Copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, sulfonated

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Feb 2017 to 28 Mar 2017

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:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Feb 2017 to 28 Mar 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Sprague Dawley Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC on 28 Feb 2017
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: minimum of 70 days of age
- Weight at study initiation: Male body weights ranged from 331 g to 401 g and female body weights ranged from 234 g to 271 g on Study Day 0.
- Fasting period before study: Not reported
- Housing: Housed 2–3 rats/cage by sex in clean, solid-bottom cages with bedding material (Bed-O' Cobs®; The Andersons, Cob Products Division, Maumee, OH).
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, provided ad libitum
- Water (e.g. ad libitum): Municipal water, treated with Reverse osmosis-purified (on-site) drinking water, provided ad libitum
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY: Feed lots used during the study were documented in the study records. Feeders were changed and sanitized once per week. Municipal water supplying the facility was sampled for contaminants according to Charles River SOPs. The results of the diet and
water analyses are maintained at Charles River. No contaminants were present in animal feed or wa
ter at concentrations sufficient to interfere with the objectives of this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): f 68°F to 78°F (20°C to 26°C)
- Humidity (%): 30% to 70%
- Air changes (per hr): 10 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod

IN-LIFE DATES: From: 24 Feb 2017 To: 28 Mar 2017

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

Polyethylene glycol (PEG) 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The vehicle suspension was dispensed approximately weekly for administration to the control group (Group 1) and for preparation of the test item formulations; aliquots were prepared for daily dispensation to the control group and stored at room temperature (18ºC to 24ºC). The vehicle was mixed throughout the preparation, sampling, and dose administration procedures.The test item formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature (18ºC to 24ºC). The test item formulations were stirred continuously throughout the
preparation, sampling, and dose administration procedures.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not reported
- Concentration in vehicle: Not reported
- Amount of vehicle (if gavage): dose volume for all groups was 5 mL/kg.
- Lot/batch no. (if required): 2EL0005
- Purity: Not reported

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days days of unsuccessful pairing: If evidence of copulation was not detected after 14 days of pairing, the animals were separated without further opportunity for mating.
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged (how): animals were separated
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for homogeneity and/or concentration determination were collected from the top, middle, and bottom strata of the first 20, 120, and 200 mg/mL dosing formulations and from the middle stratum of the first control and 70 mg/mL dosing formulations. In addition, samples for resuspension homogeneity determinations were collected from the top and bottom strata of an aliquot taken from the first 20, 120, and 200 mg/mL dosing suspensions following room temperature storage for 10 days; aliquots were mixed for a minimum of 30 minutes prior to sample collection. Samples for concentration analysis were also collected from the middle stratum from the third and last preparation of each dosing formulation (including the control group) prepared during the study. One set of samples from each collection was subjected to the appropriate analyses. All remaining samples were stored at room temperature (18ºC to 24ºC) as back-up. All analyses were conducted by the Charles River Analytical Chemistry Department using a high performance liquid chromatography method with ultraviolet absorbance detection.

Duration of treatment / exposure:

28 days

Frequency of treatment:

F0 male animals were dosed daily for a minimum of 14 days prior to mating and continuing throughout mating for a minimum of 28 days. At the end of the 28-day treatment period, males assigned to the recovery groups remained on study for 15 days without treatment.
The F0 female animals were dosed once daily for 14 days prior to mating and continuing throughout mating, gestation and lactation until 1 day prior to scheduled euthanasia.

Details on study schedule:

- F1 parental animals not mated until 14 days of treatment after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 10 days of age.
- Age at mating of the mated animals in the study: 12 weeks

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

20 mg/mL concentration

Dose / conc.:

350 mg/kg bw/day (nominal)

Remarks:

70 mg/mL concentration

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

120 mg/mL concentration

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

200 mg/mL concentration

No. of animals per sex per dose:

15/sex/dose for control and highest dose level.
10/sex/dose for all other dose levels.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosage levels were selected based on the results of a previous 14-day range-finding study in which male and female rats were administered the test substance at dosage levels of 35, 350, 500, and 1000 mg/kg/day. In that study, clinical observations included blue staining or discoloration on the fur, tail, and ears were noted at all dosage levels as a result of discolored excrement. This finding was related to the color of the neat test substance and was not considered adverse. Mean body weights, food consumption, and organ weights were unaffected by treatment. Therefore, dosage levels of 100, 350, 600, and 1000 mg/kg/day were selected for the current study.The selected route of administration for this study was oral (gavage) because this a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.
- Rationale for animal assignment (if not random): random assignment

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: mortality and moribundity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at appropriate intervals per the study report.

BODY WEIGHT: Yes
- Time schedule for examinations: at appropriate intervals per the study report.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, at appropriate intervals per the study report.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: N/A

Oestrous cyclicity (parental animals):

Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of the estrous cycle of each female for 14 days prior to randomization and continuing for females selected for the breeding phase until evidence of copulation was observed or until termination of the mating period for females with no evidence of mating. The average cycle length was calculated and reported for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P] until the detection of evidence of mating), beginning with the first day of dose administration. Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. For breeding phase females, the cycle during which evidence of mating was observed for a given animal was not included in the mean individual estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.

Sperm parameters (parental animals):

Not reported, although historical data was included in report.

Litter observations:

- The mean litter proportions (% per litter) of pup viability during the postnatal period and sex ratio at birth.
- Organ weights
- Locomotor Activity Data
- Postnatal survival

Postmortem examinations (parental animals):

Complete postmortem examinations were performed on all parental animals at the scheduled necropsies.

SACRIFICE by carbon dioxide inhalation
- Male animals: All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.]
- Maternal animals: All surviving animals [describe when, e.g. after the last litter of each generation was weaned.]

GROSS NECROPSY
- the necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal and pelvic cavities, including viscera. For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy. For F0 females that were mated, the number of former implantation sites was recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed: Adrenal glands (2), Aorta, Bone with marrow, Sternum, Brain, Coagulating glands (2), Eyes with optic nerves (2), Gastrointestinal tract, Esophagus, Stomach, Duodenum, Peyer’s patches, Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys (2), Liver (sections of 2 lobes), Lungs (including bronchi, fixed by inflation, with fixative), Lymph nodes, Axillary (2), Mandibular (2), Mesenteric, Ovaries and oviducts (2), Pancreas, Peripheral nerve (sciatic), Pituitary, Prostate, Salivary glands (mandibular [2]), Seminal vesicles (2), Skeletal muscle (rectus femoris), Skin with mammary gland, Spinal cord (cervical), Spleen, Testes with epididymides (2), and vas deferens, Thymus, Thyroid with parathyroids (2), Trachea, Urinary bladder, Uterus e with cervix and vagina, and Gross lesions (when possible).

Postmortem examinations (offspring):

SACRIFICE
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: postnatal survival, weight, developmental parameters, hormone values, necropsy.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGTHS
- same as parental organs.

Statistics:

All statistical analyses were conducted using SAS® software.

Reproductive indices:

Male/female mating index, Male/female fertility index, Male/female copulation index,

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All animals survived to the scheduled necropsies.

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:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

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):

The test substance-related microscopic findings were noted in the stomach, duodenum, jejunum, ileum, cecum, colon, and rectum of all test substance-administered groups at the primary necropsy. Findings in gastrointestinal tract organs consisted of blue discoloration and/or contents. The findings were attributed to the color of the test substance, and were not considered to be adverse. There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of effects.

Critical effects observed:

no

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):

no effects observed

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:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of effects.

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

Administration of the test substance via oral gavage once daily to F0 male rats for a minimum of 14 days prior to mating and continuing throughout mating for a minimum of 28 days and to F0 female rats for 14 days prior to mating and continuing throughout mating, gestation and lactation until 1 day prior to scheduled euthanasia was well tolerated. Test substance-related blue discoloration/contents were observed macroscopically in the stomach, duodenum, jejunum, ileum, cecum, colon, and rectum of all test substance-administered groups at the primary necropsy, which correlated microscopically with blue contents. Blue matting of skin and tail were additionally noted macroscopically at the primary and recovery necropsies. The findings were attributed to the blue color of test substance and were considered to be nonadverse. No test substance-related differences in final body weights or organ weights were noted. NOAEL for F0 systemic toxicity was considered to be 1000 mg/kg/day. The NOAEL for F1 neonatal toxicity was 1000 mg/kg/day based on the absence of effects on F1 offspring at all dosage levels.

Executive summary:

The objectives of the study were to evaluate the potential toxic effects of the test substance when administered to rats for 28 days and to evaluate the potential of the test substance to affect male and female reproductive performance such as gonadal function, mating behavior, and conception through Day 13 of postnatal life, according to OECD 422.

Administration of the test substance via oral gavage once daily to F0 male rats for a minimum of 14 days prior to mating and continuing throughout mating for a minimum of 28 days and to F0 female rats for 14 days prior to mating and continuing throughout mating, gestation and lactation until 1 day prior to scheduled euthanasia was well tolerated. Doses included 100, 350, 600 and 1000 mg/kg bw/day. Test substance-related blue discoloration/contents were observed macroscopically in the stomach, duodenum, jejunum, ileum, cecum, colon, and rectum of all test substance-administered groups at the primary necropsy, which correlated microscopically with blue contents. Blue matting of skin and tail were additionally noted macroscopically at the primary and recovery necropsies. The findings were attributed to the blue color of test substance and were considered to be nonadverse. No test substance-related differences in final body weights or organ weights were noted.

Under the conditions of this screening study, no test substance-related effects were observed on reproductive performance at any dosage level. Therefore, a dosage level of 1000 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive toxicity of the test substance when administered orally by gavage to Crl:CD(SD) rats. There were no adverse test substance-related clinical observations or effects on mean body weights, body weight gains, and food consumption noted at any dosage level. Furthermore, there were no adverse effects on organ weights, clinical pathology parameters, thyroid hormones (males only) or macroscopic/microscopic examination in the F0 males and females at any dosage level.

Therefore, the NOAEL for F0 systemic toxicity was considered to be 1000 mg/kg/day. The NOAEL for F1 neonatal toxicity was 1000 mg/kg/day based on the absence of effects on F1 offspring at all dosage levels.

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

Test material form:

solid: crystalline

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Sprague Dawley Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC on 28 Feb 2017
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: minimum of 70 days of age
- Weight at study initiation: Male body weights ranged from 331 g to 401 g and female body weights ranged from 234 g to 271 g on Study Day 0.
- Fasting period before study: Not reported
- Housing: Housed 2–3 rats/cage by sex in clean, solid-bottom cages with bedding material (Bed-O' Cobs®; The Andersons, Cob Products Division, Maumee, OH).
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, provided ad libitum
- Water (e.g. ad libitum): Municipal water, treated with Reverse osmosis-purified (on-site) drinking water, provided ad libitum
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY: Feed lots used during the study were documented in
the study records. Feeders were changed and sanitized once per week. Municipal water supplying
the facility was sampled for contaminants according to Charles River SOPs. The results of the diet
and water analyses are maintained at Charles River. No contaminants were present in animal feed or
water at concentrations sufficient to interfere with the objectives of this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): f 68°F to 78°F (20°C to 26°C)
- Humidity (%): 30% to 70%
- Air changes (per hr): 10 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod

IN-LIFE DATES: From: 24 Feb 2017 To: 28 Mar 2017

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The vehicle and test item formulations were administered orally by gavage, via appropriately sized flexible, disposable, plastic feeding tubes once daily for 14 consecutive days (Study Days 0–13). In dividual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day.

Vehicle:

polyethylene glycol

Remarks:

Polyethylene glycol (PEG) 400

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The vehicle suspension was dispensed approximately weekly for administration to the control group (Group 1) and for preparation of the test item formulations; aliquots were prepared for daily dispensation to the control group and stored at room temperature (18ºC to 24ºC). The vehicle was mixed throughout the preparation, sampling, and dose administration procedures.The test item formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature (18ºC to 24ºC). The test item formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not reported
- Concentration in vehicle: Not reported
- Amount of vehicle (if gavage): dose volume for all groups was 5 mL/kg.
- Lot/batch no. (if required): 2EL0005
- Purity: Not reported

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for homogeneity and/or concentration determination were collected from the top, middle, and bottom strata of the first 20, 120, and 200 mg/mL dosing formulations and from the middle stratum of the first control and 70 mg/mL dosing formulations. In addition, samples for resuspension homogeneity determinations were collected from the top and bottom strata of an aliquot taken from the first 20, 120, and 200 mg/mL dosing suspensions following room temperature storage for 10 days; aliquots were mixed for a minimum of 30 minutes prior to sample collection. Samples for concentration analysis were also collected from the middle stratum from the third and last preparation of each dosing formulation (including the control group) prepared during the study. One set of samples from each collection was subjected to the appropriate analyses. All remaining samples were stored at room temperature (18ºC to 24ºC) as back-up. All analyses were conducted by the Charles River Analytical Chemistry Department using a high performance liquid chromatography method with ultraviolet absorbance detection.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

20 mg/mL concentration

Doses / concentrations 2

Dose / conc.:

350 mg/kg bw/day (nominal)

Remarks:

70 mg/mL concentration

Doses / concentrations 3

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

120 mg/mL concentration

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

200 mg/mL concentration

No. of animals per sex per dose:

15/sex/dose for control and highest dose level.
10/sex/dose for all other dose levels.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosage levels were selected based on the results of a previous 14-day range-finding study in which male and female rats were administered the test substance at dosage levels of 35, 350, 500, and 1000 mg/kg/day. In that study, clinical observations included blue staining or discoloration on the fur, tail, and ears were noted at all dosage levels as a result of discolored excrement. This finding was related to the color of the neat test substance and was not considered adverse. Mean body weights, food consumption, and organ weights were unaffected by treatment. Therefore, dosage levels of 100, 350, 600, and 1000 mg/kg/day were selected for the current study.The selected route of administration for this study was oral (gavage) because this a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.
- Rationale for animal assignment (if not random): random assignment

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, once in the morning and once in the afternoon.
- Cage side observations checked: moribundity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily, prior to dose administration and at appropriate intervals per the study report.

BODY WEIGHT: Yes
- Time schedule for examinations: at appropriate intervals per the study report.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, at appropriate intervals per the study report.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes, Food consumption was normalized to the number of animals/cage and was reported as g/animal/day.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: N/A

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: N/A
- Dose groups that were examined: N/A

HAEMATOLOGY: No
- Time schedule for collection of blood: N/A
- Anaesthetic used for blood collection: N/A
- Animals fasted: N/A
- How many animals: N/A
- Parameters checked: N/A

CLINICAL CHEMISTRY: No
- Time schedule for collection of blood: N/A
- Animals fasted: N/A
- How many animals: N/A
- Parameters checked: N/A

URINALYSIS: No
- Time schedule for collection of urine: N/A
- Metabolism cages used for collection of urine: N/A
- Animals fasted: N/A
- Parameters checked: N/A

NEUROBEHAVIOURAL EXAMINATION: yes
- Time schedule for examinations: At appropriate times.
- Dose groups that were examined: all
- Battery of functions tested: motor activity

IMMUNOLOGY: No
- Time schedule for examinations: N/A
- How many animals: N/A
- Dose groups that were examined: N/A
- Parameters checked: N/A

OTHER: No

Sacrifice and pathology:

- the necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal and pelvic cavities, including viscera. For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy. For F0 females that were mated, the number of former implantation sites was recorded.

Other examinations:

The following organs were weighed from all animals at the scheduled necropsy: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries with oviducts, Pituitary, Prostate with seminal vesicles, Spleen, Testes, Thymus gland, Thyroids with parathyroids, and Uterus.

Statistics:

All statistical analyses were conducted using SAS® software.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

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:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related alterations in final body weights or organ weights at the scheduled necropsies. However, some statistically significant differences were observed when the control and test substance-treated groups were compared. Higher mean spleen weights (absolute and relative to final body weight) were noted in the 1000 mg/kg/day group females at the primary necropsy. No microscopic correlates were noted and weights were within the historical control database range; higher spleen weights were attributed to biological variability and not related to administration of the test substance. At recovery, statistically lower mean ovaries/oviducts weights (relative to final body weight) were noted in the 1000 mg/kg/day group females. Control and the test substance-administered ovaries/oviducts weights were similar at the primary necropsy, the difference in weight was of minimal magnitude, and weights were within the historical control range; therefore, the difference in weights was attributed to biological variability and not test substance-related. Statistically significantly lower mean thymus weight relative to final body weight was noted in the 1000 mg/kg/day group males at recovery. No thymus weight difference was noted at the primary necropsy and weights were within the historical control range; therefore, the difference in thymus weight was not considered to be test substance-related.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The test substance-related microscopic findings were noted in the stomach, duodenum, jejunum, ileum, cecum, colon, and rectum of all test substance-administered groups at the primary necropsy. Findings in gastrointestinal tract organs consisted of blue discoloration and/or contents. The findings were attributed to the color of the test substance, and were not considered to be adverse. There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of effects.

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Administration of the test substance via oral gavage once daily to F0 male rats for a minimum of 14 days prior to mating and continuing throughout mating for a minimum of 28 days and to F0 female rats for 14 days prior to mating and continuing throughout mating, gestation and lactation until 1 day prior to scheduled euthanasia was well tolerated. Test substance-related blue discoloration/contents were observed macroscopically in the stomach, duodenum, jejunum, ileum, cecum, colon, and rectum of all test substance-administered groups at the primary necropsy, which correlated microscopically with blue contents. Blue matting of skin and tail were additionally noted macroscopically at the primary and recovery necropsies. The findings were attributed to the blue color of test substance and were considered to be nonadverse. No test substance-related differences in final body weights or organ weights were noted. NOAEL for F0 systemic toxicity was considered to be 1000 mg/kg/day. The NOAEL for F1 neonatal toxicity was 1000 mg/kg/day based on the absence of effects on F1 offspring at all dosage levels.

Executive summary:

The objectives of the study were to evaluate the potential toxic effects of the test substance when administered to rats for 28 days and to evaluate the potential of the test substance to affect male and female reproductive performance such as gonadal function, mating behavior, and conception through Day 13 of postnatal life, according to OECD 422. Administration of the test substance via oral gavage once daily to F0 male rats for a minimum of 14 days prior to mating and continuing throughout mating for a minimum of 28 days and to F0 female rats for 14 days prior to mating and continuing throughout mating, gestation and lactation until 1 day prior to scheduled euthanasia was well tolerated. Doses included 100, 350, 600 and 1000 mg/kg bw/ day. Test substance-related blue discoloration/contents were observed macroscopically in the stomach, duodenum, jejunum, ileum, cecum, colon, and rectum of all test substance-administered groups at the primary necropsy, which correlated microscopically with blue contents. Blue matting of skin and tail were additionally noted macroscopically at the primary and recovery necropsies. The findings were attributed to the blue color of test substance and were considered to be nonadverse. No test substance-related differences in final body weights or organ weights were noted.

Under the conditions of this screening study, no test substance-related effects were observed on reproductive performance at any dosage level. Therefore, a dosage level of 1000 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive toxicity of the test substance when administered orally by gavage to Crl:CD(SD) rats. There were no adverse test substance-related clinical observations or effects on mean body weights, body weight gains, and food consumption noted at any dosage level. Furthermore, there were no adverse effects on organ weights, clinical pathology parameters, thyroid hormones (males only) or macroscopic/microscopic examination in the F0 males and females at any dosage level. Therefore, the NOAEL for F0 systemic toxicity was considered to be 1000 mg/kg/day. The NOAEL for F1 neonatal toxicity was 1000 mg/kg/day based on the absence of effects on F1 offspring at all dosage levels.