1,4-naphthoquinone

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,4-naphthoquinone

EC Number:

204-977-6

EC Name:

1,4-naphthoquinone

Cas Number:

130-15-4

Molecular formula:

C10H6O2

IUPAC Name:

1,4-naphthoquinone

Test material form:

solid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on species / strain selection:

This strain is widely used in toxicity studies using rodents, there is abundant historical data and a large number of animals are available.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc. (Hino Breeding Center)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks old
- Weight at study initiation: Males: 333.9 to 377.2 g and females 193.9 to 259.2 g
- Fasting period before study: No
- Housing: Hanging type stainless wire mesh cages. For copulated females and dams polymethylpentene cages were used.
- Diet (e.g. ad libitum): Radiation sterilised pellet diet fed freely (except during fresh urine collection and measurement of motor activity). Animals were also fasted ahead of scheduled necropsy.
- Water (e.g. ad libitum): Automatic water supply available freely (except during measurements of motor activity).
- Acclimation period: A quarantine period was set for 5 days and an acclimation period of 13 days.

DETAILS OF FOOD AND WATER QUALITY: Data for each lot of diet was routinely analysed for contaminants which were all within acceptable limits. Water was analysed twice yearly and results were within acceptable limits.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.7 to 24.1°C
- Humidity (%): 44.9 to 62.8%
- Air changes (per hr): 10 to 20 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle (7:00 to 19:00)

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test substance was administered orally using a disposable syringe attached to a gastric tube. Dosing formulations for the test substance groups were stirred with a magnetic stirrer during administration.

Vehicle:

methylcellulose

Remarks:

0.5 w/v% in water for injection

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

Dosing solutions were prepared once every 9 days as observed to be stable over this period. These were stored refridgerated ahead of use each day. For the high dose, a set amount of the substance was weighed and ground using a pestle and mortar. The vehicle was gradually added and the suspension added to a measuring cylinder. All equipment was washed with vehicle and added to the cylinder. This was brought upto a final volume in order to prepare a concentration of 0.2 mg/mL. The dosing preparations were well mixed. For the intermediate and low dose, a set amount of the high dose formulation was added to measuring cylinders and made up with vehicle in order to prepare 0.05 and 0.0125 mg/mL formulations. These were also well mixed. All dosing formulations were appropriately identified.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Selected based on work conducted in preliminary repeated dose study and a seperate homogeniety and stability test. Methyl cellulose is a standard vehicle for tests of this type.
- Concentration in vehicle: 0.0125, 0.05 and 0.2 mg/mL
- Amount of vehicle (if gavage): 10 mL/Kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At the initial preparation analytical samples were taken from the low, mid and upper layer of the whole dosing formulation at each concentration. These were then analysed by HPLC to ensure the dose formulations were homogeneous

Duration of treatment / exposure:

Males: 42 days in total
Females: 14 days before mating, until Day 13 of lactation.
Recovery females: 42 days in total

Frequency of treatment:

Once daily between 8:01 and 12:36.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.125 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

0.5 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

0.2 mg/kg bw/day (nominal)

No. of animals per sex per dose:

As per Table in 'Any other information on materials and methods incl. tables' section.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 14 day preliminary study. Doses of 30 mg/kg were deemed to severe to rats with death and changes following corrosion noted. Changes at 6 mg/kg were less severe but changes noted in the stomach's of animals were similar to that observed at 30 mg/kg.
- Rationale for animal assignment (if not random): Vaginal smears were collected from females 9 days ahead of the start of dosing and the oestrous cycles observed. Some animals presented with abnormalities and were not selected for use on study. All other animals were randomly allocated to study based on bodyweight (within 20% of the sex mean).
- Rationale for selecting satellite groups: Satellite animals were included on study in the control and high dose groups. These were included to assess the potential for recovery of effects that may have been observed on study.
- Post-exposure recovery period in satellite groups: 2 weeks

Positive control:

Not required

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice a day during the dosing periods and once a day on other periods

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to dosing and once a week during the dosing and recovery periods.

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

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 42 for test males, Day 56 for recovery males and satellite females and lactation Day 13 for the test females.
- Anaesthetic used for blood collection: Yes (intraperitoneal injection of sodium pentobarbitol).
- Animals fasted: Yes for around 18 hours
- How many animals: 5 animals per group for test males, all recovery males, all satellite females and 5 animals per group from the earlier partuition date for test females.
- Parameters examined: white blood cell, red blood cell, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, reticulocyte ratio and count, platelets, differential leukocyte ratio and count, prothrombin time and activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 42 for test males, Day 56 for recovery males and satellite females and lactation Day 13 for the test females.
- Animals fasted: Yes for around 18 hours
- How many animals: 5 animals per group for test males, all recovery males, all satellite females and 5 animals per group from the earlier partuition date for test females.
- Parameters examined: total protein, albumin, albumin/globulin ratio, aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), gamma glutamyltranspeptidase, alkaline phosphatase (ALP), total bilirubin, total bile acid, total cholesterol, triglycerides, glucose, urea nitrogen, creatinine, calcium, inorganic phosphorus, sodium, potassium and chloride.

URINALYSIS: Yes
- Time schedule for collection of urine: Day 41, 55 and 56 in test males (5 animals per dose)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: pH, protein, glucose, ketone body, occult blood, crystals, red blood cells, white blood cells, epithelial cells, casts, colour, specific gravity, sodium, potassium and chloride.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 5 males per group (week 6). For females, 5 animals with the nearer partuition date were selected and FOBs performed once during the final week during the lactation period. No examinations were performed during the recovery periods as no effects were observed.
- Dose groups that were examined: All
- Battery of functions tested: sensory activity to stimuli, grip strength measurement and motor activity measurement.

IMMUNOLOGY: Yes
- Time schedule for examinations: Males after the dosing period and after the recovery period and offspring on postnatal Day 13.
- How many animals: 5 animals per group (F0 males) and 5 offspring per group.
- Dose groups that were examined: All dose groups examined
- Parameters examined: Total T4

Sacrifice and pathology:

GROSS PATHOLOGY: All surviving animals were euthanized by exsanguination under anesthesia and subjected to necropsy. In addition, the vaginal smears were collected from the females on LD 14 (necropsy day) in the morning and the stage of the oestrous cycle was examined under a microscope.
Non-copulated females (Nos. 661 and 695) were necropsied after 14 days from the completion of the mating period. Non-delivered females (Nos. 670 and 688) were necropsied on GD 26. These animals were euthanized and necropsied in the same manner as the above-mentioned surviving animals.

HISTOPATHOLOGY: The organs/tissues of 5 animals per group of the test males and 5 animals per group from the earlier parturition date for the test females in the control and 2 mg/kg groups collected at the end of the dosing period and all gross lesions were embedded in paraffin, sectioned and stained with hematoxylin and eosin, and then examined by microscopy. The samples derived from 1 female with total litter loss (No.680) and the ovary derived from non-pregnant animals (Nos. 670 and 688) were prepared into hematoxylin-eosin stained samples and examined.

According to these results, a change suspected to be attributable to the test substance treatment was observed in the stomach of males. Therefore, the additional examination was performed for the organs of 5 males from the other dose groups collected at the end of the dosing period and that of all groups collected at the end of the recovery period.

Organ sampling and histopathology: stomach, duodenum, jujunum, ileum (including payer's patches), caecum, colon, rectum, liver, pancreas, submandibular gland, trachea, lung (including bronchus), thymus, spleen, femur/bone marrow, sternum/bone marrow, mandibular lymph node, mesenteric lymph node, heart, kidney, urinary bladder, testis, epididymis, prostate (ventral lobe), seminal vesicle (including dorsolateral lobe and coagulating gland), levator ani/bulbocavernosus muscle, comper's gland, glans penis, ovary, uterus, vagina, pituitary gland, thyroid/parathyroid, adrenal, brain (cerebrum, cerebellum and medulla oblongata/pons), spinal cord, femoral muscle/sciatic nerve, eyeball, mammary gland (dam with total litter loss) and any gross lesions.

ORGAN WEIGHTS: Selected organs were weighed (absolute weight), and the ratios of organ weight to bodyweight were calculated. The measurement was conducted in 5 animals per group of the test males, all recovery males, all satellite females and 5 animals per group from the earlier parturition date in the test females. The testis and epididymis weights were measured in all males. The measurement for non-copulated females and non-delivered females was not performed. Paired organs were weighed together.

Organs weighed: liver, thymus, spleen, heart, kidney, testis, epididymides, prostate, seminal vesicle, levator ani/bulbocavernosus muscle, cowper's gland, glans penis, ovary, uterus, thyroid/parathyroid, adrenal and the brain.

Statistics:

For the following numerical data, mean values and standard deviations were calculated in each group. Bartlett’s test was performed to compare variances among groups (significance level: 5%). When variance of data was homogeneous, Dunnett’s multiple comparison test was performed to compare with the control group. When variance of data was heterogeneous, Steel’s multiple comparison test was performed to compare with the control group. For urinalysis (qualitative data: results from reagent strip method and urinary sediment observation), Steel’s multiple comparison test was performed after the grades were converted into numeric values.

Number of rearing, grip strength, motor activity, body weights, food consumption, urinalysis (except for urine color), hematology, blood chemistry, absolute and relative and organ weights.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related change was noted in any animal throughout the dosing or recovery period. No treatment-related change was noted in any animal in the hand held observation or observation on the open field throughout the dosing or recovery period.

As a change without dose-dependency and considered as not treatment-related, high values of number of rearing were noted in males of the 0.125 and 0.5 mg/kg groups in Week 3.

Mortality:

no mortality observed

Description (incidence):

No unscheduled mortality occured.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant difference was noted in males or females between the control and test substance groups throughout the dosing or recovery period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No significant difference was noted in males or females between the control and test substance groups throughout the dosing or recovery period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related changes were noted. At the end of the dosing period, shortening of APTT was noted in females of the 0.125 mg/kg group and above. However, this was judged not to be treatment-related as the change was slight compared to that of the control group and opposite to the toxicity effect. As a change without dose-dependency and considered as not treatment-related, low values of neutrophil count and shortening of PT were noted in males and females of the 0.5 mg/kg group, respectively.

At the end of the recovery period, high values of reticulocyte count (and ratio) and basophil ratio were noted in males and females of the 2 mg/kg group, respectively. However, these were judged not to be treatment-related as the changes were slight compared to those of the control group, the similar changes were not noted at the end of the dosing period or no related change was noted in any examination.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related changes were noted. At the end of the recovery period, low values of triglyceride and glucose and high values of urea nitrogen and creatinine were noted in females of the 2 mg/kg group. However, these were judged not to be treatment-related as the changes were slight compared to those of the control group or no related change was noted in any examination.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related changes were noted. During the dosing period, ketone was biased toward positive in males of the 2 mg/kg group in the qualitative analysis (control group: 2 animals in grade 1+, 2 mg/kg group: 5 animals in grade 1+). However, this was judged not to be treatment-related as no related change was noted in any examination. No significant different was noted between the control and test substance groups in the urinary sediment and accumulated urine test.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related changes were observed for sensory reactivity to simuli, grip strength or motor activity.

As a change without dose-dependency and considered as not treatment-related, low values of motor activity between 10 and 30 minutes and total motor activity were noted in males of the 0.125 mg/kg group.

Immunological findings:

no effects observed

Description (incidence and severity):

No significant difference was noted in the plasma total T4 concentration between the control and test substance groups.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related changes were noted. At the end of the dosing period, high values of absolute kidneys weight were noted in males of the 2 mg/kg group. However, this was judged not to be treatment-related as the change was slight compared to that of the control group and no related change was noted in any examination. As a change without dose-dependency and considered as not treatment-related, high values of absolute kidneys weight and low values of relative levator ani/bulbocavernosus muscle were noted in males of the 0.125 mg/kg group and 0.5 mg/kg group, respectively.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related changes were noted. At the end of the dosing period, small of the testis was observed bilaterally in 1 male of the 2 mg/kg group. Small of the Cowper’s gland and brownish patch of the Cowper’s gland were observed unilaterally in 1 male of the 2 mg/kg group and 1 male of the 0.125 mg/kg group, respectively. However, these were judged not to be treatment-related owing to the lack of histopathological correlate or findings also observed in control animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the dosing period, minimal hyperplasia of squamous epithelium in the forestomach was observed in 1 male of the 2 mg/kg group. Minimal oedema of mucosa and minimal inflammatory cell infiltration of submucosa were observed in this site. As this was only observed in one animal, although potentially treatment related this finding is of no toxicological significance. It was also not observed at the end of the recovery period in any animals.

Various histopathological changes were noted in both sexes of the control and 2 mg/kg groups. However, these were judged not to be treatment-related as they are observed occasionally in normal rats, and there were no clear dose-differences in the incidence. In the non-pregnant females and the one dam with total litter loss, no relevant histopathological changes were noted.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Dose descriptor:

NOAEL

Effect level:

2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically relevant effects noted and accordingly NOAEL is set as the top dose.

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The substance was orally administered to rats by oral gavage at 0.125, 0.5 and 2 mg/kg/day for the required periods in the OECD 422 guideline, with an additional recovery phase included. Results indicated some effects in the stomach in one male at the high dose group which was observed in isolation and not at the end of the recovery period. As such the NOAEL for the study is deemed to be the highest dose tested of 2 mg/kg/day.