2-Naphthalenol, 1-[[4-(phenylazo)phenyl]azo]-, ar-heptyl ar',ar''-Me derivs.

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003 - 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study conducted in accordance with GLP

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003 - 2004

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source of animals: Charles River Laboratories, Portage, MI
Age at arrival: Approx 9 weeks
All animals acclimated for 1 week during which they were observed for clinical signs of disease.
Animals place on stdy had BW within the range of +/- 20% of the mean bodyweight for each sex.
Randomisation and assignment to treatment groups using a standard, by weight procedure
10 male and 10 female rats were assigned to each dose group.
Each animal was assigned a unique study identification number and microchipped.
Animal room conditions: Temperature maintained between 63 and 75 degrees F; Humidity between 32 and 69%.
Flourescent lighting was set to a 12 hour light cycle.
All animals individually housed (except during mating) in stainless steel cages containing cage liners (changed 3 times per week).
During mating animals were paierd (one male and one female) wihtin each treatment group. During parturition (from approx day 20 of gestation, females transferred to plastic cages with wood chip bedding.
Diet: Lab Diet Certified Rodent Diet #5002, sourced from PMI Nutrition International Inc. and tap water available ad-libitum throughout the study.
Water was monitored for specieid contaminants at periodic intervals according to the lab SOP.
Study director was not aware of any contaminants in the water that could have interfered with the study.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Test material administered for at lest 42 consecutive days to males and up to 54 consecutive days to females. It was administered once per day at the same time using a fixed dose volume of 5 ml/Kg bodyweight.
Control animals recieved the corn oil vehicle. Individual doses were based on the most recent bodyweight data.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Doses were analyzed for homogeneity, Stability and test material concentration. Concentration of test material in the dosing solutions was verified using a 5ml sample from each test material formulation at weeks 1, 4, 7.

Duration of treatment / exposure:

Males - 42 consecutive days
females - 54 consecutive days

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

Doses were selected based on a range finding study in non-pregnant animals, and from a previous OECD 422 study that had several issues leading to it being considered as unreliable.

Positive control:

No

Observations and examinations performed and frequency:

In-life Examinations:

Mortality and Cageside Observations:
All rats were observed twice daily for morbidity, mortality, signs of injury, and the availability of food and water.

Detailed Clinical Examinations:
A detailed clinical observation of each animal was conducted once prior to initiation of test article administration and weekly during the study.

Neurobehavioral Observations:
Neurobehavioral observations of each animal were conducted once prior to initiation of test article administration and weekly during the study.

Body Weight:
Individual body weights for the males and unmated females were recorded the day after arrival, prior to randomization, at initiation of test article administration, weekly during the study and at termination. Mated females were weighed at these same intervals prior to mating and on Day 0, 7, 14, and 20 of gestation. Females delivering litters were also weighed on Days 0 and 4 of lactation.

Food Consumption:
Individual food consumption for males was measured and recorded weekly during the study, except during the mating period. Food consumption was also measured weekly during the
premating period for females. During the mating period, food consumption was not measured because the animals were cohabited. Food consumption was measured for mated females on Days 0-7, 7-14, and 14-20 of gestation. For females with litters, food consumption was measured on Days 0-4 of lactation.

Fo Breeding Procedures:
After two weeks of treatment, the males were randomly cohabited with corresponding females in the same treatment or control group (one male with one female). Animals were paired for mating in the cage of the male.
Vaginal lavage was performed daily on females during the mating period and the presence or absence of sperm or vaginal plug was recorded. The day on which evidence of mating (presence of sperm in vaginal lavage, or vaginal plug) was observed was designated as Day 0 of gestation. The rats were paired for a maximum of 14 days. When evidence of mating was
noted, the female was removed from the cage of the male and individually housed. After the mating period, any female with no positive evidence of mating was individually housed in a
plastic cage with wood chip bedding. Any female not producing a litter within 25 days of evidence of mating or separation from the male was euthanized and necropsied as described in Section 4.4. Postmortem Study Evaluations.
After the mating period, males were individually housed and continued on treatment for approximately two weeks. At the end of this period, the males were euthanized and necropsied as described in Section 4.4. Postmortem Study Evaluations.

Fo Parturition and F1 Litter Observations:
Toward the end of the gestation period, females were examined twice daily for signs of parturition. Females (Fo) were allowed to give birth (F1). The duration of gestation was calculated. The day on which all pups were delivered was designated as Day 0 of lactation. The litters were examined as soon as possible after delivery and parameters including litter size, number of stillborn and live born pups, and gross abnormalities of the pups were recorded.
Pups were weighed, sexed, and externally examined on Days 0 and 4 of lactation. Any abnormal behavior observed in the pups was recorded daily. On Day 4 of lactation, the pups were euthanized, externally examined, and discarded without further evaluation.

Behavioral Observations (Fo):
Five males and females from each group were randomly selected for behavioral testing. Observations were conducted during the last week of treatment for males and on Day 3 of
lactation in females. When females with litters were not available for selection, females with evidence of mating were evaluated on Day 25 of gestation (Group 4). Dams were not
removed from their litters for more than 40 minutes at one time, to avoid possible hypothermia of the pups.The animals were evaluated using the following neurobehavioral evaluations.

Functional Observational Battery (FOB) evaluations were conducted without knowledge on the part of the testers of the treatment groups.

Motor Activity:
Activity was assessed by placement in a Digiscan® Activity Monitor.

Clinical Pathology:
Five animals/sex/group were randomly selected for blood collection to evaluate hematology and clinical chemistry parameters. Females with viable litters were chosen, and when not possible, additional females with evidence of mating were chosen.

Sacrifice and pathology:

Postmortem Study Evaluations:
Complete necropsy examinations were performed under procedures approved by a veterinary pathologist on all adult animals (F0) at scheduled necropsy.

Macroscopic:
All animals were euthanized by carbon dioxide inhalation and examined carefully for external abnormalities, including palpable masses. The skin was reflected from a ventral midline incision and any abnormalities were identified and correlated with antemortem findings. The abdominal, thoracic, and cranial cavities were examined for abnormalities, and the organs were removed and examined. Special emphasis was placed on organs of the reproductive system. Implantation sites (scars) along the uterus and corpora lutea per ovary, when present, were counted and recorded for all females. All tissues were fixed in neutral buffered formalin except the testes and epididymides, which were placed in Bouin's fixative. Formalin was infused into the lung via the trachea and into the urinary bladder.

Organ Weights:
Fasted body weights were recorded for all animals, and protocol-specified organ weights were recorded for five randomly-selected rats/sex/group (the same animals chosen for clinical pathology evaluations) at necropsy and appropriate organ weight ratios were calculated (absolute and relative to body weight). In addition, the epididymides, testes, and ovaries were weighed for all animals. Paired organs were weighed together.

Microscopic:
Protocol-specified organs and tissues were shipped to Dr. W. Ray Brown at Research Pathology Services Inc., New Britain, Pennsylvania, for processing and microscopic examination of fixed hematoxylin and eosin-stained paraffin sections for five randomly-selected animals/sex/group (the same animals chosen for clinical pathology evaluations and organ weight measurements). In addition, the epididymides, testes, ovaries, and liver, stomach, spleen, and tissues with gross changes were examined from all rats of all groups.

Statistics:

The sets of comparisons used in the statistical analyses are presented below.

Statistical Comparison:
Control Group Treatment Groups
1 2, 3, 4

Statistical Analysis Methods:
Group Pair-wise Comparisons
Fisher's Exact Test
Log Transformation
Arcsin-Square-Root Transformation
Covariate Analysis
Descriptive Statistics

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test article-related effects noted in clinical findings in either sex at dose levels up to and including 80 mg/kg/day.

Mortality:

no mortality observed

Description (incidence):

There were no test article-related effects noted in mortalities in either sex at dose levels up to and including 80 mg/kg/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was no test article-related effect on body weight among males at dose levels up to and including 80 mglkg/day during the study or in females at 2 mglkg/day. During the premating period, a test article-related decrease (11-13%) in mean body weight was noted among females. Females at 20 and 80 mglkg/day also had a test article-related decrease (11-25%) in gestation body weights throughout the gestation period. A corresponding dose-related decrease in gestation body weight change over the entire period (Days 0-20 gestation [0]) was apparent at 20 and 80 mg/kg/day. Mean body weight was decreased (14-21 %) on Lactation Day 0 and 4 at 20 and 80 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was no test article-related effect on food consumption in males at dose levels up to and including 80 mg/kg/day or in females at 2 mg/kg/day. Food consumption was decreased during the premating and gestation periods in females at 20 and/or 80 mg/kg/day. These decreases included: premating period, (25-41%); Days 0-7 (23-26%); Days 7-14 (17-22%); and Days 14-20 (18%) at 80 mg/kg/day only. During the lactation period (Days 0-4), food consumption was decreased 51-55% at 20 and 80 mg/kg/day.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test article-related effects noted in hematology in either sex at dose levels up to and including 80 mg/kg/day.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no test article-related effects noted in clinical chemistry in either sex at dose levels up to and including 80 mg/kg/day.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no test article-related effects noted in neurobehavioral observations, FOB, motor activityin either sex at dose levels up to and including 80 mg/kg/day.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There was no test article-related organ weight effects noted in either sex at 2 mg/kg/day. Statistically significant increases in absolute and relative liver weights (18-29%) and in absolute and relative spleen weights (24-34%) among males at 80 mg/kg/day were considered test article-related. A test article-related decrease (26-30%) in absolute ovary weights at 20 and 80 mg/kg/day corresponded to the ovarian atrophy seen microscopically at these levels.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Pink staining of tissues due to red dye.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were no test article-related microscopic effects noted in either sex at 2 mg/kg/day. An increased incidence ofthe amount of hemosiderin pigment in the red pulp of the spleen was evident in both sexes at 20 and 80 mg/kg/day. Several male rats at 20 and 80 mg/kg/day were noted with hepatocellular cytoplasmic vacuolation. The ovaries of several female rats at 20 and 80 mg/kg/day were small due to generalized atrophy and decreased corpora lutea.

Test article effects in the stomach were somewhat varied and equivocal. The effects observed included: diffuse hyperplasia and hyperkeratosis of the squamous mucosa with associated submucosal edema and inflammation of the nonglandular area in one female at 20 and 80 mg/kg/day; focal erosion of the affected mucosa in the female affected at 20 mg/kg/day; and, hyperplasia and hyperkeratosis ofthe squamous mucosa ofthe limiting ridge in both sexes at 80 mg/kg/day. These changes are consistent with a local irritating effect of the test article on this area of the stomach.

Dose descriptor:

NOEL

Remarks:

Systemic toxicity and fertility

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects on BW, FC, organ wt, histopathology at 20 and 80 mg/kg bw Decreased fertility index at 20 and 80 mg/kg bw - reduced no. of litters and viable pups

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

kidney

liver

ovary

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Reproductive Effects:

There was no test article-related effect on any endpoint of mating or fertility at 2 mg/kg/day, including male and female mating and fertility indices, and time to mating. Mating indices (percent of paired animals that mated) were comparable among all groups. A test article-related decrease in the fertility index (percent of matings that resulted in pregnancy) was noted at 20 (60%) and 80 mg/kg/day (40%) compared to controls (90%). A corresponding test article-related decrease in the fecundity index was noted at these levels as well.

There was no effect in copulatory interval or gestation length at dose levels up to and including 80 mg/kglday. There was no effect on the number of females that delivered live offspring at 2 mg/kglday. There were no females that delivered an entirely stillborn litter.

A test article-related decrease was noted in the mean number oflive pups/litter compared to control values on Day 0 at 20 mg/kglday (9.2 vs. 14.7, respectively) and at 80 mg/kglday (5.8 vs. 14.7, respectively). Of the four females that delivered viable litters at 80 mg/kglday, three of the four litters had four or fewer pups.

There was no effect on pup survival or pup sex ratio at dose levels up to and including 80 mg/kglday. There were no test article-related clinical or external findings noted in the pups at any dose level.

There was no effect in pup body weight at 2 mg/kglday and no statistically significant differences noted at any level. However, pup body weight gain at 20 mg/kglday between Days 0 and 4 was reduced compared to the control value (1.97 vs. 3.59 g, respectively). A similar effect was not noted at 80 mg/kglday, but this is believed due to the small litter size (four or fewer pups) in three of the four females that delivered. The reduction in body weight gain at 20 mg/kglday was considered a test article-related effect.

Analytical Results:

Concentration analysis of formulations prepared at 2, 20, and 80 mglkg/day indicated that all preparations were generally within acceptable target levels (± 15%). The formulation was homogenous and stable for 14 days.

Conclusions:

Under the conditions of this study, Automater Red BXL SS Dye produced clear parental and reproductive toxicity at doses of 20 mg/kg bw and higher. Consequently the NOEL for parental toxicity and reproductive toxicity is 2 mg/kg bw/day.

Executive summary:

This nonclinical study was conducted for Rohm and Haas Company, to evaluate the effects of the test article, Automate™ Red BXL SS Dye, including its potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition, and early postnatal development. This study also placed emphasis on neurological effects as a specific endpoint in order to identify neurotoxic potential. This study was comprised of three treatment groups and a vehicle control group. Each group contained 10 male and 10 female Sprague-Dawley rats [Crl: CD® (SD)IGS BR]. The test article was administered to the treated groups via oral gavage at respective dose levels of 2, 20, and 80 mg/kg/day at a dose volume of 5 ml/kg. Males were treated for at least 42 consecutive days, while females were treated for up to 54 days depending on reproductive performance. The control animals received the vehicle, com oil, at the same volume and dosing regimen as the treated groups.

Observations of the animals included clinical signs, neurobehavioral observations, body weights, and food consumption. After two weeks of treatment, the animals were cohabited, one male and one female within the same treatment group for up to 14 days. Females were allowed to deliver litters and maintain the pups until Day 4 of lactation. Litters were evaluated for the number of live born or stillborn pups, survival indices, body weights, and any external gross abnormalities on Days 0 and 4. Evaluations for motor activity and functional observational battery (FOB), as well as blood collection for clinical pathology evaluations, were conducted prior to scheduled euthanasia. Complete necropsies were performed on all parental animals and protocol-designated organs and tissues were weighed and microscopically examined. Pups were euthanized and externally examined on Day 4 of lactation, and the carcasses were discarded without further evaluation.

Concentration analysis of formulations prepared at 2, 20, and 80 mg/kg/day indicated that all preparations were generally within acceptable target levels (± 15%). The formulation was homogenous and stable for 14 days.

There were no test article-related effects noted in mortalities, clinical findings, neurobehavioral observations, FOB, motor activity, hematology, clinical chemistry or macroscopic observations in either sex at dose levels up to and including 80 mg/kg/day.

There was no test article-related effect on body weight among males at dose levels up to and including 80 mg/kg/day during the study or in females at 2 mglkg/day. During the premating period, a test article-related decrease (11-13%) in mean body weight was noted among females. Females at 20 and 80 mg/kg/day also had a test article-related decrease (11-25%) in gestation body weights throughout the gestation period. A corresponding dose-related decrease in gestation body weight change over the entire period (Days 0-20 gestation [0]) was apparent at 20 and 80 mg/kg/day. Mean body weight was decreased (14-21 %) on Lactation Day 0 and 4 at 20 and 80 mg/kg/day.

There was no test article-related effect on food consumption in males at dose levels up to and including 80 mg/kg/day or in females at 2 mg/kg/day. Food consumption was decreased during the premating and gestation periods in females at 20 and/or 80 mg/kg/day. These decreases included: premating period, (25-41%); Days 0-7 (23-26%); Days 7-14 (17-22%); and Days 14-20 (18%) at 80 mg/kg/day only. During the lactation period (Days 0-4), food consumption was decreased 51-55% at 20 and 80 mg/kg/day.

There was no test article-related organ weight effects noted in either sex at 2 mg/kg/day. Statistically significant increases in absolute and relative liver weights (18-29%) and in absolute and relative spleen weights (24-34%) among males at 80 mg/kg/day were considered test article-related. A test article-related decrease (26-30%) in absolute ovary weights at 20 and 80 mg/kg/day corresponded to the ovarian atrophy seen microscopically at these levels.

There were no test article-related microscopic effects noted in either sex at 2 mg/kg/day. An increased incidence ofthe amount of hemosiderin pigment in the red pulp ofthe spleen was evident in both sexes at 20 and 80 mg/kg/day. Several male rats at 20 and 80 mg/kg/day were noted with hepatocellular cytoplasmic vacuolation. The ovaries of several female rats at 20 and 80 mg/kg/day were small due to generalized atrophy and decreased corpora lutea.

Test article effects in the stomach were somewhat varied and equivocal. The effects observed included: diffuse hyperplasia and hyperkeratosis of the squamous mucosa with associated submucosal edema and inflammation of the nonglandular area in one female at 20 and 80 mg/kg/day; focal erosion of the affected mucosa in the female affected at 20 mg/kg/day; and, hyperplasia and hyperkeratosis ofthe squamous mucosa ofthe limiting ridge in both sexes at 80 mg/kg/day. These changes are consistent with a local irritating effect of the test article on this area of the stomach.

There was no test article-related effect on any endpoint of mating or fertility at 2 mg/kg/day, including male and female mating and fertility indices, and time to mating. Mating indices (percent of paired animals that mated) were comparable among all groups. A test article-related decrease in the fertility index (percent of matings that resulted in pregnancy) was noted at 20 (60%) and 80 mg/kg/day (40%) compared to controls (90%). A corresponding test article-related decrease in the fecundity index was noted at these levels as well.

There was no effect in copulatory interval or gestation length at dose levels up to and including 80 mg/kglday. There was no effect on the number of females that delivered live offspring at 2 mg/kglday. There were no females that delivered an entirely stillborn litter.

A test article-related decrease was noted in the mean number oflive pups/litter compared to control values on Day 0 at 20 mg/kglday (9.2 vs. 14.7, respectively) and at 80 mg/kg/day (5.8 vs. 14.7, respectively). Of the four females that delivered viable litters at 80 mg/kglday, three of the four litters had four or fewer pups.

There was no effect on pup survival or pup sex ratio at dose levels up to and including 80 mg/kglday. There were no test article-related clinical or external findings noted in the pups at any dose level.

There was no effect in pup body weight at 2 mg/kg/day and no statistically significant differences noted at any level. However, pup body weight gain at 20 mg/kg/day between Days 0 and 4 was reduced compared to the control value (1.97 vs. 3.59 g, respectively). A similar effect was not noted at 80 mg/kg/day, but this is believed due to the small litter size (four or fewer pups) in three of the four females that delivered. The reduction in body weight gain at 20 mg/kg/day was considered a test article-related effect.

Under the conditions of this study, Automater Red BXL SS Dye produced clear parental and reproductive toxicity at doses of 20 mg/kg bw and higher. Consequently the NOEL for parental toxicity and reproductive toxicity is 2 mg/kg bw/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

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 (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-Naphthalenol, 1-[[4-(phenylazo)phenyl]azo]-, ar-heptyl ar',ar''-Me derivs.

EC Number:

296-120-8

EC Name:

2-Naphthalenol, 1-[[4-(phenylazo)phenyl]azo]-, ar-heptyl ar',ar''-Me derivs.

Cas Number:

92257-31-3

Molecular formula:

Not applicable

IUPAC Name:

2-​Naphthalenol, 1-​[2-​[4-​(2-​phenyldiazenyl)​phenyl]​diazenyl]​-​, ar-​heptyl ar',​ar''-​Me derivatives *

Details on test material:

Lot no. RWS1-51

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source of animals: CHarles River Laboratories, Portage, MI
Age at arrival: Approx 9 weeks
All animals acclimated for 1 week during which they were observed for clinical signs of disease.
Animals place on stdy had BW within the range of +/- 20% of the mean bodyweight for each sex.
Randomisation and assignment to treatment groups using a standard, by weight procedure
10 male and 10 female rats were assigned to each dose group.
Each animal was assigned a unique study identification number and microchipped.
Animal room conditions: Temperature maintained between 63 and 75 degrees F; Humidity between 32 and 69%.
Flourescent lighting was set to a 12 hour light cycle.
All animals individually housed (except during mating) in stainless steel cages containing cage liners (changed 3 times per week).
During mating animals were paierd (one male and one female) wihtin each treatment group. During parturition (from approx day 20 of gestation, females transferred to plastic cages with wood chip bedding.
Diet: Lab Diet Certified Rodent Diet #5002, sourced from PMI Nutrition International Inc. and tap water available ad-libitum throughout the study.
Water was monitored for specieid contaminants at periodic intervals according to the lab SOP.
Study director was not aware of any contaminants in the water that could have interfered with the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Test material administered for at lest 42 consecutive days to males and up to 54 consecutive days to females. It was administered once per day at the same time using a fixed dose volume of 5 ml/Kg bodyweight.
Control animals recieved the corn oil vehicle. Individual doses were based on the most recent bodyweight data.

Details on mating procedure:

After 2 weeks of treatment, the males were randomly assigned a female from the same dose group to co-habit duering the breeding period. Vaginal lavage was performed daily on the females during the breeding phase to assess for the presence or absence of sperm and vaginal plug. the rats were paired for a maximum of 14 days. Once evidence of mating had been collected, the male and female rats were separated and returned to being individually housed. Females that showed no evidecne of mating were individually housed in a plastic cage with wood chip bedding. Females not producing a litter within 25 days of evidence of mating or separation from the male were Euthenized and necropsied.
FOllowing mating the males were housed individually for a further 2 weeks (Approximately) after which they were euthanized and necropsied.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Doses were analhysed for homogeneity, Stability and test material concentration. Concentration of test material in the dosing solutions was verified using a 5ml sample from each test material formulation at weeks 1, 4, 7.

Duration of treatment / exposure:

Males - 42 consecutive days
females - 54 consecutive days

Frequency of treatment:

daily

Details on study schedule:

2 weeks pre-breed, up to 2 weeks breeding, males dosed for a further 2 weeks (to reach the required approx 42 days dosing), females dosed through gestation until day 4 of lactation (study termination)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

Doses were selected based on a range finding study in non-pregnant animals, and from a previous OECD 422 study that had several issues leading to it being considered as unreliable.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

daily assessment of morbidity, mortality, signs of injury and availability of food/water.
once prior to and then once per week during the study, the animals were subject to a detailed clinical observation
Neurobehavioural observations were conducted once prior to study initiation and weekly during the study. Observations were made in outside the home cage in a standard arena. Observations included (but not limited to) change in the skin, mucous membranes, occurrance of secretions/excretions, autonimic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait and posture as well as the presence of clonic or tonic movements, stereotypy (excessive groomng etc) or bizzare behaviour (self mutilation) were also recorded.
Body weights were recorded the day after arrival in the laboratory, prior to randomization, and then weekly during the study and at termination. Mated females were also weighed at day 0, 7, 14 and 20 of gestation. Females delivering litters were also weighed on days 0 and 4 of lactation.
Food consumption was measured and recorded weekly duirng the study and for mated females on days 0-7, 7-14, 14-20 (gestation) and females delivering litters were assessed on days 0-4 of lactation.
Toward the end of gestation females were examined daily for evidence of parturition and allowed to give birth. gestation duration was subsequently calculated.

During the last week of dosing for males and on lactation day 3 for females, 5 rats per sex from each dose were subjected to a Functional Observational Battery (dams were not removed from litters for more than 40 minutes at a time).
Motor activity was also assessed for 3x 10 minute periods

Oestrous cyclicity (parental animals):

not assessed

Sperm parameters (parental animals):

not assessed

Litter observations:

Pups were weighed, sexed and externally examined on days 0-4 of lactation. Abnormal behaviour was recoreded and the pups were euthenized on day 4 of lactation.External examinations were made and then the pups were discarded.

Postmortem examinations (parental animals):

Clinical Pathology on 5 animals per sex per dose
All animals euthenized by CO2 inhalation - external examination for abnormalities. gross necropsy - observations of the abdominal and thoracic cavities for any abnormalities, secial focus on the reproductive system organs. Implantation sites along with the uterus, and corpora lutea per ovary were counted and recorded for all females where present. Tissues collected and stored in Bouin's fixative. Formalin was infused into the lung via the trachea and into the urinary bladder.
Fasted BW recorded for all animals. the 5 animals chosen for clin chem were also used to assess absolute and relative organ weights. In addition the epididymes, testes and ovaries were weighed for all animals.

Histopathology of the any organs that had gross lesions. In addition, epididymides, testes, ovaries, liver, stomach, spleen were examined.

Postmortem examinations (offspring):

external examination only

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

decrease in mean BW in females (mid + high dose) during pre-mating; also a test material related decrease in gestation BW throughout gestation in these two groups. Mean Lactation BW also decreased on day 0 and 4 of lactation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

decrease in mean BW in females (mid + high dose) during pre-mating; also a test material related decrease in gestation BW throughout gestation in these two groups. Mean Lactation BW also decreased on day 0 and 4 of lactation.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver, Spleen, stomach Ovaries - see below

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

mid and high dose group - fertility index reduced in mid (60%) and high (40%) versus controls (90%). no effect in low dose

Details on results (P0)

No Neurological effects observed in any dose group, no test article related effects on FOB parameters or moteor activity.
Organ weight changes
80 mkd males:
18-29% increase in absolute and relative liver weights; 24-34% increase in spleen weights (absolute and relative)

20 and 80 mkd females:
decrease in absolute ovary weight in 20 and 80 mkd dose group
Histopathology:
Spleen: increase in hemosiderin pigment in the red pulp of the spleen - males and females in 20 and 80 mkd groups.
Increased hepatocellular cytoplasmic vaccuolation - males at 20 and 80 mkd
Ovaries: Small ovaries in the 20 and 80 mkd females, associated with generalized atrophy and decreased corpra lutea

Reproductive performance:
decrease in fertility index - 60% in mid dose group and 40% in high dose group, versus 90% in control.
No effects on other reproductive parameters
No effects on gestation length
no females delivered an entirely still born litter. Test article related decrease noted in the mean number of live pups per litter Day 0 compared to control values at 20 mkd (9.2 vs 14.7 respectively) and at 80 mkd (5.8 vs 14.7 respectively). Only four of the high dose females delivered a litter, of these viable litters, three of the litters had four or fewer pups.

Effect levels (P0)

Dose descriptor:

NOEL

Remarks:

Systemic toxicity and fertility

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects on BW, FC, organ wt, histopathology at 20 and 80 mg/kg bw Decreased fertility index at 20 and 80 mg/kg bw - reduced no. of litters and viable pups

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

reduced in mid and top dose, but pup survival to LD 4 not affected

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

reduced weight gain in mid and high dose considered likely to be treatment related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Effect levels (F1)

Dose descriptor:

NOEC

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: decreased bwg in 20 and 80 mg/kg bw pups

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Summary of effects:

2 mg/kg bw/day:

No test article related effects

20 mg/kg bw:

Decreased BW and FC - females

Decreased ovary wt

microscopic changes in spleen (both sex) liver (males), ovary and stomach (both sexes)

Decreased fertility and fecundity indices

reduced litter size at birth

Decreased pup BWG

80 mg/kg bw

Decreased BW and FC - females

Increased liver and spleen weight (males)

Decreased ovary wt

microscopic changes in spleen (both sex) liver (males), ovary, and stomach (both sexes)

Decreased fertility and fecundity indices

reduced litter size at birth

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test material produced clear parental and reproductive toxicity at doses of 20 mg/kg bw and higher. Consequently the NOEL for parental toxicity and reproductive toxicity is 2 mg/kg bw/day

Executive summary:

This nonclinical study was conducted to evaluate the effects of the test article, including its potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition, and early postnatal development. This study also placed emphasis on neurological effects as a specific endpoint in order to identify neurotoxic potential. This study was comprised of three treatment groups and a vehicle control group. Each group contained 10 male and 10 female Sprague-Dawley rats [Crl: CD® (SD)IGS BR]. The test article was administered to the treated groups via oral gavage at respective dose levels of 2, 20, and 80 mglkg/day at a dose volume of 5 mLlkg. Males were treated for at least 42 consecutive days, while females were treated for up to 54 days depending on reproductive performance. The control animals received the vehicle, com oil, at the same volume and dosing regimen as the treated groups. Observations of the animals included clinical signs, neurobehavioral observations, bodyweights, and food consumption. After two weeks of treatment, the animals were cohabited, one male and one female within the same treatment group for up to 14 days. Females were allowed to deliver litters and maintain the pups until Day 4 of lactation. Litters were evaluated for the number of live born or stillborn pups, survival indices, body weights, and any external gross abnormalities on Days 0 and 4. Evaluations for motor activity and functional observational battery (FOB), as well as blood collection for clinical pathology evaluations, were conducted prior to scheduled euthanasia. Complete necropsies were performed on all parental animals and protocol-designated organs and tissues were weighed and microscopically examined. Pups were euthanized and externally examined on Day 4 of lactation, and the carcasses were discarded without further evaluation. Concentration analysis of formulations prepared at 2, 20, and 80 mglkg/day indicated that all preparations were generally within acceptable target levels (± 15%). The formulation was homogenous and stable for 14 days. There were no test article-related effects noted in mortalities, clinical findings, neurobehavioral observations, FOB, motor activity, hematology, clinical chemistry or macroscopic observations in either sex at dose levels up to and including 80 mglkg/day. There was no test article-related effect on body weight among males at dose levels up to and including 80 mglkg/day during the study or in females at 2 mglkg/day. During the premating period, a test article-related decrease (11-13%) in mean body weight was noted among females. Females at 20 and 80 mglkg/day also had a test article-related decrease (11-25%) in gestation body weights throughout the gestation period. A corresponding dose-related decrease in gestation body weight change over the entire period (Days 0-20 gestation [0]) was apparent at 20 and 80 mg/kg/day. Mean body weight was decreased (14-21 %) on Lactation Day 0 and 4 at 20 and 80 mg/kg/day. There was no test article-related effect on food consumption in males at dose levels up to and including 80 mg/kg/day or in females at 2 mg/kg/day. Food consumption was decreased during the premating and gestation periods in females at 20 and/or 80 mg/kg/day. These

decreases included: premating period, (25-41%); Days 0-7 (23-26%); Days 7-14 (17-22%); and Days 14-20 (18%) at 80 mg/kg/day only. During the lactation period (Days 0-4), food consumption was decreased 51-55% at 20 and 80 mg/kg/day. There was no test article-related organ weight effects noted in either sex at 2 mg/kg/day. Statistically significant increases in absolute and relative liver weights (18-29%) and in absolute and relative spleen weights (24-34%) among males at 80 mg/kg/day were considered test article-related. A test article-related decrease (26-30%) in absolute ovary weights at 20 and 80 mg/kg/day corresponded to the ovarian atrophy seen microscopically at these levels. There were no test article-related microscopic effects noted in either sex at 2 mg/kg/day. An increased incidence ofthe amount of hemosiderin pigment in the red pulp ofthe spleen was evident in both sexes at 20 and 80 mg/kg/day. Several male rats at 20 and 80 mg/kg/day were noted with hepatocellular cytoplasmic vacuolation. The ovaries of several female rats at 20 and 80 mg/kg/day were small due to generalized atrophy and decreased corpora lutea. Test article effects in the stomach were somewhat varied and equivocal. The effects observed included: diffuse hyperplasia and hyperkeratosis of the squamous mucosa with associated submucosal edema and inflammation of the nonglandular area in one female at 20 and 80 mg/kg/day; focal erosion of the affected mucosa in the female affected at 20 mg/kg/day; and, hyperplasia and hyperkeratosis ofthe squamous mucosa ofthe limiting ridge in both sexes at 80 mg/kg/day. These changes are consistent with a local irritating effect of the test article on this area of the stomach. There was no test article-related effect on any endpoint of mating or fertility at 2 mg/kg/day, including male and female mating and fertility indices, and time to mating. Mating indices (percent of paired animals that mated) were comparable among all groups. A test article-related decrease in the fertility index (percent of matings that resulted in pregnancy) was noted at 20 (60%) and 80 mg/kg/day (40%) compared to controls (90%). A corresponding test article-related decrease in the fecundity index was noted at these levels as well. There was no effect in copulatory interval or gestation length at dose levels up to and including 80 mg/kglday. There was no effect on the number of females that delivered live offspring at 2 mg/kglday. There were no females that delivered an entirely stillborn litter. A test article-related decrease was noted in the mean number oflive pups/litter compared to control values on Day 0 at 20 mg/kglday (9.2 vs. 14.7, respectively) and at 80 mg/kg/day (5.8 vs. 14.7, respectively). Of the four females that delivered viable litters at 80 mg/kglday, three of the four litters had four or fewer pups. There was no effect on pup survival or pup sex ratio at dose levels up to and including 80 mg/kglday. There were no test article-related clinical or external findings noted in the pups at any dose level. There was no effect in pup body weight at 2 mg/kglday and no statistically significant differences noted at any level. However, pup body weight gain at 20 mg/kglday between Days 0 and 4 was reduced compared to the control value (1.97 vs. 3.59 g, respectively). A similar effect was not noted at 80 mg/kglday, but this is believed due to the small litter size (four or fewer pups) in three of the four females that delivered. The reduction in body weight gain at 20 mg/kglday was considered a test article-related effect.

The overall NOEL for both parental systemic and reproductive toxicity from this study is 2 mg/kg bw/day.