each of male and female during the mating period, and 1 each cage after delivery.

- Diet: ad libitum
- Water: ad libitum
- Acclimation period: two weeks

DETAILS OF FOOD AND WATER QUALITY
Food supplied by Oriental yeast Co., Ltd. Feed type CRF-1. Animals fasted before necropsy. Each lot of feed was analyzed for the presence or absence of contaminants etc. that may adversely affect the test. Analysis was conducted by Eurofins Food and Product Testing Co., Ltd. (Analysis Report: AR - 14 - JP - 005160 - 01, AR - 14 - JP - 007307 - 01) and a feed manufacturer (Analytical Test Report: 14G03-151, 14G03-164), and analytical data were obtained from the feed manufacturer on a lot-by-lot basis. Analysis items and tolerance values conformed to the standard operating procedure manual of Compound Safety Research Institute Co., Ltd. As a result of the analysis, no value exceeding the allowable value was found in any item.

Sapporo city tap water ad libitum using automatic water supply device.
Analyses for contaminants that may adversely affect the test were performed. Duplicate samples were taken from the terminal end (Room 306) of the same breeding room and the same system piping as that breeding room on July 1, 2014 and January 7, 2015. Analysis was conducted by Nippon Sanitary Co., Ltd. (Water quality test result table: No. A 261406, A 265505). Analysis items and tolerance values conformed to the standard operating procedure manual of the performing laboratory (Compound Safety Research Institute Co., Ltd). As a result of the analysis, no value exceeding the allowable value was found in any item.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): measured range 40 to 49%
- Air changes (per hr): 10 to 15 changes/hr
- Photoperiod (hrs dark / hrs light): 12 light/12 hr dark with artificial lighting

IN-LIFE DATES: From: To: December 24, 2014 to February 5th, 2015

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% CMC in water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

OTHER SPECIFICS
Gavage solutions for administration were prepared at intervals of 4 to 6 days and used within 6 days. The gavage dosing solutions were stored in a refrigerator in the test substance storage room from December 16, 2014 (preparation for uniformity and stability analysis) until February 13, 2015 (final dose administration).

CONFIRMATION OF UNIFORMITY AND STABILITY OF ADMINISTRATION SOLUTION
Solutions were prepared at concentrations of 10 and 100 mg/mL, stability was confirmed for homogeneity and refrigeration storage 6 days (the preparation day was calculated as 0 day) + room temperature for 4 hours. In the results of the homogeneity analysis, the 10 and 100 mg/mL preparation had relative standard deviations of 1.5% and 0.8%, respectively, less than 5.0% of the criteria. As a result of the stability analysis, it was confirmed that the preparation solutions of 10 and 100 mg/mL had residual ratios of 94.9% and 97.1%, respectively, within 100 ± 10.0% of the judgment criteria.

CONFIRMATION OF CONCENTRATION OF DOSING SOLUTION
The concentration of the test substance in the dosing solution with respect to the total concentration was confirmed with respect to the preparation solution at the time of preparation of the final round, which is used for the first and all groups. In the analysis results, the contents of the 10, 30 and 100 mg/mL dosing solutions were 93.3%, 90.7% and 95.2% for the first time, 104.0%, 107.0% and 107.0% for the last round, respectively, and the relative standard deviations were, 0.6% and 0.8% at the first round, 1.4%, 1.0% and 0.6% at the final round, which were within 100 ± 10% and 5.0% of the criteria.

Frequency of treatment:

Daily from 9:10 until 11:46
Males: 28-days
Females: 14 days before mating, during the mating period until mating, during gestation and 4 days after delivering F1 generation

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment: random
- Rationale for selecting satellite groups: Guideline
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale: random

Positive control:

None

Observations and examinations performed and frequency:

General condition observation: all animals were observed from Day 1 of administration through the terminal sacrifice. Observations during the administration period were twice a day before and after administration, twice a day in the morning and afternoon during the recovery period, and at the necropsy day once in the morning. The life and death, appearance, behavior, etc. of individual animals were observed.

Detailed examinations were performed for females in the main test group on days 7, 14, 21 and 28 of administration, in addition to the above, during recovery periods at study days 35 and 42 days (recovery days 7 and 14).

Observation items and methods included: [1] Position / attitude, respiratory state, tremor · convulsions, stereotyped behavior (rotation · turning), abnormal behavior (e.g., self-bite) were observed from outside the cage. [2] Remove it from the cage regarding ease of removal, ease of handling, muscle contractility, piloerection, condition of hair, appearance of skin, eye / eyeball and mucosa, pupil diameter, lacrimation, salivation, other secretions Sometimes observed. [3] walking, motor coordination, response to environmental stimulus, search behavior (smell of sniffing), excretion state (urination / drainage), stereotyped behavior (grooming / jerking), abnormal behavior (e.g., backward gait / abnormal vocalization), and attackability in the open field.

Functional Observation Battery (FOB) inspection: Five females of each group selected. Females of the main test group were selected earlier on the day of delivery on the 21st day of administration in each group. Timing: 4 weeks of administration (23 days of dosing) and recovery day 8. Observation items and methods included: [1] Sensory - motor response to stimuli: The following were observed on the examination table. Visual stimulation, tactile stimulation, auditory stimulation, pain sensation stimulation, intrinsic receptor stimulation, airborne forward reflection, [2] Grip strength: Using a CPU gauge (Aikoh Engineering Co., Ltd.), the forelimb and hindlimbs were each measured three times and recorded in units of 1 g., [3] Locomotor activity: spontaneous momentum measuring device (Supermex and Compact, Muromachi Machinery Co., Ltd.) for 1 hour at intervals of 10 minutes following the above.

Weight measurement: Females and males and satellites were weighed before test substance administration and at days 1, 4, 7, 14, 21 and 28, on recovery days 1, 7 and 14 and necropsy days. The females in the main test group were weighed before test substance administration and on days 1, 4, 7 and 14 days of administration, on days 0, 7, 14, and 20 days of gestation, and on days 0 and 4 of lactation, and day 5 postpartum. However, the weights for females with delayed parturition was taken on 26th of potential pregnancy (autopsy date), for cases where copulation failed, before administration on 21, 28, 35, 42 and 49 days of administration and on the day of necropsy (day after administration 51 days).
Measurement method: Measured using an electronic scale balance (GX-2000, A-Day Day Co., Ltd.) and recorded to the nearest 1 g unit.

Feeding amount measurement: The same day as the body weight measurement, except for the week of the mating and on both autopsy days.

Urinalysis test: Males for each group in the same group as functional test, female for recovery in satellite group at end of recovery. Urinalysis was performed after 4 weeks of test substance administration (26-27 days of administration) and during the 2 weeks of recovery (recovery Days 9-10). Urine was collected using a metabolic cage for rats (KN-646, B-1 type, Natsume Seisakusho) under non-fasting conditions, and stored urine analyzed within approximately 3 hours after collection. Analyses were carried out with accumulated urine of 21 hours. [9] For sediments, inspection was performed after centrifugation at 1500 rpm for 5 minutes. Urine measurements included: pH, protein, glucose, ketone bodies, urobilinogen, bilirubin, occult blood, color, sediment, volume, specific gravity. The collected urine was discarded after the examination was completed.

Hematology: 5 males from each group (males in the main test group were selected in order of younger animal number than non-animals used for functional test, females in the main test group are the same as in the functional test, all cases in the satellite group). At the time of necropsy (males in the main test group and female in the satellite group (postmortem examination at the end of the treatment) was the day after dosing completed 28 days, females in the main test group 5 days after parturition, males and females in the satellite group Autopsy cases) were bled on the day after recovery 14 days]. Rats were anesthetized with sodium pentobarbital (intraperitoneally) under fasting overnight (16 to 22 hours) from evening and blood was collected from the abdominal aorta. The obtained blood and plasma were discarded after the examination was completed. Because white blood cell fractions of all cases were obtained, white blood cell smears were not prepared. Hematology included: RBC, HCT, HGB, MCV, MCH, MCHC, platelets, WBC, reticulocyte count, Differentials (Neu, Eos, Bas, Mon, Lym), PT and APTT. Clinical chemistry included: AST, ALT, ALP, gamma-GTP, Glucose, total cholesterol, triglyceride, total bilirubin, total bile acid, blood urea nitrogen, creatinine, Na, K, Cl, Ca, IP, total protein, A/G ratio, albumin, and protein fraction (albumin alpha 1-globulin, alpha 2-globulin, beta-globulin, and gamma-globulin).

Sacrifice and pathology:

Males in the main test group and females in the satellite group (autopsy at the end of treatment) were sacrificed the day after administration 28 days, females of the main test group 5 days after delivery (4th day after nursing), males and females of the satellite group (Autopsy at the end of recovery) was autopsied the next day after the 14th day of recovery. However, the example of delayed parturition was conducted on 26th day of pregnancy, and the cases where copulation failed were conducted 24 days after the end of the mating period (the day after administration 51 days).

Necropsy performed after intraperitoneal administration of sodium pentobarbital. For blood test examples, blood was taken from the abdominal aorta, followed by euthanasia by exsanguination, and the entire organs and tissues were observed macroscopically. In addition, according to guidelines, organs / tissues were fixed and stored in 10% neutral buffered formalin solution for histological evaluation. The eyes and Harderian glands were fixed and stored with Davidson's solution, testis and epididymis were fixed in Bouin's solution and stored in 70% ethanol. For the lungs, the fixative was injected and immobilized after injection. Regarding the left and right organs, left and right fixed and preserved.

CONFIRMATION OF SEX CYCLE
For all females in the satellite group, vaginal plaques were collected before necropsy on the autopsy day, subjected to Giemsa staining, and observed under an optical microscope to confirm the sex cycle phase. This data was aided in organ weight or histopathological evaluation.

ORGAN/TISSUE NAME
brain (cerebrum, cerebellum and pons), spinal cord, pituitary gland, thymus, thyroid, parathyroid gland, adrenal gland, spleen, heart, esophagus, stomach, liver, pancreas, submandibular gland, duodenum, jejunum, The ileum (including Peyer's patch), cecum, colon, rectum, trachea, lung, kidney, bladder, testis, epididymis, prostate, seminal vesicle (including coagulated gland), ovary, uterus (corner and neck), vagina , Eye and Harderian gland, mammary gland (female only, right abdomen), femur (including bone marrow, right), mesenteric lymph node, mandibular lymph node, skeletal muscle (gastrocnemius, right) and sciatic nerve.

ORGAN WEIGHTS
Organ Weight Measurements from all animals were taken during necropsy. The weight of the following organs was measured using an electronic scale balance (GR-200, A & D Co., Ltd.). In addition, some organs on the left and right were measured in left and right. Organ name: pituitary, thyroid (including parathyroid gland), spleen, thymus, adrenal gland, prostate, ovary, uterus; and so on; brain, heart, liver, kidney, testis, epididymis, seminal vesicle. Relative weight was calculated from absolute weight and body weight measured on the day of necropsy.

HISTOPATHOLOGICAL EXAMINATION
Samples were prepared for all organs / tissues in the control group and high dose group fixed and preserved at the time of necropsy for males and females of the main test group (necropsy). In addition, the testis and epididymis of one male (animal number 10211) in the 100 mg/kg group who had abnormal findings at necropsy were also examined. As a result of the microscopic examination, since the influence of administration of the test substance was not observed in the high dose group, the examination of the low dose and middle dose groups was not carried out. As for the necropsy cases at the end of the recovery, no test was performed due to the fact that no influence of administration of the test substance was observed.

After embedding tissues in paraffin blocks, they were cut into thin sections, and hematoxylin / eosin stained specimens were prepared and examined microscopically.

Other examinations:

REPRODUCTIVE AND DEVELOPMENTAL TOXICITY
Sex cycle inspection for all females in the main test group.
Period: From the administration start date to the mating success. For cases where copulation failed up to the 14th day of breeding.
Methods: Vaginal plaque smears by Giemsa staining were prepared and the sex cycle phase was observed under an optical microscope.
Judgment: Those repeating each step of the sex cycle (estrus first period, estrus period, estrus late period and estrus period) at intervals of 4 days to 6 days was regarded as normal. The estrus period interval, the number of estrous cycles and the number of animals with abnormal sexual cycle abnormality were calculated for 14 days from the first day of administration to the 14th day of administration.

REPRODUCTIVE ABILITY TEST
Number: All males and females in main test group
Timing Method: 14 days of administration as the start of cohabitation, the next day of the day of mating is set as a day of mating, from the evening of the day of mating, within a limit of 14 days .
Mating combination: Animals paired
Confirmation of copulation: Judged by sperm confirmation in vaginal plug or vaginal plaque smear specimen that dropped in vagina or saucer. The day on which one was recognized was taken as the Day 0 of pregnancy. The number of days required from the start of cohabitation to the established day of copulation was counted.

FERTILITY
Confirmation of pregnancy was made based on the presence or absence of delivery and the presence or absence of implantation traces in the uterus at necropsy. The conception rate was calculated for each group.
Observing labor and nursing conditions: All female animals confirmed to copulate were allowed to deliver spontaneously. The labor condition was observed at least 3 times (9:00, 13:00 and 17:00) every day from the 21st to 25th day of gestation.
Confirmation of end of delivery: If it is observed that the mother collects the child in the nest and holds it under the belly at 9:00, end of calving was made, and that day was taken as nursing day 0 (0 day after birth). Nursing was given to those who gave birth to one or more living babies. The birth rate was calculated for each group.
Calculation of sex ratio of litters: The sex of individual pediatric animals was judged on length between anus and reproductive protrusions on 0 and 4 days after birth. For each surviving child on 0 postnatal day without including deceased offspring
General condition observation of newborns: Every day from postnatal day 0 to postnatal day 4 (necropsy day). Confirmation of survival or death and general condition was observed. The death cases were necropsy promptly after discovery and the whole body was fixed and stored with 10% neutral buffered formalin solution. Calculation of Neonatal Survival Ratio Neonatal survival rates on 0 and 4 days after birth were calculated per dam..
Weight measurement of neonates: Weights were measured for all surviving pups on days 0 and 4 after birth.
Uterine weights were obtained for each dam.

NEONATAL NECROPSY
Performed on day 4 after birth. External (including the inside of the oral cavity) observations, euthanized by sodium pentobarbital overdose, and the organs and tissues of the whole body were observed macroscopically.

Statistics:

MiTOX program for statistical analysis of grip strength, locomotor activity, bw, bw gain, fc, urine volume, hematology, blood chem., abs. and rel. weights, estrus period, number of estruses, days required for mating. Mean and SD were calculated for: implantation sites, number of births, number of live births, dead pups, birth rate, gestation period and externals. For equal variance (p >0.05), analysis by one-way ANOVA, and in case of unequal variance (p <0.05) it was analyzed by the Kruskal-Wallis. As a result of the one-way ANOVA, comparison with the control group using Dunnett's test method when a significant difference was (p <0.1). As a result of the analysis of the Kruskal-Wallis method, comparison with the control group using Steel's method when (p <0.1). Number corpus leutea, implantation and survival rates on 0 and 4 days after birth were separately subjected to the same test as above using Sanken Systems and SAS programs. Detailed observations, sensory motor response, urinalysis were analyzed by the Kruskal-Wallis; when a significant difference was observed (p <0.1), Steel's Comparison with the control group. Fisher's exact probability: sexual cycle, mating, conception, birth, sex ratio, frequency of litters with external abnormalities and histopathology. Group mean and standard deviation are calculated for grip strength, locomotor activity, weight, body weight gain, food intake, urine volume, hematological examination, blood chemistry test, performance of absolute weight and relative weight of the organ in the satellite group, Analysis of equal variance by F test was conducted. In the case of equal variance (p >0.05), Student's t-test was performed, and in the case of unequal variance (p <0.05) Welch's test was used to compare with the control group. Detailed condition observation in the satellite group, sensory motor response to stimulation, urinalysis excluding urine volume were compared with control group using Wilcoxon test.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

During both the treatment and recovery periods of the main test, no treatment related effects on general condition were observed. In addition, during detailed clinical observations, no treatment-related effects were observed in any treatment group.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

For males, during both the treatment and recovery periods, there were no statistically significant or biologically relevant changes in body weight or body weight gain.

For females there were no statistically significant or biologically-relevant changes in body weight or body weight gain in any treatment group, during treatment Days 1-14.

For females dams during gestation, there were no statistically significant or biologically-relevant changes in body weight during gestation (F0 gestation Days 1-20). For female dams during gestation, there were no statistically significant or biologically-relevant changes in body weight gain during gestation. However, there was an overall decrease in cumulative body weight gain in the high-dose group (1000 mg/kg/day) at the p <0.05 significance level (cumulative body weight gain in control, 100, 300, and 1000 mg/kg/day groups was 174.9, 159.9, 167.2, and 152.5 g, respectively). This decrease was not considered to be either treatment-related or biologically relevant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

This was a gavage study, but food consumption was measured and no statistically significant or biologically-relevant differences in food consumption were observed in the treatment groups, when compared to controls.

Food efficiency:

not examined

Description (incidence and severity):

Food efficiency was not calculated, but there were no statistically significant or biologically-relevant differences in either body weight or food consumption.

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):

In males, at the end of the treatment period (Day 29), a statistically significant higher platelet count was observed in the high dose group (104.56, 116.76, 115.56, and 125.18 in controls, 100, 300 and 1000 mg/kg/day, respectively). However, since there were no other hematological effects in this dose group, the values were within historical control limits, and there was no histopathological findings in the bone marrow, this finding was not considered treatment-related by the study authors.

In females, at the end of the treatment period (Day 5 of lactation), a statistically significantly low reticulocyte count was observed in the high dose group (8.230, 8.832, 8.006, and 5.738 in controls, 100, 300 and 1000 mg/kg/day groups, respectively). Additionally, in the female high dose satellite group, at the end of treatment period (Day 29, a statistically significant decrease in red blood cell numbers was noted (871.6 versus 786.4 10000/µL, in control and high dose group, respectively), along with correspondingly high calculated values for MCV and MCH. The effects on red blood cell maturity, absolute numbers, volume, and hemoglobin amount were considered treatment-related because these effects were consistent with regard to RBC and effects on the erythroid lineage were also observed in a preliminary test with 2,6-naphthalene dicarboxylic acid.

No statistically significant or biologically-relevant hematological differences were observed in either the male or female recovery groups.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the high dose males in the 1000 mg/kg/day group at the end of the treatment period (Day 29), ALP was statistically significantly higher (338.4, 417.2, 398.5, and 431.0 IU/L in control, 100, 300, and 1000 mg/kg/day groups, respectively). However, all values were lower than the historical control data and there was no histopathological correlate in liver histopathology. As such, the difference was not considered treatment-related.

In the high dose main study group females, at the end of the treatment period (lactation Day 5), a statistically significantly lower inorganic phosphorus was noted in the high dose group (9.42, 9.16, 8.88, and 8.12 mg/dL in the control, 100, 300, and 1000 mg/kg/day groups, respectively). Also there was a statistically significantly lower percent of alpha2-globulin in the high dose group (8.34, 7.76, 7.52, and 7.18% in the control, 100, 300, and 1000 mg/kg/day groups, respectively). Both of these higher values were within the historical control range for this facility, there was no change in calcium or A/G ratios or other globulin fractions, and histopathological examination of the kidney or liver showed no abnormalities. As such, these differences were considered with the normal range of variability and were not considered treatment-related or biologically relevant.

There were no biologically relevant or treatment-related changes in serum chemistry noted in the female satellite group (sacrifice on Lactation Day 5, or in the recovery groups.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were several statistically significantly increases in absolute and/or relative organ weights that were either not dose related, were within historical control ranges and/or were not considered biologically relevant by the study authors (i.e., increased epididymis weights in the 300 mg/kg/day males, increased kidney weights in the 100 mg/kg/day females at lactation Day 5, and increased uterine weights in the female satellite group).

A statistically significantly increased relative liver weight (2.570 g/100 g versus 2.736 g/100 g in control versus 1000 mg/kg/day females, respectively) was observed in the satellite group (Day 29). Although the absolute liver weight was not statistically significantly higher, it was higher and considered biologically relevant (6.980 grams in controls versus 7.234 grams in high dose). The increased absolute and relative liver weights were considered treatment-related due to corresponding effects on the erythroid lineage and potential metabolism of the test substance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The only notable gross pathological finding was a small (one side) testis and epididymis in one male in the 100 mg/kg/day dose group. Since this finding was a single finding and not dose related, it was considered incidental and not related to treatment.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

During the Functional Observation Battery testing: main study group: A statistically significantly greater value for locomotor activity in the high dose male group (1000 mg/kg/day) was observed during one assessment interval (50-60 minutes). However, since there was no statistically significant increase in locomotor activity during the total observation period (0-60 minutes) the increase was considered incidental (i.e., not considered treatment-related). No other statistically significant or biologically relevant changes were observed during the FOB testing of the main test group.

During the Functional Observation Battery testing: Recovery group: A statistically significantly greater value for locomotor activity in the high dose male group (1000 mg/kg/day) was observed during one assessment interval (50-60 minutes). However, since there was no statistically significant increase in locomotor activity during the total observation period (0-60 minutes) the increase was considered incidental (i.e., not considered treatment-related). No other statistically significant or biologically relevant changes were observed during the FOB testing of males and females the recovery test group.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathological evaluations were reported for: lung, trachea, submandibular gland, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, pancreas, liver, heart, kidney, urinary bladder, ovary, uterine horn, cervix, vagina, cerebrum, cerebellum, pons, spinal cord, sciatic nerve, spleen, thymus, femur bone marrow, submandibular lymph node, mesenteric lymph node, Peyer's patch, pituitary gland, thyroid, parathyroid, adrenal, eyeball, Harderian gland, skeletal muscle, femur, mammary gland.

In the main study test group males (Day 29 sacrifice), only the high dose (1000 mg/kg/day) group was evaluated, since no findings were considered treatment-related. Findings that occurred in both control and high-dose groups, at similar incidence and severity include: pulmonary alveolar macrophage colonization in the lung, renal tubular ossification in the kidney, interstitial inflammation in the prostate, anterior lobe cyst in the pituitary gland, and ultimobranchial body in the thyroid gland.

In the main study female satellite group (Day 29 sacrifice), there were isolated cases of minimal severity findings of lung macrophage alveolar aggregation, liver microgranuloma, kidney cortico-medullary junction mineralization, thyroid ultimobranchial body, and adrenal cortical mineralization that occurred in similar incidences in both the control and high dose groups. Due to the similar incidence and severity in control and 1000 mg/kg/day groups, none of the findings were considered treatment-related or biologically relevant.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

This was not a carcinogenesis study. However, tissues were examined for any histopathological change and no neoplastic or pre-neoplastic findings were reported.

Other effects:

no effects observed

Description (incidence and severity):

There were no statistically significant or treatment-related effects on estrus cycles or reproductive performance (copulation and fertility indices) in any of the treatment groups (n=12, doses of 100, 300, and 1000 mg/kg/day)

There was no statistically significant or biologically relevant findings related to mating rate, conception rate, or mating period,

There was no statistically significant differences in gestation period, number of implantations, birth index, number of offspring, number of live or dead newborns, gestation index, number of corpra leutea, implantation index, or nursing index. There was a statistically significant difference in the sex ratio in the 300 mg/kg/day dose group (46.6, 50.6, 60.0, and 52.5 in the control, 100, 300, and 1000 mg/kg/day groups, respectively). However, due to a lack of dose response this finding was not considered biologically relevant or treatment-related.

No external abnormalities were noted in any offspring. There were no clinical signs in any offspring and no differences in survival rates between Days 0 and 4 after birth. There were no statistically significant differences in pup body weights between Days 0 and 4 after birth.

The only internal finding in offspring during terminal necropsy on Day 4 after birth was an isolated finding of 1/135 high dose pups with bilateral dilation of the renal pelvis of the kidney. Since this was an isolated occurrence of a relatively common finding, it was not considered treatment-related.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No effects were observed at highest dose tested (1000 mg/kg/day)

Key result

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

haematology

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: This is the No Adverse Effect Level. Treatement related effects observed at 1000 mg/kg/day are listed above in the LOAEL row

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat. (total fraction)

Sex:

male/female

Basis for effect level:

other: No treatement-related or biologically relevant effects were obsered at the highest dose tested (1000 mg/kg/day)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

System:

haematopoietic

Organ:

erythrocyte development

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

Conclusions:

For parental generation males and females, there were no treatment-related effects in either the 100 or 300 mg/kg/day groups. There were also no treatment-related effects in the top dose (1000 mg/kg/day) male dose group. In female parental generation animals, there were treatment-related decreased body weight gain in the main test group (Day 29); higher liver weights in females on Day 4 of lactation; lower reticulocyte counts in the main test group (Day 29); and lower red blood cell counts, and higher mean red blood cell volume and mean red blood cell hemoglobin at four days on Day 4 of lactation. There were no effects on fertility or offspring development at the highest dose tested (1000 mg/kg/day). There were no effects on either FOB or Motor Activity in any male or female animals in the main study. As such, the NOAEL for systemic toxicity in males was 1000 mg/kg/day, the NOAEL for female systemic toxicity was 300 mg/kg/day, and the NOAEL for reproductive and developmental toxicity was 1000 mg/kg/day.

Executive summary:

SYSTEMIC TOXICITY

In the 100 and 300 mg/kg group, neither sex nor effect of test substance administration was observed. In the 1000 mg/kg group, there was a significant low value in body weight gain in female gestation period, significant high value in female liver weight in non-crossing group, significant low value in reticulocyte count in main test group, non-crossing group, meaningfully low values for red blood cell count, mean red blood cell volume and mean red blood cell hemoglobin level were found to be significantly high. Therefore, the no-effect level (NOEL) and the non-toxicity level (NOAEL) for repeated dose toxicity of 2,6-naphthalene dicarboxylic acid under this test condition were 1000 mg/kg/day for male and 300 mg/kg/day for female, respectively.

 

NEUROTOXICITY

No treatment-related effects in the FOB and Motor Assessments were observed in any dose group. As such the NOAEL for FOB and Motor Assessments is 1000 mg/kg/day.

 

REPRODUCTIVE AND DEVELOPMENTAL TOXICITY

In the fertility examination, up to the 1000 mg/kg group, the incidence of sexual cycle abnormality, the estrus period interval and the number of estrus, the copulation rate, the conception rate, the number of days required for copulation, the number of pregnant lutea body, the number of implantation, the implantation rate, the birth rate , gestation period, birth rate, number of births, number of surviving pups and number of dead pups in childbirth, and nursing rate on 4th day of nursing did not show any changes related to the administration of the test substance. In the newborn's body weight, general condition, survival rate, sex ratio and necropsy findings, no changes related to the administration of the test substance were observed up to the 1000 mg/kg group.

As described above, no changes related to administration of the test substance were observed in the reproductive capacity and neonatal development of the parent animal up to the 1000 mg/kg group, so that the parent animal of 2,6-naphthalene dicarboxylic acid (NOEL) and non - toxic level (NOAEL) for reproduction and NOEL and non-toxic dose (NOAEL) for neonatal development were all considered to be 1000 mg/kg/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

12 animals per sex/dose

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Naphthalene-2,6-dicarboxylic acid

EC Number:

214-527-0

EC Name:

Naphthalene-2,6-dicarboxylic acid

Cas Number:

1141-38-4

Molecular formula:

C12H8O4

IUPAC Name:

naphthalene-2,6-dicarboxylic acid

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: J82SE
- Concentration and homogeneity of the dosing solutions: confirmed
- Purity test date: Test Substance received October 20, 2014 with purity of 99.7%, Initial confirmation
of purity November 17, 2014 (100.3%), final confirmation of purity February 3, 2015 (100%)
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (measured as 5.0 to 9.4 degrees celcius) in airtight
bottles, dark
- Stability under test conditions: Stable, no measured degradation
- Solubility and stability of the test substance in the solvent/vehicle: Soluble, stability in dosing solu
tion analytically verified
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Prepared in gavage dosing solution: (1% w/v) methyl cellu
lose aqueous solution
- Preliminary purification step (if any): none
FORM AS APPLIED IN THE TEST: prepared and administered in gavage dosing solution

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Japan Charles River Company, Ltd.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks
- Weight at study initiation: males 240 to 330 g, females 160 to 230 g
- Housing: In general, suspended wire mesh cages. With respect to mating female animals, from the
17th day of pregnancy to 4th day of nursing, the wire mesh floor of the same cage was replaced with
a small saucer as a floor covering for experimental animals (white Flake, Japan Charles River Co.,
Ltd.) was used.
Number of housed animals per cage: two animals during the quarantine and acclimatization period,
one mouse after group assignments, one each of male and female during the mating period, and 1
each cage after delivery.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: two weeks
DETAILS OF FOOD AND WATER QUALITY
Food supplied by Oriental yeast Co., Ltd. Feed type CRF-1. Animals fasted before necropsy. Each lot
of feed was analyzed for the presence or absence of contaminants etc. that may adversely affect the
test. Analysis was conducted by Eurofins Food and Product Testing Co., Ltd. (Analysis Report: AR -
14 - JP - 005160 - 01, AR - 14 - JP - 007307 - 01) and a feed manufacturer (Analytical Test Report:
14G03-151, 14G03-164), and analytical data were obtained from the feed manufacturer on a lot-bylot
basis. Analysis items and tolerance values conformed to the standard operating procedure manual
of Compound Safety Research Institute Co., Ltd. As a result of the analysis, no value exceeding the
allowable value was found in any item.
Sapporo city tap water ad libitum using automatic water supply device.
Analyses for contaminants that may adversely affect the test were performed. Duplicate samples wer
e taken from the terminal end (Room 306) of the same breeding room and the same system piping as
that breeding room on July 1, 2014 and January 7, 2015. Analysis was conducted by Nippon Sanitary
Co., Ltd. (Water quality test result table: No. A 261406, A 265505). Analysis items and tolerance valu
es conformed to the standard operating procedure manual of the performing laboratory (Compound
Safety Research Institute Co., Ltd). As a result of the analysis, no value exceeding the allowable valu
e was found in any item.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): measured range 40 to 49%
- Air changes (per hr): 10 to 15 changes/hr
- Photoperiod (hrs dark / hrs light): 12 light/12 hr dark with artificial lighting
IN-LIFE DATES: From: To: December 24, 2014 to February 5th, 2015

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% CMC in water

Details on analytical verification of doses or concentrations:

OTHER SPECIFICS
Gavage solutions for administration were prepared at intervals of 4 to 6 days and used within 6 days.
The gavage dosing solutions were stored in a refrigerator in the test substance storage room from D
ecember 16, 2014 (preparation for uniformity and stability analysis) until February 13, 2015 (final dose
administration).
CONFIRMATION OF UNIFORMITY AND STABILITY OF ADMINISTRATION SOLUTION
Solutions were prepared at concentrations of 10 and 100 mg/mL, stability was confirmed for homog
eneity and refrigeration storage 6 days (the preparation day was calculated as 0 day) + room temper
ature for 4 hours. In the results of the homogeneity analysis, the 10 and 100 mg/mL preparation had
relative standard deviations of 1.5% and 0.8%, respectively, less than 5.0% of the criteria. As a result
of the stability analysis, it was confirmed that the preparation solutions of 10 and 100 mg/mL had re
sidual ratios of 94.9% and 97.1%, respectively, within 100 ± 10.0% of the judgment criteria.
CONFIRMATION OF CONCENTRATION OF DOSING SOLUTION
The concentration of the test substance in the dosing solution with respect to the total concentration
was confirmed with respect to the preparation solution at the time of preparation of the final round, wh
ich is used for the first and all groups. In the analysis results, the contents of the 10, 30 and 100 mg/
mL dosing solutions were 93.3%, 90.7% and 95.2% for the first time, 104.0%, 107.0% and 107.0%
for the last round, respectively, and the relative standard deviations were, 0.6% and 0.8% at the first
round, 1.4%, 1.0% and 0.6% at the final round, which were within 100 ± 10% and 5.0% of the criteria.

Duration of treatment / exposure:

Males: 28-days
Females: 14 days before mating, during the mating period until mating, during gestation and 4 days
after delivering F1 generation

Frequency of treatment:

Daily administration (from 9:10 until 11:46)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment: random
- Rationale for selecting satellite groups: Guideline
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale: random

Examinations

Maternal examinations:

GENERAL CONDITION OBSERVATION
all animals were observed from Day 1 of administration through the terminal sacrifice. Observations during the administration period were twice a day before and after administration, twice a day in the morning and afternoon during the recovery period, and at the necropsy day once in the morning. The life and death, appearance, behavior, etc. of individual animals were observed.
Detailed examinations were performed for females in the main test group on days 7, 14, 21 and 28 of administration, in addition to the above, during recovery periods at study days 35 and 42 days (recovery days 7 and 14).

OBSERVATION ITEMS AND METHODS INCLUDED
[1] Position / attitude, respiratory state, tremor · convulsion s, stereotyped behavior (rotation · turning), abnormal behavior (e.g., self-bite) were observed from outside the cage. [2] Remove it from the cage regarding ease of removal, ease of handling, muscle contractility, piloerection, condition of hair, appearance of skin, eye / eyeball and mucosa, pupil diameter, lacrimation, salivation, other secretions Sometimes observed. [3] walking, motor coordination, response to environmental stimulus, search behavior (smell of sniffing), excretion state (urination / drainage), stereotyped behavior (grooming / jerking), abnormal behavior (e.g., backward gait / abnormal vocalization), and attackability in the open field.

FUNCTIONAL OBSERVATION BATTERY (FOB)
Five females of each group selected. Females of the main test group were selected earlier on the day of delivery on the 21st day of administration in each group. Timing: 4 weeks of administration (23 days of dosing) and recovery day 8. Observation items and methods included: [1] Sensory - motor response to stimuli: The following were observed on the examination table. Visual stimulation, tactile stimulation, auditory stimulation, pain sensation stimulation, intrinsic receptor stimulation, airborne forward reflection, [2] Grip strength: Using a CPU gauge (Aikoh Engineering Co., Ltd.), the forelimb and hindlimbs were each measured three times and recorded in units of 1 g., [3] Locomotor activity: spontaneous momentum measuring device (Supermex and Compact, Muromachi Machinery Co., Ltd.) for 1 hour at intervals of 10 minutes following the above.

WEIGHT MEASUREMENT
Females and males and satellites were weighed before test substance administration and at days 1, 4, 7, 14, 21 and 28, on recovery days 1, 7 and 14 and necropsy days. The females in the main test group were weighed before test substance administration and on days 1, 4, 7 and 14 days of administration, on days 0, 7, 14, and 20 days of gestation, and on days 0 and of lactation, and day 5 postpartum. However, the weights for females with delayed parturition was taken on 26th of potential pregnancy (autopsy date), for cases where copulation failed, before administration on 21, 28, 35, 42 and 49 days of administration and on the day of necropsy (day after administration 51 days).
Measurement method: Measured using an electronic scale balance (GX-2000, A-Day Day Co., Ltd.) and recorded to the nearest 1 g unit.
Feeding amount measurement: The same day as the body weight measurement, except for the week of the mating and on both autopsy days.
Urinalysis test: Males for each group in the same group as functional test, female for recovery in satellite group at end of recovery. Urinalysis was performed after 4 weeks of test substance administration (26-27 days of administration) and during the 2 weeks of recovery (recovery Days 9-10). Urine was 5 KS.Repeated dose toxicity with Repro/Dev (2015) collected using a metabolic cage for rats (KN-646, B-1 type, Natsume Seisakusho) under non-fasting conditions, and stored urine analyzed within approximately 3 hours after collection. Analyses were carried out with accumulated urine of 21 hours. [9] For sediments, inspection was performed after centrifugation at 1500 rpm for 5 minutes. Urine measurements included: pH, protein, glucose, ketone bodies, urobilinogen, bilirubin, occult blood, color, sediment, volume, specific gravity. The collected urine was discarded after the examination was completed.

HEMATOLOGY
Five males from each group (males in the main test group were selected in order of younger animal number than non-animals used for functional test, females in the main test group are the same as in the functional test, all cases in the satellite group). At the time of necropsy (males in the main test group and female in the satellite group (postmortem examination at the end of the treatment) was the day after dosing completed 28 days, females in the main test group 5 days after parturition, males and females in the satellite group Autopsy cases) were bled on the day after recovery 14 days]. Rats were anesthetized with sodium pentobarbital (intraperitoneally) under fasting overnight (16 to 22 hours) from evening and blood was collected from the abdominal aorta. The obtained blood and plasma were discarded after the examination was completed. Because white blood cell fractions of all cases were obtained, white blood cell smears were not prepared. Hematology included: RBC, HCT, HGB, MCV, MCH, MCHC, platelets, WBC, reticulocyte count, Differentials (Neu, Eos, Bas, Mon, Lym), PT and APTT. Clinical chemistry included: AST, ALT, ALP, gamma-GTP, Glucose, total cholesterol, triglyceride, total bilirubin, total bile acid, blood urea nitrogen, creatinine, Na, K, Cl, Ca, IP, total protein, A/G ratio, albumin, and protein fraction (albumin alpha 1-globulin, alpha 2-globulin, beta-globulin, and gamma-globulin).

SACRIFICE AND PATHOLOGY
Males in the main test group and females in the satellite group (autopsy at the end of treatment) we re sacrificed the day after administration 28 days, females of the main test group 5 days after delivery (4th day after nursing), males and females of the satellite group (Autopsy at the end of recovery) was autopsied the next day after the 14th day of recovery. However, the example of delayed parturition was conducted on 26th day of pregnancy, and the cases where copulation failed were conducted 24 days after the end of the mating period (the day after administration 51 days).
Necropsy performed after intraperitoneal administration of sodium pentobarbital. For blood test examp les, blood was taken from the abdominal aorta, followed by euthanasia by exsanguination, and the entire organs and tissues were observed macroscopically. In addition, according to guidelines, organs / tissues were fixed and stored in 10% neutral buffered formalin solution for histological evaluation.
The eyes and Harderian glands were fixed and stored with Davidson's solution, testis and epididymis were fixed in Bouin's solution and stored in 70% ethanol. For the lungs, the fixative was injected and immobilized after injection. Regarding the left and right organs, left and right fixed and preserved.

CONFIRMATION OF SEX CYCLE
For all females in the satellite group, vaginal plaques were collected before necropsy on the autopsy day, subjected to Giemsa staining, and observed under an optical microscope to confirm the sex cycle phase. This data was aided in organ weight or histopathological evaluation.

ORGAN/TISSUE NAME
brain (cerebrum, cerebellum and pons), spinal cord, pituitary gland, thymus, thyroid, parathyroid gland, adrenal gland, spleen, heart, esophagus, stomach, liver, pancreas, submandibular gland, duodenum, jejunum, The ileum (including Peyer's patch), cecum, colon, rectum, trachea, lung, kidney , bladder, testis, epididymis, prostate, seminal vesicle (including coagulated gland), ovary, uterus (corner and neck), vagina , Eye and Harderian gland, mammary gland (female only, right abdomen), femur (including bone marrow, right), mesenteric lymph node, mandibular lymph node, skeletal muscle (gastrocnemius, right) and sciatic nerve.

ORGAN WEIGHTS
Organ Weight Measurements from all animals were taken during necropsy. The weight of the follow ing organs was measured using an electronic scale balance (GR-200, A & D Co., Ltd.). In addition, some organs on the left and right were measured in left and right. Organ name: pituitary, thyroid (including parathyroid gland), spleen, thymus, adrenal gland, prostate, ovary, uterus; and so on; brain, heart, liver, kidney, testis, epididymis, seminal vesicle. Relative weight was calculated from absolute weight and body weight measured on the day of necropsy.

HISTOPATHOLOGICAL EXAMINATION
Samples were prepared for all organs / tissues in the control group and high dose group fixed and preserved at the time of necropsy for males and females of the main test group (necropsy). In addition, the testis and epididymis of one male (animal number 10211) in the 100 mg/kg group who had abnormal findings at necropsy were also examined. As a result of the microscopic examination, since the influence of administration of the test substance was not observed in the high dose group, the examination of the low dose and middle dose groups was not carried out. As for the necropsy cases at the end of the recovery, no test was performed due to the fact that no influence of administration of
the test substance was observed.
After embedding tissues in paraffin blocks, they were cut into thin sections, and hematoxylin / eosin stained specimens were prepared and examined microscopically.

OTHER EXAMINATIONS

REPRODUCTIVE AND DEVELOPMENTAL TOXICITY
Sex cycle inspection for all females in the main test group.
Period: From the administration start date to the mating success. For cases where copulation failed up to the 14th day of breeding.
Methods: Vaginal plaque smears by Giemsa staining were prepared and the sex cycle phase was observed under an optical microscope.
Judgment: Those repeating each step of the sex cycle (estrus first period, estrus period, estrus late period and estrus period) at intervals of 4 days to 6 days was regarded as normal. The estrus period interval, the number of estrous cycles and the number of animals with abnormal sexual cycle abnormal ity were calculated for 14 days from the first day of administration to the 14th day of administration.

REPRODUCTIVE ABILITY TEST
Number: All males and females in main test group
Timing Method: 14 days of administration as the start of cohabitation, the next day of the day of mating is set as a day of mating, from the evening of the day of mating, within a limit of 14 days .
Mating combination: Animals paired Confirmation of copulation: Judged by sperm confirmation in vaginal plug or vaginal plaque smear specimen that dropped in vagina or saucer. The day on which one was recognized was taken as the Day 0 of pregnancy. The number of days required from the start of cohabitation to the established day of copulation was counted.

FERTILITY
Confirmation of pregnancy was made based on the presence or absence of delivery and the presence or absence of implantation traces in the uterus at necropsy. The conception rate was calculated for each group.
Observing labor and nursing conditions: All female animals confirmed to copulate were allowed to deliver spontaneously. The labor condition was observed at least 3 times (9:00, 13:00 and 17:00) every day from the 21st to 25th day of gestation.
Confirmation of end of delivery: If it is observed that the mother collects the child in the nest and holds it under the belly at 9:00, end of calving was made, and that day was taken as nursing day 0 (0 day after birth). Nursing was given to those who gave birth to one or more living babies. The birth rate was calculated for each group.
Calculation of sex ratio of litters: The sex of individual pediatric animals was judged on length between anus and reproductive protrusions on 0 and 4 days after birth. For each surviving child on 0 postnatal day without including deceased offspring General condition observation of newborns: Every day from postnatal day 0 to postnatal day 4 (necropsy day). Confirmation of survival or death and general condition was observed. The death cases were necropsy promptly after discovery and the whole body was fixed and stored with 10% neutral buffer
ed formalin solution. Calculation of Neonatal Survival Ratio Neonatal survival rates on 0 and 4 days after birth were calculated per dam.
Weight measurement of neonates: Weights were measured for all surviving pups on days 0 and 4 after birth.
Uterine weights were obtained for each dam.

NEONATAL NECROPSY
Performed on day 4 after birth. External (including the inside of the oral cavity) observations, euthanized by sodium pentobarbital overdose, and the organs and tissues of the whole body were observed macroscopically.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data

Statistics:

MiTOX program for statistical analysis of grip strength, locomotor activity, bw, bw gain, fc, urine volume, hematology, blood chem., abs. and rel. weights, estrus period, number of estruses, days required for mating. Mean and SD were calculated for: implantation sites, number of births, number of live births, dead pups, birth rate, gestation period and externals. For equal variance (p >0.05), analysis by one-way ANOVA, and in case of unequal variance (p <0.05) it was analyzed by the Kruskal-Wallis. As a result of the one-way ANOVA, comparison with the control group using Dunnett' s test method when a significant difference was (p <0.1). As a result of the analysis of the Kruskal-Wallis method, comparison with the control group using Steel's method when (p <0.1). Number corpus leutea, implantation and survival rates on 0 and 4 days after birth were separately subjected to the same test as above using Sanken Systems and SAS programs. Detailed observations, sensory
motor response, urinalysis were analyzed by the Kruskal-Wallis; when a significant difference was observed (p <0.1), Steel's Comparison with the control group. Fisher's exact probability: sexual cycle, mating, conception, birth, sex ratio, frequency of litters with external abnormalities and histopathology. Group mean and standard deviation are calculated for grip strength, locomotor activity, weight, body weight gain, food intake, urine volume, hematological examination, blood chemistry test, performance of absolute weight and relative weight of the organ in the satellite group, Analysis of equal variance by F test was conducted. In the case of equal variance (p >0.05), Student's t-test was performed, and in the case of unequal variance (p <0.05) Welch's test was used to compare with the control group. Detailed condition observation in the satellite group, sensory motor response to stimulation, urinalysis excluding urine volume were compared with control group using Wilcoxon test.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

During both the treatment and recovery periods of the main test, no treatment related effects on gen
eral condition were observed. In addition, during detailed clinical observations, no treatment-related
effects were observed in any treatment group.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

For males, during both the treatment and recovery periods, there were no statistically significant or biologically relevant changes in body weight or body weight gain. For females there were no statistically significant or biologically-relevant changes in body weight or body weight gain in any treatment group, during treatment Days 1-14. For females dams during gestation, there were no statistically significant or biologically-relevant changes in body weight during gestation (F0 gestation Days 1-20). For female dams during gestation, there were no statistically significant or biologically-relevant changes in body weight gain during gestation. However, there was an overall decrease in cumulative body weight gain in the high-dose group (1000 mg/kg/day) at the p <0.05 significance level (cumulative body weight gain in control, 100, 300, and 1000 mg/kg/day groups was 174.9, 159.9, 167.2, and 152.5 g, respectively). This decrease was not considered to be either treatment-related or biologically relevant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

This was a gavage study, but food consumption was measured and no statistically significant or
biologically-relevant differences in food consumption were observed in the treatment groups, when
compared to controls.

Food efficiency:

not examined

Description (incidence and severity):

Food efficiency was not calculated, but there were no statistically significant or biologically-relevant
differences in either body weight or food consumption.

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):

In males, at the end of the treatment period (Day 29), a statistically significant higher platelet count was observed in the high dose group (104.56, 116.76, 115.56, and 125.18 in controls, 100, 300 and 1000 mg/kg/day, respectively). However, since there were no other hematological effects in this dose group, the values were within historical control limits, and there was no histopathological findings in the bone marrow, this finding was not considered treatment-related by the study authors.
In females, at the end of the treatment period (Day 5 of lactation), a statistically significantly low reticulocyte count was observed in the high dose group (8.230, 8.832, 8.006, and 5.738 in controls, 100, 300 and 1000 mg/kg/day groups, respectively). Additionally, in the female high dose satellite group, at the end of treatment period (Day 29, a statistically significant decrease in red blood cell numbers was noted (871.6 versus 786.4 10,000/μL, in control and high dose group, respectively), along with correspondingly high calculated values for MCV and MCH. The effects on red blood cell maturity, absolute numbers, volume, and hemoglobin amount were considered treatment-related because these effects were consistent with regard to RBC and effects on the erythroid lineage were also observed in a preliminary test with 2,6-naphthalene dicarboxylic acid.
No statistically significant or biologically-relevant hematological differences were observed in either the male or female recovery groups.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the high dose males in the 1000 mg/kg/day group at the end of the treatment period (Day 29), ALP was statistically significantly higher (338.4, 417.2, 398.5, and 431.0 IU/L in control, 100, 300, and 1000 mg/kg/day groups, respectively). However, all values were lower than the historical control data and there was no histopathological correlate in liver histopathology. As such, the difference was not considered treatment-related.
In the high dose main study group females, at the end of the treatment period (lactation Day 5), a statistically significantly lower inorganic phosphorus was noted in the high dose group (9.42, 9.16, 8.88, and 8.12 mg/dL in the control, 100, 300, and 1000 mg/kg/day groups, respectively). Also there was a statistically significantly lower percent of alpha2-globulin in the high dose group (8.34, 7.76, 7.52,
and 7.18% in the control, 100, 300, and 1000 mg/kg/day groups, respectively). Both of these higher values were within the historical control range for this facility, there was no change in calcium or A/G ratios or other globulin fractions, and histopathological examination of the kidney or liver showed no abnormalities. As such, these differences were considered with the normal range of variability and
were not considered treatment-related or biologically relevant.
There were no biologically relevant or treatment-related changes in serum chemistry noted in the female satellite group (sacrifice on Lactation Day 5, or in the recovery groups.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were several statistically significantly increases in absolute and/or relative organ weights that were either not dose related, were within historical control ranges and/or were not considered biologically relevant by the study authors (i.e., increased epididymis weights in the 300 mg/kg/day males, increased kidney weights in the 100 mg/kg/day females at lactation Day 5, and increased uterine weights in the female satellite group).
A statistically significantly increased relative liver weight (2.570 g/100 g versus 2.736 g/100 g in control versus 1000 mg/kg/day females, respectively) was observed in the satellite group (Day 29). Although the absolute liver weight was not statistically significantly higher, it was higher and considered biologically relevant (6.980 grams in controls versus 7.234 grams in high dose). The increased absolute and
relative liver weights were considered treatment-related due to corresponding effects on the erythroidlineage and potential metabolism of the test substance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The only notable gross pathological finding was a small (one side) testis and epididymis in one male in the 100 mg/kg/day dose group. Since this finding was a single finding and not dose related, it was considered incidental and not related to treatment.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

During the Functional Observation Battery testing: main study group: A statistically significantly greater value for locomotor activity in the high dose male group (1000 mg/kg/day) was observed during one assessment interval (50-60 minutes). However, since there was no statistically significant increase in locomotor activity during the total observation period (0-60 minutes) the increase was considered incidental (i.e., not considered treatment-related). No other statistically significant or biologically relevant changes were observed during the FOB testing of the main test group.
During the Functional Observation Battery testing: Recovery group: A statistically significantly greater value for locomotor activity in the high dose male group (1000 mg/kg/day) was observed during one assessment interval (50-60 minutes). However, since there was no statistically significant increase in locomotor activity during the total observation period (0-60 minutes) the increase was considered incidental (i.e., not considered treatment-related). No other statistically significant or biologically relevant changes were observed during the FOB testing of males and females the recovery test group.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathological evaluations were reported for: lung, trachea, submandibular gland, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, pancreas, liver, heart, kidney, urinary bladder, ovary, uterine horn, cervix, vagina, cerebrum, cerebellum, pons, spinal cord, sciatic nerve, spleen, thymus, femur bone marrow, submandibular lymph node, mesenteric lymph node, Peyer's patch, pituitary gland, thyroid, parathyroid, adrenal, eyeball, Harderian gland, skeletal muscle, femur, mammary gland.
In the main study test group males (Day 29 sacrifice), only the high dose (1000 mg/kg/day) group was evaluated, since no findings were considered treatment-related. Findings that occurred in both control and high-dose groups, at similar incidence and severity include: pulmonary alveolar macrophage colonization in the lung, renal tubular ossification in the kidney, interstitial inflammation in the prostate, anterior lobe cyst in the pituitary gland, and ultimobranchial body in the thyroid gland.
In the main study female satellite group (Day 29 sacrifice), there were isolated cases of minimal severity findings of lung macrophage alveolar aggregation, liver microgranuloma, kidney cortico-medullary junction mineralization, thyroid ultimobranchial body, and adrenal cortical mineralization that occurred in similar incidences in both the control and high dose groups. Due to the similar incidence and severity in control and 1000 mg/kg/day groups, none of the findings were considered treatmentrelated or biologically relevant.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

This was not a carcinogenesis study. However, tissues were examined for any histopathological change and no neoplastic or pre-neoplastic findings were reported.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: Developmental toxicity

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

There was a statistically significant difference in the sex ratio in the 300 mg/kg/day dose group (46.6, 50.6, 60.0, and 52.5 in the control, 100, 300, and 1000 mg/kg/day groups, respectively). However, due to a lack of dose response this finding was not considered biologically relevant or treatment-related.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There was no significant difference in body weight between 0 and 4 days after birth between the test substance-administered group and the control group in both males and females.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

Changes in the general condition caused by administration of the test substance were not observed in any administration group.
There was no significant difference between survival rates on 0 and 4 days after birth between the test substance administered group and the control group.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Developmental toxicity

Overall developmental toxicity

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

For parental generation males and females, there were no treatment-related effects in either the 100 or 300 mg/kg/day groups. There were also no treatment-related effects in the top dose (1000 mg/kg/day) male dose group. In female parental generation animals, there were treatment-related decreased body weight gain in the main test group (Day 29); higher liver weights in females on Day 4 of lactation; lower reticulocyte counts in the main test group (Day 29); and lower red blood cell counts, and higher mean red blood cell volume and mean red blood cell hemoglobin at four days on Day 4 of lactation. There were no effects on fertility or offspring development at the highest dose tested (1000 mg/kg/day). There were no effects on either FOB or Motor Activity in any male or female animals in the main study. As such, the NOAEL for systemic toxicity in males was 1000 mg/kg/day, the NOAEL for female systemic toxicity was 300 mg/kg/day, and the NOAEL for reproductive and developmental toxicity was 1000 mg/kg/day.

Executive summary:

In the fertility examination, up to the 1000 mg/kg group, the incidence of sexual cycle abnormality, the estrus period interval and the number of estrus, the copulation rate, the conception rate, the number of days required for copulation, the number of pregnant lutea body, the number of implantation, the implantation rate, the birth rate , gestation period, birth rate, number of births, number of surviving

pups and number of dead pups in childbirth, and nursing rate on 4th day of nursing did not show any changes related to the administration of the test substance. In the newborn's body weight, general condition, survival rate, sex ratio and necropsy findings, no changes related to the administration of the test substance were observed up to the 1000 mg/kg group.

As described above, no changes related to administration of the test substance were observed in the reproductive capacity and neonatal development of the parent animal up to the 1000 mg/kg group, so that the parent animal of 2,6-naphthalene dicarboxylic acid (NOEL) and non - toxic level (NOAEL) for reproduction and NOEL and non-toxic dose (NOAEL) for neonatal development were all considered to be 1000 mg/kg/day.