1-amino-4-hydroxy-2-phenoxyanthraquinone

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

from March 24 to October 15, 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

The complete read across justification is detailed in section 13. Source study has reliability 1.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 03

IUPAC Name:

Similar Substance 03

Test material form:

solid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source : Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: males: 9-10 weeks old, females: 9-10 weeks old.
Body weight at the allocation of the animals to the experimental groups (together with study 140271):
males: 253 - 281 g (mean: 268.93 g, ± 20 % = 215.14 – 322.71 g)
females: 171 - 191 g (mean: 180.34 g, ± 20 % = 144.27 – 216.41 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.
The animal study was authorized by local government under file no. 55.2-1-54-2532.2-11-11.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10/hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 1526)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at
regular intervals)
- Animals were housed in groups of 2 animals/sex/cage in IVC cages (type III H, polysulphone cages) during the premating period in both males and females and during postmating period in males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period and males were returned to its original cage. Each cage was provided with Altromin saw fibre bedding (lot no. 131113)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days) under laboratory conditions


Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. None of the animals showed any adverse clinical signs before the study initiation. Before the first administration all animals to be used for the study were weighed.
Mean body weight of the group housed animals was used to assign all animals to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively, while ensuring to keep each animal with its initial cage partner if possible. Each animal was marked with its identification number by individual ear tattoo.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Test item was ground to a fine powder with the help of a mortar and pestle. Afterwards it was weighed into a tared plastic vial on a precision balance and suspended in corn oil. The formulation was homogenized using an ultra turrax (homogenizer).
The test item formulations were prepared once in every ten days and stored at 2-8 °C. Every day before the dose administration the formulation samples were allowed to reach the room temperature and the homogeneity was ensured by vortexing the sample on vortex machine.

The dose range finding study (14 days repeated dose, BSL study no. 140259) was performed at dose levels 100, 300 and 1000 mg/kg/d with no overt toxicity observed up to the highest dose tested. According to these results and in consultation with the sponsor the following doses are selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C).

Control: 0 mg/kg bw/d
Low Dose: 100 mg/kg bw/d
Medium Dose: 300 mg/kg bw/d
High Dose: 1000 mg/kg bw/d

The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. This dose also happens to be a limit dose. A descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL. Animals in the control group were handled in an identical manner to the test group subjects and received corn oil using the same volume as
used for the test dose groups.

The test item formulation and vehicle were administered at a single dose to the animals by oral gavage with disposable polypropylene feeding tubes for rats (78 mm long; 3.0 mm diameter; Instech Laboratories Inc). The application volume for all groups was 5 mL/kg body weight. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each dosing concentration was analysed with respect to the target nominal concentration. Stability and homogeneity of the test item in the vehicle were analysed for the low and high dose concentrations.
Samples for the nominal concentration verification were taken in study week 1 (first week of pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation/lactation) from all groups (16 samples).
Samples for homogeneity analysis were taken from the top, middle and bottom of the high dose and the low dose formulation in study week 1 and 5 (12 samples).
Samples for stability analysis were taken in the first week of the study [0 hours, 6 hours (at RT) and 10 days after the preparation (stored at 2-8 °C)], from high and low dose formulations (6 samples).
All formulation samples were analysed on the day of sample collection and were stored at -20 °C. These samples were analysed after the completion of the toxicity study at BSL BIOSERVICE Scientific Laboratories GmbH under the BSL study no. 140262.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 54 days, i.e. during 14 days of pre-mating and 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Frequency of treatment:

Once daily.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).
This included the control animals (10 males and 10 females) which were shared with BSL study 140271.

Control animals:

yes, concurrent vehicle

Details on study design:

The dose range finding study (14 days repeated dose, BSL study no. 140259) was performed at dose levels 100, 300 and 1000 mg/kg/d with no overt toxicity observed up to the highest dose tested.

Examinations

Observations and examinations performed and frequency:

Clinical Observations
General clinical observations were made once a day, preferably at the same time each day after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Once before the first exposure, and once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size.
Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

Functional Observations
Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and on day 3 of the lactation period in 5 randomly selected females (only lactating females were evaluated) outside the home cage using a functional observational battery of tests. Except 1/5 selected females of LD group was non-pregnant and the FOB in the last week of treatment was not performed.
Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

Body Weight and Food Consumption
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups.
Food consumption was measured weekly on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

Haematology
Haematological parameters were examined from 5 randomly selected males and females (only lactating females were evaluated) of each group at the end of the treatment prior to or as part of the sacrifice of the animals. Except 1/5 selected females of LD group was non- pregnant and hematology parameters was not examined on this animal. Blood from the abdominal aorta of the animals was collected in EDTA-coated tubes. The following haematological parameters were examined: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV),
mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso), large unstained cells (Luc)

Blood Coagulation
Coagulation parameters from 5 randomly selected males and females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. Except 1/5 selected females of LD group was non-pregnant and blood coagulation parameters was not examined on this animal. Blood from the abdominal aorta of the animals was collected in citrate-coated tubes. The following coagulation parameters were examined: prothrombin time (PT), activated partial thromboplastin time (aPTT).

Clinical Biochemistry
Parameters of clinical biochemistry from 5 randomly selected males and females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. Except 1/5 selected females of LD group was non pregnant and clinical biochemistry parameters was not examined on this animal. Blood from the abdominal aorta of the animals was collected in serum separator tubes. The following parameters of clinical biochemistry were examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na), potassium (K).

Urinalysis
A urinalysis was performed with samples collected from 5 randomly selected males prior to or as part of the sacrifice of the animals. Additionally, urine colour/appearance were recorded. Urinalysis was also performed on 4 females of control and dose groups.
The following parameters were measured using qualitative indicators (Heiland Urine Stripes URI 10SL): specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood, leukocytes.

Sacrifice and pathology:

Pathology
All male animals were sacrificed after the completion of the mating period (total dosing period: 28 days) on study day 29 or 30, while female animals were sacrificed on post-natal day 4 using an anaesthesia.
All surviving pups were killed by decapitation on post natal day 4.
Dead pups and pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.
Non-pregnant females (females 42 and 71) were sacrificed on day 26 from day of sperm positive vaginal smear or from the last day of mating period. All animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), and all organs showing macroscopic lesions of all adult animals were preserved in 10 % neutral buffered formalin, except for testes and epididymides which were fixed in modified Davidson’s Solution for 24 hours and then transferred to 70 % ethanol.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Organ Weights
The wet weight of the organs (liver, uterus with cervix, kidneys, thymus, adrenals, thyroid/parathyroid glands, testes, spleen, epididymides, brain, prostate, seminal vesicles and coagulating glands, pituitary gland, ovaries, heart) of 5 sacrificed males and 5 females (only lactating females were evaluated) randomly selected from each group was recorded as soon as possible. Paired organs were weighed together. In addition reproductive organs of all animals were weighed.
The following tissues (brain (cerebrum, cerebellum and pons), heart, spinal cord, ovaries (females), liver, uterus with cervix (females), kidneys, vagina (females), adrenal glands, testes (males), stomach, epididymides (males), small and large intestines (including Peyer´s patches), prostate and seminal vesicles with coagulating glands as a whole (males), thymus, urinary bladder, thyroid, lymph nodes (mesentric and axillary), spleen, peripheral nerve (e.g. sciatic nerve) with skeletal muscle, lung and trachea, bone with bone marrow (sternum), mammary glands, pituitary gland, gross lesions, oesophagus, skin) of the same selected animals from each group were preserved in 10 % neutral buffered formalin except for testes and epididymides that were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70% ethanol.

Histopathology
A full histopathology was carried out on the preserved organs and tissues (see above) of 5 randomly selected male and female animals (only lactating females were evaluated) of the control and high dose groups which were sacrificed at the end of the treatment period.
All gross lesions from all groups, were examined by light microscopy. Furthermore, testes, epididymides, prostate, seminal vesicles with coagulating glands, ovaries, uterus with cervix and vagina were examined in all animals, and, on the testes, special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Because of possible treatment-related findings noted in the high dose group, spleen and bone marrow (sternum) from 5 selected male and female animals of the low and medium dose groups (groups 2 and 3, respectively) were examined to establish a no-effect level.
Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and eosin and were examined in all animals.
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle for the evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.6.01 software (p<0.05 was considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicological relevance. However, red feces in HD group, due to test item colour, were noted. Other clinical signs (e.g. alopecia, slight abnormal breathing, nasal discharge, salivation and regurgitation) in a few animals of control and/ or dose groups, were considered incidental.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In MD and HD groups. In males, there was statistically significantly lower mean RBC, HCT and Hb in MD and HD groups; statistically significantly higher mean MCV and reticulocyte count in MD and HD group; statistically significantly higher mean MCH in HD group. In females, there was statistically significantly lower mean RBC, Hb, HCT value in HD group and statistically significantly higher mean MCV, MCH and reticulocyte count in HD group.
Statistically significantly lower MCHC value in male MD group, higher eosinophil and lower LUC count in female MD group were no dose- or tretment-related. In males and females, no effect on the blood coagulation parameters.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In males and females, there was statistically significantly higher absolute and relative (to terminal body weight) spleen weight in MD and HD groups, when compared to controls. The change in spleen weight correlated with the increase in mean severity of erythrocytic extramedullary hemopoiesis in the spleen recorded in both sexes of MD and HD groups. These findings are considered to be adverse events attributable to treatment with the test item.
In addition, statistically significantly lower absolute and relative (to terminal body weight) pituitary gland weight in MD group of male animals, higher relative kidney weight in HD group. No treatment-related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Enlarged spleen was recorded in 3/10 males of MD group, in 4/10 males and one female of HD group, was considered to be treatment-related change.
Discoloured pale or discoloured of the kidney was observbed macroscopically in all male groups including the control group and in one female of MD group. However, there were no corresponding microscopic findings in any animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopically, the treatment-related findings were recorded in the spleen and bone marrow. The increase in mean severity of erythrocytic extramedullary hemopoiesis in the spleen was recorded in both sexes of MD and HD groups, along with an increase in incidence and/or severity of hemosiderin deposition in the spleen and erythropoiesis in the bone marrow. These findings are most likely to be the histomorphologic indicator of hemolytic anemia and were considered to be adverse event attributable to treatment with the test item.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No dose response relationship in any findings recorded in the male reproductive organs including testes, epididymides, prostate glands, coagulating glands and seminal vesicles which were examined in this study, and therefore, all histologic findings were considered to be spontaneous changes which may be recorded in animals of this strain and age.
All other findings within the range of normal background lesions possibly recorded in animals of this strain and age, or accidental changes possibly occurring in toxicological studies with rodents.

Effect levels

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

haematopoietic

Organ:

blood

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Based on results in male and female Wistar rats exposed to dose levels of 100, 300, and 1000 mg/kg bw/d of test substance for at least 28 days, no observed adverse effect level (NOAEL) is 100 mg/kg bw/d for systemic toxicity in males and females and 1000 mg/kg bw/d for reproduction/developmental toxicity.

Executive summary:

Method

Combined repeated dose toxicity and reproductive/developmental toxicity study in male and female Wistar rats.

Test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period up to 54 days, i.e. 14 days pre-mating and maximum 14 days of mating in both males and females, and during the gestation period up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 -29 days. Animals of an additional control group were handled identically as the dose groups but received corn oil, the vehicle used in this study. Each group comprised 10 male and 10 female Wistar rats, dosed as follows:

Control (C):                  0        mg/kg/d

Low Dose (LD):            100     mg/kg/d

Medium Dose (MD):     300     mg/kg/d

High Dose (HD):           1000   mg/kg/d

Test item formulation was prepared once in every ten days and stored at 2-8 °C. Test item was suspended in corn oil and dose volumes were adjusted individually based on weekly body weight measurements administered as 5 ml/kg bw/d.

During administration period, animals were observed daily for signs of toxicity. At the conclusion of the test, animals were sacrificed and observed macroscopically.

Changes in body weight, food consumption, haematological and clinical biochemistry, urinalysis, functional signs including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behavior observations were recorded during the study.

After 14 days of treatment, animals were mated (1:1) for a maximum of 14 days. Vaginal smears of females were checked daily to see evidence of mating. After confirmation of mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum.

Males were sacrificed on treatment days 29 and 30 and females along with their pups were sacrificed on post natal day 4. Non-pregnant females (females 42 and 71) were sacrificed on day 26 from day of sperm positive vaginal smear or from the last day of mating period.

A full histopathological evaluation of the tissues was performed of 5 randomly selected male and female animals of the control and high dose groups as well as all gross lesions from all groups, were examined by light microscopy. Furthermore, testes, epididymides, prostate, seminal vesicles with coagulating glands, ovaries, uterus with cervix and vagina were examined in all animals, and, on the testes, special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Because of possible treatment-related findings noted in the high dose group, spleen and bone marrow (sternum) from 5 selected male and female animals of the low and medium dose groups were examined to establish a no-effect level.

Results

No mortality occurred in the control or any dose group during study period.

No clinical signs of toxicological relevance in dose groups when compared to control. However, there were red feces in HD group and signs of moving the bedding in males and/ or females of MD and/ or HD groups. The red feces were due to test item colour and the moving the bedding was considered due to local effect of the test item.

During the weekly detailed clinical observation, no significant changes or differences between the groups were found.

No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period.

In males and female, no adverse changes of toxicological relevance on body weight, body weight gain and food consumption in dose groups when compared to the concurrent control. 

In males and females, there were findings of toxicological relevance on haematological parameters in MD and HD groups. In males, there was statistically significantly lower mean RBC, HCT and Hb in MD and HD groups; statistically significantly higher mean MCV and reticulocyte count in MD and HD group; statistically significantly higher mean MCH in HD group. In females, there was statistically significantly lower mean RBC, Hb, HCT value in HD group and statistically significantly higher mean MCV, MCH and reticulocyte count in HD group.

In males and females, there was no effect on the blood coagulation, clinical biochemistry and urinary parameters. Enlarged spleen, which correlated microscopically with increased extramedullary erythrocytic hemopoiesis, was recorded in 3/10 males of MD group, in 4/10 males and one female of HD group, was considered to be treatment-related change.

All other gross lesions recorded were within the range of normal background alterations which may be recorded in animals of this strain and age.

In males and females, there was statistically significantly higher absolute and relative (to terminal body weight) spleen weight in MD and HD groups, considered to be adverse events attributable to treatment with the test item.

Microscopically, treatment-related findings in MD and HD groups were recorded in the spleen and bone marrow. Increase in mean severity of erythrocytic extramedullary hemopoiesis in the spleen along with an increase in incidence and/or severity of hemosiderin deposition in the spleen and erythropoiesis in the bone marrow were seen. These findings are histomorphologic indicator of hemolytic anemia and are attributed to treatment with the test item.

Conclusion

NOAEL of 100 mg/kg/d and LOAEL of 300 mg/kg/d were found for systemic toxicity; NOAEL of 1000 mg/kg/d was identified for reproduction/developmental toxicity (see section 7.8.1 for details).