Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
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
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
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)

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.
Dose / conc.:
0.125 mg/kg bw/day (nominal)
Dose / conc.:
0.5 mg/kg bw/day (nominal)
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
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.
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
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.
Critical effects observed:
no
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.

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

Constituent 1
Chemical structure
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)
Sex:
male/female
Details on test animals or test system and environmental conditions:
This strain is widely used in toxicity studies using rodents, there is abundant historical data and a large number of animals are available.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Methylcellulose
Remarks:
0.5 w/v% in water for injection
Details on 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
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Up to 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Individually in polymethylpentene cages
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.
Details on study schedule:
- Age at mating of the mated animals in the study: approximately 11 weeks (parental). No mating of the F1 generation occured as is not a requirement of the testing guideline.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
0.125 mg/kg bw/day (nominal)
Dose / conc.:
0.5 mg/kg bw/day (nominal)
Dose / conc.:
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

Parental animals: Observations and examinations:
Details included in the Endpoint study record for the repeated dose aspect of this study (cross referenced).
Oestrous cyclicity (parental animals):
For females, vaginal smears were collected with a swab for 9 days from the end of quarantine period, and the oestrous cycle was observed. This information was used to assist in the selection of suitable females for use on study.

Vaginal smears were also collected with swabs from all test females in the morning (same time every day) from the initial dosing day to the day of successful copulation or end of the mating period to confirm the estrous cycle. The obtained smears were collected on a plate for each animal, and stained with Giemsa. The estrous cycle was classified into diestrus (D), proestrus (P), estrus (E) and metestrus (M).

The mean estrous cycle (number of days from the estrous period to the next estrous period) and the number of the estrous period during the test period were calculated.
Sperm parameters (parental animals):
Parameters examined in male parental generation:

Testis weight and epididymis weight were measured in parental males. No further assessment of sperm parameters were taken.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: On PND 4, the litter size was standardized to 8 (4/sex/litter) by random removal of offspring. Even when the number of either males or females per litter was less than 4, the litter size was adjusted to 8 regardless of the sex ratio. Litter with less than 8 offspring was maintained as is.

The offspring culled at the litter size adjustment (offspring excluded from blood collection) were euthanized by overdose with pentobarbital sodium (undiluted solution, 0.05 to 0.1 mL/body) via an intraperitoneal injection and discarded. Offspring not culled after standardization were
allocated for the collection of blood samples.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Anogenital distance, nipple development, number of litter (numbers of live newborn and stillborn), sex, the presence of external abnormalities, mortality and bodyweight.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external abnormalities; possible cause of death was not determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Not conducted in pups (not a guideline requirement)

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Not conducted in pups (not a guideline requirement)
Postmortem examinations (parental animals):
Details included in the Endpoint study record for the repeated dose aspect of this study (cross referenced).
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed on postnatal day 13.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external examinations including the cervical, thoracic, and abdominal viscera. The offspring subjected to blood sampling were necropsied after blood sampling by decapitation after anesthetizing by inhalation of isoflurane. Other animals were euthanized by exsanguination from the lateral iliac artery under anesthesia with pentobarbital sodium via the intraperitoneal injection and necropsied. Furthermore, the thyroid glands of 1 male and 1 female (animals subjected to blood sampling for hormone level measurement) per litter were collected and fixed and preserved in 10 vol% phosphate buffered formalin.

Statistics:
For the following numeral data, mean values and standard deviations were calculated in each group. Bartlett’s test was performed to compare variances among groups (significance level: 5%). When variance of data was homogeneous, Dunnett’s multiple comparison test was performed to compare with the control group. When variance of data was heterogeneous, Steel’s multiple comparison test was performed to compare with the control group.

Oestrous cycle, number of oestrus, days until copulation, gestation length, number of implantations, number of delivered offspring, number of live offspring, body weight of offspring and AGD.

The following items were tested with the chi-square test for comparison between the control and each test substance group.

Copulation index, fertility index, gestation index, delivery index and sex ratio.

The following items were tested with the Wilcoxon’s rank sum test for comparison between the control and each test substance group.

Stillborn index, external anomaly index, external anomaly typing index, birth index, viability index on Days 4 and 13 and nipple development anomaly index.
Reproductive indices:
Days until copulation, copulation index (%), fertility index (%), gestation length, gestation index (%) and delivery index (%).
Offspring viability indices:
Birth index (%), stillborn index (%), viability index on Days 4 and 13 (%), sex ratio, external anomaly index (%), external anomaly typing index (%) and nipple development anomaly index (%).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Details included in the Endpoint study record for the repeated dose aspect of this study (cross referenced).
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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):
Details included in the Endpoint study record for the repeated dose aspect of this study (cross referenced).
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Details included in the Endpoint study record for the repeated dose aspect of this study (cross referenced).
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Details included in the Endpoint study record for the repeated dose aspect of this study (cross referenced).
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Details included in the Endpoint study record for the repeated dose aspect of this study (cross referenced).
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Details included in the Endpoint study record for the repeated dose aspect of this study (cross referenced).
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
No significant difference was noted in the mean estrous cycle or count of oestrus, indicating no effect of the test substance on prolongation or shortening of the oestrous cycle.

Besides, irregular oestrous cycle was noted in 1 female of the 2 mg/kg group. However, this was judged not to be treatment-related as the copulation and conception were confirmed. Irregular oestrous cycle was also noted in 1 female of the control group; in which abnormalities were noted in the ovary and uterus
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No effects were noted on the organ weights of the testis or epididymides. No effects were noted pathologically.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
No significant difference was noted in the copulation index or fertility index between the control and test substance groups.

Besides, shortening of duration of copulation was noted in the 2 mg/kg group. However, this was judge not to be treatment-related as the mating was confirmed in 7/12 animals at the 1st oestrus in the 2 mg/kg group. No mating was confirmed in 1 female in the 2 mg/kg group. At necropsy, no abnormality was noted in the genital organs of this animal and the male counterpart. Although the cause of non-copulation was not clear, the change was judged not to be treatment-related for its incidence. Non-copulated female was also noted in 1 female of the control group. The cause of non-copulation was abnormal findings in the ovary and uterus. Non-pregnancy was noted in 1 female of the 2 mg/kg group. In the male counterpart, abnormalities were observed in the testis and epididymis; indicating spontaneous changes. Therefore, non-pregnancy was judged not to be treatment-related. Non-pregnancy was also noted in 1 female of the 0.125 mg/kg group. However, this was judged not to be treatment-related as the state of incidence.

No treatment-related change was noted in the delivery or nursing. Total litter loss was observed in 1 dam of the 0.5 mg/kg group on LD 3. This was judged not to be treatment-related.

No significant difference was noted in the gestation length, number of implantations and litter, delivery index or gestation index.

Details on results (P0)

Overall, there were no treatment related effects on paternal animals when considering reproductive performance.

Effect levels (P0)

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 (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related change was noted in anogenital distance or nipple development.

Low values of relative AGD value were noted in male offspring of the 2 mg/kg group. However, this was judged not to be treatment-related as the change was within mean ± 2 S.D. of the historical data at the test facility and no related change was noted in the plasma total T4 concentration.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related change was noted in any offspring.

Visceral inversion was observed in 1 male of the 0.125 mg/kg group, indicating spontaneous change.
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
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.

Details on results (F1)

Overall no treatment-related effects were noted in the offspring of parental animals treated with the test substance.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
2 mg/kg bw/day (nominal)
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 (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
A reliable OECD 422 study is available providing data on fertility of parental animals and offspring development. This study showed no effects at the highest feasible dose (due to the corrosive nature of the substance) on parental animal reproductive performance or offspring indices and accordingly the high dose is set as the NOAEL.