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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 19, 2006 - February 26, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2,3-Dichloro-1,4-naphthoquinone
- Physical state: Yellow powder
- Analytical purity: 99.9% (GC area ratio)
- Storage condition of test material: in a dark place at room temperature

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hino Breeding Center, Charles River Laboratories, Japan
- Age at study initiation: 9 weeks
- Weight at study initiation: from 327.6 to 363.7 g in the males and from 206.1 to 239.9 g in the females
- Housing: Individually in wire-floored stainless steel cages; during the mating period, 1 male and 1 female were housed together in each cage
- Diet: solid food, MF, lot Nos. 060802, 060907 and 061004, Oriental Yeast Co., Ltd, ad libitum
- Water: ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.4 to 24
- Humidity (%): 47.6 to 59.9
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: sodium carboxymethyl cellulose
Details on mating procedure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed accurately and suspended in 0.5 w/v% CMC-Na solution to prepare 1.0 w/v% solution. 0.1 and 0.01 w/v% solutions were prepared by dilution the 1.0 w/v% solution with 0.5 w/v% CMC-Na solution. These solutions were prepared once every 14 days under a sodium lamp and stored in light-resistant containers in a cool, dark place.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the test substance was insoluble in purified water but a favorable suspension was obtained in an aqueous solution of sodium carboxymethyl cellulose
- Amount of vehicle (if gavage): 5 mL per kg body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A study to examine the stability of 2,3-dichloro-1,4-naphthoquinone, uniformity, stability and concentration of the preparation solution (Study code X02-0161)” was conducted at Hita Office of the institute. The uniformity of the test substance in the preparation solutions was checked by sampling from the upper, middle and lower layers (n=1 each) of 25.0 and 0.01 w/v% solutions and, after pretreatment, measuring the concentration of the test substance by high-performance liquid chromatography (HPLC). For confirmation of the stability of the test substance in the preparation solutions, the solution used in the uniformity test was stored in a cool, dark place. The samples were collected from the middle layer at the measurement points (8 and 15 days after preparation) (n=1 each) and, after pretreatment, measuring the concentration of the test substance once by high-performance liquid chromatography (HPLC). The concentration of the test substance in the preparation solution was confirmed by sampling from the middle layers of 1.0, 0.1 and 0.01 w/v% solutions immediately after the initial preparation and, after pretreatment, and measuring the concentration once by high-performance liquid chromatography. It was confirmed that 20.0 and 0.01 w/v% solutions were uniform and stable for 14 days in a dark, cool place. It was also confirmed that the concentrations of the test substance in the preparation solutions used in the initial administration were all within 100 ± 10% to the designated values of 1.0, 0.1 and 0.01 w/v%.
Duration of treatment / exposure:
males 42 days, females 41-45 days (until day 4 of lactation)
Frequency of treatment:
daily
Details on study schedule:
In the afternoon of Day 15 of treatment, the females except for those in the recovery group were moved to the cages of the males at the same dose level for cohabitation in 1:1 ratio. The animals were mated every day until copulation was confirmed setting a period of 14 days as the limit. During the mating period, copulation was checked every day and establishment of copulation was judged by the presence of vaginal plug or vaginal smear setting that date as Day 0 of gestation.
Doses / concentrationsopen allclose all
Dose / conc.:
0.5 mg/kg bw/day (actual dose received)
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
50 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: based on a 14-day range finder
- Post-exposure recovery period in satellite groups: 5 males of the control and the high dose group were examined for reversibility of the effects after 14-day withdrawal of test substance.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
The males were observed twice a day from the first day of administration to the day before dissection and once on the dissection day. The females were observed twice a day from the first day of administration to Day 4 after parturition including the parturition condition and nursing condition and once on Day 5 of parturition (day of dissection). During the recovery period, the animals were observed once every day until the day before dissection.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All males and females were observed once before the start of administration and once a week thereafter (except for the mating period) under blinding and the results were scored. The females were also examined on Day 4 after parturition. The examination from Day 18 of gestation to the day of parturition was not conducted to avoid causing unnecessary stress to female animals.

BODY WEIGHT: Yes
Both the males and females were weighed once a week during the acclimatization period and on the day of group allocation. Thereafter, the males were weighed on Days 1, 3, 7, 14, 21, 28, 35, 42 and 43 (day of taking out, under fasting) of treatment and the females were weighed on Days 1, 3, 7and 14 of treatment, Days 0, 7, 14, 17 and 20 of gestation, and Day 0 (day of parturition), 4 and 5 (day of taking out, under fasting) after parturition. The females in the recovery group were weighed on the same days as males. During the recovery period, both the males and females were weighed on Days 1, 3, 7, 14 and 15 (day of taking out, under fasting) of recovery.

FOOD CONSUMPTION
Food consumption was measured on Days 1, 3, 7, 14, 28, 35 and 42 of treatment for males, and on Days 1, 3, 7 and 14 before gestation, Days 0, 7, 14, 17 and 20 of gestation, and Days 0 (day of parturition) and 4 after parturition for females. Food consumption by the females of the recovery group was measured on Days 1, 3, 7, 14, 21, 28, 35 and 42. During the recovery period, food consumption was measured on Days 1, 3, 7 and 14 of recovery for both males and females. Food consumption was determined in intervals from the measurement day to the next measurement day for both males and females and mean daily food consumption was calculated.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment and at the end of recovery
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: 5 of the surviving males at each dose level in the ascending order of animal number and for 5 of the females at each dose level in the order of closer date of parturition.
- Parameters examined: Red blood cell count, White blood cell count, Hemoglobin concentration, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Reticulocyte ratio, Prothrombin time, Activated partial thromboplastin time, Differential leukocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment and at the end of recovery
- Animals fasted: Yes
- How many animals: 5 of the surviving males at each dose level in the ascending order of animal number and for 5 of the females at each dose level in the order of closer date of parturition.
- Parameters examined: Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Cholinesterase, Gamma-glutamyl transpeptidase, Total cholesterol, Triglyceride, Blood glucose, Total protein, Albumin, A/G ratio, Urea nitrogen, Creatinine, Total bilirubin, Calcium, Inorganic phosphorus, Sodium, Potassium, Chlorine

NEUROBEHAVIOURAL EXAMINATION: Yes
Five males at each dose level in the ascending order of animal numbers were examined for the following items in the final week of administration. Of females expected for parturition, 5 females in each group in the order of closer date of parturition were examined on the day before dissection (Day 4 after parturition) before the start of fasting. No examinations were performed during the recovery period because no abnormalities were noted in the treatment period.
- Battery of functions tested: reactivity / sensory activity / grip strength / motor activity
Oestrous cyclicity (parental animals):
Vaginal smears were collected from all females from the first day of administration to the day confirming copulation, stained with Geimsa stain, and examined under a light microscope to calculate the mean sexual cycle (number of days) from each estrus to the next estrus.
Litter observations:
The neonates were examined for the number of births, number of stillbirths, number of live births, sex, and external appearance of live births on the day of birth (defined as Day 0 after birth). Subsequently, general health conditions, number of live neonates and number of dead neonates were checked up to Day 3 after birth and, on Day 4 after birth (day of sacrifice), general health conditions, number of live neonates, sex, external appearance of live neonates, number of dead neonates, and external appearance of dead neonates were checked and the following items were calculated. Body weights were measured in litter unit and for males and females on the date of birth and Day 4 after birth.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
Macroscopic observation including observation of the body surface, openings, subcutaneous, cranial cavity, thoracic cavity, abdominal cavity, pelvic cavity and their contents was performed for all animals at the end of the treatment period (except for the recovery group) and at the end of the recovery period.

HISTOPATHOLOGY: Yes
The trachea, lungs, stomach, intestines (duodenum to rectum including Peyer’s patches), liver, heart, kidneys, bladder, testes, epididymides, prostate, seminal vesicles, ovaries, uterus, vagina, brain (including cerebrum, cerebellum and pons), spinal cord, sciatic nerves, bone marrow (femur), axillary lymph nodes, mesenteric lymph nodes, spleen, thymus, pituitary gland, thyroid gland (including parathyroid) and adrenal glands were examined in 5 males in each group in the ascending order of animal number and 5 females in each of the vehicle control group and 50 mg/kg group in the order of closer date of parturition. The kidneys were examined in all females in the vehicle control recovery group and 50 mg/kg recovery group as well as 5 females in each group in the order of closer date of parturition as abnormality indicative of the effect of the test substance was noted in the females of the 50 mg/kg group. The anterior stomach was examined in the males and females of the vehicle control recovery group, 0.5 and 5 mg/kg groups, and 50 mg/kg recovery group as abnormality suggesting the effect of the test substance administration was found in the 50 mg/kg group at autopsy.
The epididymides were examined in 2 males (Nos. 28 and 29) of the 5 mg/kg group that showed macroscopic lesion, hepatic lymph nodes in 4 males (Nos. 38, 39, 40 and 41) of the 50 mg/kg group and 2 females (Nos. 145 and 146), and glandular stomach in 1 male (No. 45) of the 50 mg/kg recovery group.
In the animal that died (No. 142), stomach, intestines, liver, heart, kidneys, spleen, thymus and adrenal glands were examined to investigate the cause of death.
In 2 dams (Nos. 102 and 149) that showed total litter death, stomach, intestines, liver, heart, kidneys, spleen, thymus and adrenal glands were examined to check the pathological condition and, further, the skin was examined in 1 animal (No. 102).
For 3 females (Nos. 124, 133 and 147) that did not start parturition even after 25 days from confirmation of copulation, the ovaries, uterus and vagina were examined to investigate the cause of infertility.
Statistics:
- Body weight (except for the time of taking out), food consumption, hematology, blood biochemistry, organ weight, mean sexual cycle, time to copulation, gestational period, number of corpus lutea verum, number of implantation sites, grip strength, locomotor activity, number of births, number of live births, 0-day sex ratio of live births, 4-days sex ratio of live neonates, body weight of neonates: Bartlett method and, if equal variance was noted at 5% significance level, the one-way analysis of variance was performed. When a significant difference was noted in the analysis of variance, the vehicle control group and each dose group were compared by the Dunnett’s test. If equal variance was not detected, the Kruskal-Wallis rank-sum test was performed to examine the significance in all groups followed by the Dunnett’s multiple comparison test.
- Defecation frequency (number of feces) and urination frequency (number of urine pools): Kruskal-Wallis test and, if a significant difference was noted, Dunnett’s multiple comparison test.
- Copulation rate, fertility rate, conception rate, fertility rate and 0-day sex ratio of live births and 4-days sex ratio of live neonates: chi-square test (by Fisher’s exact test if any of the marginal frequencies was 10 or less).
- Implantation rate, pre-implantation embryo loss rate, post-implantation embryo loss rate of parent animals and delivering rate, birth rate of live births, fertility rate of live births, external appearance abnormality rate, and 4-day survival rate of neonates: Kruskal-Wallis rank sum test for the tested for significance among all groups followed by the Dunnett’s multiple comparison test. The body weights of neonates were analyzed by litter unit and for males and females.
Reproductive indices:
Copulation rate, Fertility rate, Conception rate, Time (number of days) to copulation, Implantation rate, Pre-implantation embryo loss rate, Post-implantation embryo loss rate
Offspring viability indices:
Birth rate, Birth rate of live births, Sex ratio of live births on Day 0, External appearance abnormality rate, 4-day survival rate, 4-day sex ratio of live neonates

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the 50 mg/kg group, all the males showed soft feces, 11 showed black soft feces and 4 showed diarrhea. No abnormalities were noted in the 0.5 and 5 mg/kg groups. In the females before establishment of copulation, 9 showed soft feces, 6 showed black soft feces and 6 showed diarrhea in the 50 mg/kg group. During the gestational period, 1 female of the 5 mg/kg group showed pallor, 6 females of the 50 mg/kg group showed soft feces and 4 showed black soft feces. Soft feces, black soft feces and diarrhea were noted in all animals of the 50 mg/kg recovery group. No abnormalities were noted in the 0.5 mg/kg group. One female (No. 142) in the 50 mg/kg group showed soft feces before establishment of copulation and black soft feces in the gestational period and died on Day 23 of gestation.
During the recovery period, two males of the 50 mg/kg recovery group showed soft feces and 3 males showed black soft feces until Day 1 of recovery but the symptoms resolved on the following day and thereafter. Four females of the 50 mg/kg recovery group showed soft feces and 1 female showed black soft feces until Day 1 of recovery but the symptoms resolved on the following day and thereafter.
One female died in the 50 mg/kg group.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No significant changes were noted in any of the males of any test substance group. In the females, significantly low body weight was observed in the 5 mg/kg group on Days 17 and 20 of gestation but the changes were not dose-dependent. No significant changes were noted in the 0.5 and 50 mg/kg groups. During the recovery period, no significant changes were noted in any of the males and females in the 50 mg/kg recovery group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No significant changes were noted in any of the males of any test substance group. A significant increase was noted in the females of the 50 mg/kg group on Day 14 of treatment before establishment of copulation. The significant decrease found in the 5 mg/kg group on Day 17 of gestation was not a dose-dependent change. No significant changes were noted in the 0.5mg/kg group. During the recovery period, no significant changes were noted in the males of the 50 mg/kg recovery group. A significant increase was noted in the females of the 50 mg/kg recovery group on Day 7 of recovery.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In the males, the white blood cell count increased significantly in the 50 mg/kg group. No significant changes were noted in the 0.5 and 5 mg/kg groups. In the females, the red blood cell count, hemoglobin and hematocrit increased significantly but these were not dose-dependent changes. No significant changes were noted in the 5 and 50 mg/kg groups. At the end of the recovery period, no significant changes were noted in the males of the 50 mg/kg recovery group. In the females, the platelet count increased significantly in the 50 mg/kg recovery group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In the males, creatinine levels decreased significantly in the 5 mg/kg group but not dose-dependently. No significant changes were noted in the 0.5 and 50 mg/kg groups. No significant changes were noted in any of the females of any test substance group. At the end of the recovery period, in the males of the 50 mg/kg recovery group, creatinine levels decreased significantly although this decrease was not noted at the end of the treatment period. In the females, no significant changes were noted in the 50 mg/kg recovery group.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Two males in the 0.5 mg/kg group showed excessive reaction to pain sensation but the change was not dose-dependent. No abnormalities were noted in the 0.5 and 5 mg/kg groups. No abnormalities were noted in any of the females of any test substance group. As neither the males nor females showed changes suspected of the influence of the test substance administration during the treatment period, the examination was not performed during the recovery p
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In the males, the relative weight of the kidneys increased significantly in the 5 mg/kg and higher and the absolute weight of the kidneys increased significantly in the 50 mg/kg group. No significant changes were noted in the 0.5 mg/kg group. In the females, significant increases in the absolute and relative kidney weights, and absolute spleen weight in the 50 mg/kg group. No significant changes were noted in the 0.5 and 5 mg/kg groups. At the end of the recovery period, no significant changes were noted in any of the males and females of the 50 mg/kg recovery group.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The female that died in the 50 mg/kg group showed mucosal depression in the anterior stomach, mucosal blackening in the glandular stomach, discoloration of the kidneys, reduction of the thymus size and swelling of the adrenal glands.
At the end of the treatment period, mucosal depression in the anterior stomach, nodulation of the epididymides, and whitening of the epididymides were found in 1 male of the 5 mg/kg group. In the 50 mg/kg group, border elevation of the anterior stomach was noted in 3 males, mucosal roughening and wall thickening in the anterior stomach in 7, and swollen hepatic lymph nodes in 6. No abnormalities were found in the 0.5 mg/kg group.
In the females, reduction of thymus size was noted in the vehicle control group and in 2 females of the 0.5 mg/kg group. In the 5 mg/kg group, mucosal depression of the anterior stomach was found in 2 females and reduction in thymus size in 1 female. In the 50 mg/kg group, border elevation of the anterior stomach was found in 1 female, mucosal roughening of the anterior stomach in 8 females, wall thickening in 9 females, and swollen hepatic lymph nodes in 3 females. At the end of the recovery period, border elevation of the anterior stomach was found in 1 male of the 50 mg/kg recovery group and mucosal depression of the glandular stomach in another male. No abnormalities were noted in the females of the 50 mg/kg recovery group.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The female of the 50 mg/kg group that died showed diffuse hyperplasia of the squamous epithelium and ulcer in the anterior stomach, mucosal necrosis in the fundic area of the glandular stomach, vacuolization of the renal tubular epithelium in the kidneys, atrophy of the spleen and thymus, and localized necrosis of the adrenal cortex.
In the males, localized myocarditis in the heart was found in 1 male, sperm granuloma in the epididymides in another male, and round cell infiltration in the prostate in 2 males in the vehicle control group. In the 5 mg/kg group, ulcer in the anterior stomach was found in 1 male, and sperm granuloma of the epididymides was found in 2 males. In the 50 mg/kg group, cell infiltration in the lamina propria and submucosa of the anterior stomach in 3 animals, diffuse hyperplasia of the squamous epithelium in the anterior stomach in 5 animals, border squamous cell hyperplasia in the anterior stomach in 2 animals, mineral deposition in the jejunal Peyer’s patches in 1 animal, localized myocarditis of the heart in 2 animals, round cell infiltration of the prostate in 1 animal, germinal center development in the hepatic lymph nodes in 4 animals, and ultimobranchial rest in the thyroid gland in 1 animal. No abnormalities were found in the 0.5 mg/kg group. In the females, mineral deposition in the renal papilla was found in 1 female and acceleration of extramedullar hemopoiesis was found in another female in the vehicle control group. In the 0.5 mg/kg group, isolated cysts were found in the kidney cortex of 1 animal. In the 5 mg/kg group, ulcer of the anterior stomach and isolated cysts in the kidney cortex were found in 1 animal, and localized hypertrophy of the renal tubular epithelium was found in another animal. In the 50 mg/kg group, cell infiltration in the lamina propria and submucosa of the anterior stomach in 1 animal, diffuse hyperplasia of the squamous epithelium in the anterior stomach of 5 animals, squamous cell hyperplasia in the border of the anterior stomach in 2 animals, severe vacuolization of the renal tubular epithelium in 2 animals, mineral deposition in the cortico-medullary junction and isolated cysts in the medulla of the kidneys in 1 animal, germinal center development in the hepatic lymph nodes in 2 animals, and ultimobranchial rest in the thyroid gland in 1 animal.
At the end of the recovery period, diffuse hyperplasia of the squamous epithelium in the anterior stomach was found in all animals of the 50 mg/kg recovery group, squamous cell hyperplasia in the border of the anterior stomach in 2 animals, and mucosal necrosis in the fundic gland area of the glandular stomach in 1 animal. In the females, diffuse hyperplasia of the squamous epithelium in the anterior stomach was found in 4 animals of the 50 mg/kg recovery group.

Reproductive function / performance (P0)

Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
No abnormalities were noted in the sexual cycle in any test substance group.
Twelve, 11, 11 and 11 animals became pregnant in the vehicle control group, and 0.5, 5 and 50 mg/kg groups, respectively. No abnormalities were noted in the time to copulation, copulation rate, fertility rate, conception rate, gestational period, number of corpus luteum verum, number of implantation sites, implantation rate, pre-implantation embryo loss rate, post-implantation embryo loss rate, fertility rate and delivering rate in any test substance group.
One animal in the vehicle control group showed depilation in the lower abdomen and unexpelled placenta resulting in the total litter death on Day 1 after parturition. One animal in the 0.5 mg/kg group abandoned nursing, and 1 animal in the 5 mg/kg group showed maldevelopment of nipples and abandoned nursing. One animal in the 50 mg/kg group showed decreases in the locomotor activity and body temperature, and abandoned nursing resulting in total litter death on Day 4 after parturition. The vacuolization of the tubular epithelium observed in a high dose female was also seen in a control group female, therefore this effect is of questionable relevance.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
0.5 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: mucosal depression and ulcer in the anterior stomach were observed in the males and females given 5 mg/kg and higher
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
50 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect of the test substance was noted in any of the examination items regarding repeoduction toxicity.

Results: F1 generation

Details on results (F1)

The decrease in body temperature observed in all neonates of one litter in the 5 mg/kg group was not a dose-dependent change. The body temperature dropped in 9 of 13 neonates in one litter in the 50 mg/kg group due to leakage of the watering bottle. No abnormalities were found in the number of births, number of live births, birth rate, birth rate of live births, sex ratio, external appearance abnormality rate or 4-day survival rate.
No significant changes were noted in the body weights of male and female neonates in any test substance group on the day of parturition and Day 4 after parturition.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse findings observed

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOEL concerning the reproductive and developmental toxicity was judged to be 50 mg/kg/day, which was the highest dose level, as no abnormalities were noted in any of the examination items.
Executive summary:

A combined repeated-dose oral toxicity and reproductive and developmental toxicity study of 2,3-dichloro-1,4-naphthoquinone was conducted at 0, 0.5, 5 and 50 mg/kg/day using 12 male and 12 female Crl:CD(SD) rats per dose group in accordance with the OECD TG422. The test substance was administered orally in males for 42 days from 2 weeks prior to mating and in females until Day 4 of lactation after parturition to examine repeated-dose toxicity and reproductive toxicity in parent animals and developmental toxicity in offspring. Autopsy was performed for 12 males and 12 females in the 0.5 and 5 mg/kg groups, and for 7 of 12 males and 7 of 12 females in the vehicle control group and the 50 mg/kg group at the end of the treatment period. Five remaining males and 5 females in the satellite group were examined for reversibility of the effects after 14-day withdrawal as the recovery group. The females in the recovery group were not mated in accordance with the test guideline.

Effects of the test substance were noted in the stomach and kidneys. The autopsy results revealed mucosal depression in the anterior stomach in the males and females of the 5 mg/kg group, and border elevation, mucosal roughening and wall thickening in the anterior stomach in the 50 mg/kg group. The histopathological examination showed ulcer in the anterior stomach in the males and females of the 5 mg/kg group, and cell infiltration in the lamina propria and submucosa and diffuse hyperplasia of squamous epithelium in the anterior stomach in the 50 mg/kg group. In addition, germinal center development in the hepatic lymph nodes in the males and females of the 50 mg/kg group and increased white blood cell count in the males of the 50 mg/kg group were noted as the secondary changes attributed to the inflammation of the stomach. In the kidneys, severe vacuolization of the renal tubular epithelium was observed in the females of the 50 mg/kg group. The relative weight of the kidneys increased in the males given 5 mg/kg and higher and females of the 50 mg/kg group and the absolute weight of the kidneys increased in the males and females of the 50 mg/kg group. In the surviving animals, soft feces, black soft feces and diarrhea were observed in the males and females of the 50 mg/kg group and pallor in the females of the 5 mg/kg group during the gestational period. One female in the 50 mg/kg group died on Day 23 of gestation and this animal also showed changes in the stomach and kidneys similar to the surviving animals. As the cause of death, bleeding from the stomach due to the administration of the test substance was considered.

In the recovery group, soft feces and black soft feces were observed until Day 1 of recovery but these resolved on the next day and thereafter. Diffuse hyperplasia of the squamous epithelium in the anterior stomach, squamous cell hyperplasia on the border and mucosal necrosis in the fundic gland area of the glandular stomach found in the stomach were alleviated compared to the findings at the end of the treatment period. No effect of the test substance was noted in the detailed observation, function test, body weight, food consumption, hematological examination or blood biochemical examination.

 

No effect of the test substance was noted in any of the examination items regarding repeoduction toxicity, including the sexual cycle, reproductive competence, fertility rate, delivering rate and nursing condition in parent animals and the birth rate, survival rate, sex ratio on Postnatal Day, sex ratio on 4 postnatal day, external appearance and body weight in offspring.

Based on the above, the no observed adverse effect level (NOAEL) for the repeated-dose toxicity of dichlone was estimated to be 0.5 mg/kg/day in both males and females because mucosal depression and ulcer in the anterior stomach were observed in the males and females given 5 mg/kg and higher. The NOAEL for the reproductive and developmental toxicity was judged to be 50 mg/kg/day, which was the highest dose level, as no abnormalities were noted in any of the examination items. The no observed effect level (NOEL) concerning the repeated-dose toxicity, on the other hand, was estimated to be 0.5 mg/kg/day for males as the relative weight of the kidneys increased, though without organic changes, in the groups given 5 mg/kg and higher and 0.5 mg/kg for females due to effects on the stomach in the groups given 5 mg/kg and higher similarly to the NOAEL. The NOEL concerning the reproductive and developmental toxicity was judged to be 50 mg/kg/day, which was the highest dose level, as no abnormalities were noted in any of the examination items.