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

Endpoint summary

Description of key information

Only reliable data via the oral route are available

Key study: OECD 422 in rats

Supporting studies: 14 day range finding study (in rats); 4 week repeated dose toxicity study in rats with 2 week recovery (top dose group only)

Disregarded studies: OECD 422 in rats - disregarded due to apparent errors in dosing throughout the study

A dermal carcinogenicity study is available, however it was performed by Industrial Biotest who were extensivly investigated for scientific fraud. Consequently this study is disregarded.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

2 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

good - several studies with highly consistent results and dose responses

System:

hepatobiliary

Organ:

blood

liver

ovary

spleen

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose oral toxicity studies

This substance has been assessed in several repeated dose oral studies in rats. The key study for this endpoint is an OECD 422 combined repeated dose/reproductive toxicity study. This is supported by a 4-week repeated dose study with a 2 week recovery group (top dose and control), and a 2 week range finding study. A second OECD 422 study is available, but this has been disregarded due to errors in dosing invalidating the study.

 

Key study: OECD 422 in Sprague dawley rats (reproductive effects reported separately)

 

The dose levels used in this study were set based on the 2-week range finding study and the disregarded OECD 422. Dose levels were 0, 2, 20 and 80 mg/kg bw/day.

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 mg/kg/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) 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 of the 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 non-glandular 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 of the squamous mucosa of the 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.

Based on the effects on organ weights, (liver, spleen, ovaries) and histopathological observations, the no observed effect level for systemic toxicity was 2 mg/kg bw/day.

 

The findings observed in this study are correlated well with those from the 4 week study and the range finding study. However, due to higher dose levels in these studies, additional findings involving hematological parameters (indicative of anemia) were also recorded in these studies.

 

4 Week oral study in Sprague Dawley rats

 

Two groups of five male and five female CD rats received 2-Naphthalenol [(phenyl azo)phenyl] azo heptyl derivative (hereafter referred to as 2-Naphthalenol) by oral gavage at dosages of 40 or 200 mg/kg/day for four weeks. A third group of ten male and ten female CD rats received 2-Naphthalenol at a dosage of 1000 mg/kg/day for four weeks: five males and five females of this group were held for a subsequent two week period without treatment for reversibility studies. A control group constituted similarly to the high dosage group received the vehicle (corn oil) during the period of treatment.

During the study one female receiving 1000 mg/kg/day was accidentally killed during routine blood sampling on Day 14 of the reversibility period; this death was not attributed to treatment.

In the top dose group (1000 mg/kg/day there was generalised pink staining of the coat, including perianal staining and red stained faeces in all animals. Low food intake and bodyweight gains and inferior food conversion ratios in males. Low haemoglobin concentrations, erythrocyte counts and packed cell volumes and high platelet counts in males and females. Low mean cell volumes in males and low mean cell haemoglobin in males and females. In addition, polychromasia and slight reticulocytosis was recorded for males and females. In comparison with controls high plasma urea and total protein concentrations were recorded in males and females after 27 days of treatment. Orange coloured urine in males and females and low urinary specific gravity and protein output in males was recorded after 24 days of treatment.

Organ weight changes that were attributed to treatment comprised high bodyweight-relative liver weights in males and females, high absolute and bodyweight-relative adrenal weights in males and low bodyweight relative and absolute ovary weights in females.

Macroscopic pathology indicated pink or red staining of the coat, the keratinised region of the stomach and the abdominal fat pads. All internal organs of one male were also stained pink.

Histopathological changes attributed to treatment were confined to the liver and comprised intrahepatocytic basophilic aggregates and periacinar hepatocytic pallor in males and females, and pigment-laden Kupffer cells and centriacinar hepatocytic hyperplasia in females.

Staining of the coat persisted throughout the reversibility period. Recovery was incomplete in respect of low haemoglobin concentrations and erythrocyte counts in males and females, low urinary protein output, high adrenal weights in males and low packed cell volumes and high plasma urea concentrations in females. Pink staining of the coat, keratinised region of the stomach and abdominal fat pads was still evident, and pigment-laden Kupffer cells were still present in one female. All other affected parameters showed complete resolution.

 

In the mid dose group (200 mg/kg bw/day) effects associated with treatment were a generalised pink staining of the coat, including perianal staining and red faeces in all animals. Low food intake and reduced bodyweight gains in males. Low haemoglobin concentrations, erythrocyte counts and packed cell volumes and slightly high platelet counts in females. Slightly high plasma urea concentrations in males and slightly high total plasma protein concentrations in males and females. Dark yellow urine was observed in four males.

Organ weight changes that were attributed to treatment comprised high bodyweight-relative liver weights in males and females, high absolute and bodyweight-relative adrenal weights in males, and low absolute and bodyweight-relative ovary weights in females.

Macroscopic pathology indicated pink or red staining of the coat, the keratinised region of the stomach and the abdominal fat pads. There were no histopathological changes that were attributable to treatment at this dosage.

 

In the low dose group (40 mg/kg/day) a generalised pink staining of the coat, including occasional perianal staining and red stained faeces in all animals. Dark yellow urine in one male. Macroscopic pathology indicated pink or red staining of the coat and of the keratinised region of the stomach

 

Treatment of CD rats with 2-Naphthalenol for four weeks resulted in clear changes at 200 or 1000 mg/kg/day. The liver was the major target organ for toxicity although there were also effects observed on the ovaries (reduced relative weight in top and mid dose group, and hematological effects indicative of an induced anemia). Reversibility of effects seen at the high dosage was demonstrated although recovery for some was incomplete two weeks following cessation of treatment. Changes in coat and urine colour are attributed to the pigment nature of the test material. The dosage of 40 mg/kg/day is considered to be the no-toxic-effect level in this study.

 

2-week range finding study in Sprague Dawley rats

Four dose levels were used in this study, 40, 100, 400 and 1000 mg/kg bw/day plus controls.

Test article-related organ weight effects were noted at 100 mg/kg bw/day and above. These included: increased absolute and relative liver weights in females at 100 mg/kg bw/day and in both sexes at 400 and 1000 mg/kg bw/day; increased absolute and relative spleen weights in both sexes at 400 and 1000 mg/kg bw/day; and decreased absolute and relative ovary weights in females at 1000 mg/kg bw/day. Not all of these changes were statistically significant but were judged test article-related. A statistically significant increase in relative liver weight was noted in females at 40 mg/kg bw/day as well. The toxicological significance of this finding is unclear since terminal body weight was also decreased significantly. There were no macroscopic findings noted in this study. A key finding in this study was the decrease in erythrocytic parameters which was considered to be a possible effect of test article administration. However, there was decreased food consumption and lower body weights in males at 400 and 1000 mg/kg bw/day, and in females administered doses of 40 mg/kg bw/day and higher. In this short term study, it is therefore difficult to assess the impact of decreased food consumption versus test article administration on the decrease in erythrocytic parameters of these rats. Despite the increase in reticulocyte counts, the hematopoietic response did not compensate for the decrease in erythrocytic parameters. This may be due to the short duration of the study and insufficient time for the hematopoietic response to have a regenerative effect. Alterations in clinical chemistry values that were considered to be possible effects of test article administration included higher total bilirubin, and higher ALT and AST values. The higher albumin values were considered most likely due to hemoconcentration secondary to decreased food and probably water consumption, but the larger increase in globulin values was considered to be a possible response to inflammation. Due to the presence of treatment related effects at all dose levels in this study a no adverse effect level could not be identified.

 

Based on the available studies target organs for this substance appear to be the liver, ovaries and hematopoetic system. There is a high degree of consistency over all available studies in terms of the effects and dose response, and the apparent no effect level is 2 mg/kg bw/day. This value will be taken forward for DNEL derivation.

Justification for classification or non-classification

In the key repeated dose toxicity study effects on the liver, spleen (increase in absolute and relative weight) and ovaries (decrease in absolute weight, with evidence of atrophy) were seen at 20 and 80 mg/kg bw/day. The atrophy of the ovaries is clearly an adverse effect, however given the reproductive toxicity findings addressed in the reproductive toxicity assessment, it is considered that the ovarian atrophy should not be considered in the assessment of whether a repeated dose toxicity classification is warrented. Considering therefore the organ weight effects on the liver and spleen, tt is unclear if these effects indicated a disruption of the normal functioning of these organs, however given the degree of change and the evidence of some histopathological correlates, it is concluded that these effects were adverse rather than adaptive. Consequently, due to the presence of these effects at dose levels lower than 100 mg/kg in a short term repeated dose toxicity study and the likelihood that these effects would be more pronounced in a longer term study it is concluded that the criteria for classification for specific target organ toxicity, Repeated dose exposure category 2 have been met.