Reaction mass of 2-(1,1-dimethylpropyl)anthraquinone and 2-(1,2-dimethylpropyl)anthraquinone

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 February 2016 - 28 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 February 2016-28 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Objective of study:

metabolism

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

Study performed as a part of a 90-day repeated dose toxicity study, conducted according to OECD Guideline 408 (May 2008)

Deviations:

no

Principles of method if other than guideline:

The metabolite identification was performed as part of a 90-day repeated dose toxicity study with male and female Wistar rats, exposed to 0 (concurrent vehicle controls), 7.5, 25 and 75 mg 2-amylanthraquinone/kg bw/day. The metabolites were identified in the pooled urine samples collected overnight at the end of the study from 5 control and 5 high-dose (75 mg/kg bw/day) rats using LC-PDA-MS method, with detection in the positive ionization mode. More than 60 m/z values of possible metabolites were detected in the pooled urine samples of high-dose males. The identification of such large number of metabolites was not withint the scope of the study and therefore only major metabolites, based on the UV signal, were selected for identification purposes.

GLP compliance:

yes

Remarks:

However, analytical part of metabolite identification in urine was not performed under GLP.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature protected from light
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: Stability for at least 24 hours at room temperature and for at least 3 weeks in freezer is confirmed over the concentration range 1 to 200 mg/mL.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On some occasions it was considered necessary to heat the test item prior to weighing to a maximum temperature of 91.1°C for maximally 82 minutes.

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:(WI) BR

Details on species / strain selection:

Recognized by international guidelines as the recommended test system for repeated dose toxicity studies (e.g. EPA, FDA, OECD and EC).

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: not applicable
- Age at study initiation: ca. 6 weeks
- Weight at study initiation: males/females, mean (SD): controls 144 (10.0)/128 (6.3), low dose 142 (8.3)/128 (7.2), mid-dose 144 (5.8)/126 (4.9), high dose 143 (7.0)/127 (5.5)
- Fasting period before study: no
- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment
(Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom). During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals did not have access to food for a maximum of 2 hours.
- Water: Free access to tap water
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY:
Diet and water evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23 February 2016 To: 28 June 2016

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Days 1-14: Formulations (w/w) were prepared daily within 6 hours prior to dosing, and were homogenized to visually acceptable levels.
Days 15-92: Formulations (w/w) were prepared daily within 3 weeks prior to dosing and were homogenized to a visually acceptable level.

Formulations were heated to a maximum temperature of 65°C for maximally 2 hours to obtain visual homogeneity. Formulations were released for dosing when they obtained a temperature of 40°C or lower. Adjustment was made for specific gravity/density of the test item and vehicle. No correction was made for the purity/composition of the test item.
On some occasions it was considered necessary to heat the test item prior to weighing to a maximum temperature of 91.1°C for maximally 82 minutes. Formulations for all dosing days were be heated to a maximum temperature of 47.5°C for maximally
38 minutes to obtain visual homogeneity. Formulations were released for dosing when they obtained a temperature of 40°C or lower. Adjustment was made for specific gravity/density of the test item and vehicle. No correction was made for the
purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch and on information from the sponsor.
- Concentration in vehicle: 1.5, 5 and 15 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw

Duration and frequency of treatment / exposure:

Once daily, 7 days/week, for 13 weeks

Dose / conc.:

0 mg/kg bw/day

Remarks:

vehicle controls

Dose / conc.:

75 mg/kg bw/day

No. of animals per sex per dose / concentration:

5 males/dose (for urine sampling for (non-GLP) metabolite investigation

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

Not used.

Details on study design:

- Dose selection rationale:

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine. Urine was collected into a specimen vial, using a metabolism cage. After sampling the urine was pooled per dose level and stored at -80 °C until further analysis. Constant fractions of urine from the males per dose group were combined and homogenized in order to obtain one urine sample per dose (control and high-dose group).
The urine sample of the high-dose group 4 and the (blank) urine sample of concurrent vehicle controls were homogenized and centrifuged for 5 min at 16000 g at room temperature. For analysis, the urine samples were injected undiluted.
- Time and frequency of sampling: once overnight (approximately 15-20 hrs) at the end of treatment
- From how many animals: first 5 control males and first 5 high-dose males, pooled. During the sampling period animals were deprived of food but water was provided.
- Method type for identification: LC-PDA-MS, with detection in the positive ionization mode. The test substance is known to be a mixture, with 2-(1,1-dimethylpropyl)anthraquinone and 2-(1,2-dimethylpropyl)anthraquinone being the major components (together 92%) and therefore two major peaks were observed in the chromatograms. More than 60 m/z values of possible metabolites were detected in the pooled urine samples of high-dose males. The identification of such large number of metabolites was not withint the scope of the study and therefore only major metabolites, based on the UV signal, were selected for identification purposes.

- Other: Structural identification:
Using PDA chromatograms and accurate MS data, the investigation of metabolites in the sample was evaluated according to the following approach:
1. The spectra obtained from the pooled urine (high-dose group) sample were screened for m/z values that were absent in the blank urine sample.
2. For identification purposes, additional MSn analyses were performed if necessary to generate MS2 and MS3 spectra.
3. The spectra obtained were interpreted to determine the molecular weight and (partial) identity of the major metabolites.
For screening, software MetWorks 1.2.0 (Thermo Fisher Scientific Inc.) was used.

MS detection:
LTQ Orbitrap mass spectrometer: electrospray probe position x = 0.075 inch, y = 0, z = C; capillary temperature 400°C. The MS was operated in positive ion mode with 2 scan events. Mass accuracy was determined from external calibration.
Scan event:
(1) Full FTMS scan m/z 100-1000 at resolution of 30000 (at m/z 400).
(2) Data dependent MS2 of the 3 most intense ions at unit resolution.
Additional analysis to generate MSn were performed using:
Scan event:
(1) Full FTMS scan m/z 100-1000 at resolution of 15000 (at m/z 400).
(2) Data dependent MS2 at resolution of 7500 (at m/z 400)
(3) Data dependent MS3 at resolution of 7500 (at m/z 400)
For MS2 and MS3 a normalized collision energy of 25% and 35%, respectively, with an isolation width of 1.0 were used. A tune file optimized for test item 2-amylanthraquinone was used.

TREATMENT FOR CLEAVAGE OF CONJUGATES: not applicable

Statistics:

Not used in the metabolite determination

Preliminary studies:

Not applicable

Type:

metabolism

Results:

Major metabolites were result of combinations of one or two oxidations with ketone formation, sulfonation and glucuronice acid conjugation

Details on absorption:

Not applicable

Details on distribution in tissues:

Not applicable

Details on excretion:

Not applicable

Metabolites identified:

yes

Details on metabolites:

An overview of some of the major metabolites detected in the pooled urine sample of high-dose males is presented in the section "Any other information on results incl. tables". These metabolites are the result of combinations of one or two
oxidations with ketone formation, sulfonation and glucuronic acid conjugation. In addition, the parent compound 2-amylanthraquinone was detected in the pooled urine sample but at very low level.

Bioaccessibility (or Bioavailability) testing results:

Not applicable.

Table 1. Characterized metabolites of 2 -amylanthraquinone detected in the pooled urine sample of high dose males.

m/z of [M+H]^+ion	Retention times^1(min)	Mass shift^2(Da)	Possible metabolic reaction
279.138	22.2 22.4	0	= 2-amylanthraquinone = 2-(1,1-dimethylpropyl)anthraquinone+ 2-(1,2-dimethylpropyl)anthraquinone
309.112	13.0 13.7 15.7 16.1	+29.974	Hydroxylation and ketone on aliphatic chain
325.107	12.1 14.0 14.5	+45.969	Two hydroxylations and ketone on aliphatic chain
375.090	24.7	+95.952	Hydroxylation and sulfonation on aliphatic chain
471.165	11.9	+192.027	Hydroxylation and glucuronic acid conjugation on aliphatic chain

1) Retention time in MS chromatograms

2) Mass shift compared to parent compound

Conclusions:

A number of metabolites were identified in the pooled urine samples of male rats who were exposed for 13 weeks to 75 mg/kg bw/day 2-amylanthraquinone. These metabolites are the result of combinations of one or two oxidations with ketone formation, sulfonation and glucuronic acid conjugation. In addition, the parent compound 2-amylanthraquinone was detected in the pooled urine sample but at very low level.

Executive summary:

The metabolite identification in the pooled urine samples from high-dose males was conducted as a part of a GLP-compliant OECD guideline 408 study with Wistar rats. In the study, groups of male and female rats received 0 (concurrent vehicle controls), 7.5, 25 and 75 mg/kg bw/day test substance once a day, 7 days/week for 13 weeks. The urine samples for metabolite identification were collected at the end of the study overnight in metabolite cages from the first five control and high-dose male rats. An LC-PDA-MS method was developed for the analysis of the samples, with MS detection in the positive ionization mode. The accurate mass spectral data were screened for m/z values of possible metabolites. More than 60 m/z values were detected in the pooled urine samples, and only major metabolites, based on the UV signal, were selected for identification purposes. A number of major metabolites were identified, which were the the result of combinations of one or two oxidations of the aliphatic chain with ketone formation, sulfonation and glucuronic acid conjugation. In addition, the parent compound 2-amylanthraquinone was detected in the pooled urine sample but at very low level. This indicates that the parental substance is readily metabolized, undergoing primarily the oxidation of the aliphatic chain, followed by subsequent sulphonation and/or glucuronic acid conjugation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1998

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Version / remarks:

1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Chemical Substances Control Law 1973, Notification of Mar. 31 2011 by MHLW (0331 No.7), METI (H23.03.29 SeiKyoku No. 5) and MOE (No. 110331009).

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

liquid: viscous

Details on test material:

- Appearance: Yellow viscous liquid
- Storage conditions: At room temperature protected from light

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature protected from light
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: Stability for at least 24 hours at room temperature and for at least 3 weeks in freezer is confirmed over the concentration range 1 to 200 mg/mL.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On some occasions it was considered necessary to heat the test item prior to weighing to a maximum temperature of 91.1°C for maximally 82 minutes.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:(WI) BR (outbred, SPF-quality)

Details on species / strain selection:

Recognized by international guidelines as the recommended test system (e.g. EPA, FDA, OECD and EC).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: ca. 6 weeks
- Weight at study initiation: males/females, mean (SD): controls 144 (10.0)/128 (6.3), low dose 142 (8.3)/128 (7.2), mid-dose 144 (5.8)/126 (4.9), high dose 143 (7.0)/127 (5.5)
- Fasting period before study: no
- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment
(Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom). During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals did not have access to food for a maximum of 2 hours.
- Water: Free access to tap water
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY:
Diet and water evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23 February 2016 To: 28 June 2016

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage, using a plastic feeding tube. A dose control system (DCS) was used to verify the dosing procedure.

Vehicle:

polyethylene glycol

Remarks:

specific gravity 1.125

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Days 1-14: Formulations (w/w) were prepared daily within 6 hours prior to dosing, and were homogenized to visually acceptable levels.
Days 15-92: Formulations (w/w) were prepared daily within 3 weeks prior to dosing and were homogenized to a visually acceptable level.

Formulations were heated to a maximum temperature of 65°C for maximally 2 hours to obtain visual homogeneity. Formulations were released for dosing when they obtained a temperature of 40°C or lower. Adjustment was made for specific gravity/density of the test item and vehicle. No correction was made for the purity/composition of the test item.
On some occasions it was considered necessary to heat the test item prior to weighing to a maximum temperature of 91.1°C for maximally 82 minutes. Formulations for all dosing days were be heated to a maximum temperature of 47.5°C for maximally
38 minutes to obtain visual homogeneity. Formulations were released for dosing when they obtained a temperature of 40°C or lower. Adjustment was made for specific gravity/density of the test item and vehicle. No correction was made for the
purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch and on information from the sponsor.
- Concentration in vehicle: 1.5, 5 and 15 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations, in Weeks 1, 3, 6 and 13). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Analysis was based on the analytical method validated for the test item based on UPLC method.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Once daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Vehicle controls

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of a 14-day oral gange finding study
- Rationale for animal assignment (if not random): By computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

Positive control:

Not used

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily for mortality/viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals after dosing. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: yes
- Time schedule for examinations: weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at pretest (all animals, including spares) and at week 13 (groups 1 and 4)


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment between 7.00 and 10.30 am
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight (maximum of 24 hours)
- How many animals: all animals
- The following parameters were examined: white blood cells, differential leucocyte count, neutrophils, lymphocytes, monocytes, eosinophils, basophils, red blood cells, reticulocytes, red blood cell distribution width, haemaglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concnetration, platelets, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment between 7.00 and 10.30 am
- Animals fasted: yes, overnight (maximum of 24 hours)
- How many animals: all animals
- The following parameters were examined: alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate, bile acids

URINALYSIS: Yes
- Time schedule for collection of urine: overnight (ca. 15-20 hr) at the end of treatment period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, water provided
- How many animals: first five control males and the first five males treated at the highest dose level

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 12-13 of treatment
- Dose groups that were examined: all groups, first 5 animals/sex/group
- Battery of functions tested: sensory activity (hearing ability, pupillary reflex, static righting reflex)/ grip strength (fore- and hind-limb, recorded as mean of three measurements per animal) / motor activity (recording period 1 hour under normal laboratory light conditions)

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The following organ weights and terminal body weight were recorded from all animals on the
scheduled day of necropsy:
Adrenal glands
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries
Prostate
Seminal vesicles including coagulating glands
Spleen
Testes
Thymus
Thyroid including parathyroid
Uterus (including cervix)

HISTOPATHOLOGY: Yes. The samples of following tissues and organs were collected:
Adrenal glands
Aorta
Brain [cerebellum, mid-brain, cortex] (7 levels)
Caecum
Cervix
(Clitoral gland)
Colon
Duodenum
Epididymides
Eyes with optic nerve [if detectable] and Harderian gland
Female mammary gland area
(Femur including joint)
Heart
Ileum
Jejunum
Kidneys
Larynx
(Lacrimal gland, exorbital)
Liver
Lung, infused with formalin
Lymph nodes - mandibular, mesenteric
(Nasopharynx)
Oesophagus
Pancreas
Peyer's patches [jejunum, ileum] if detectable
Pituitary gland
(Preputial gland)
Prostate gland
Rectum
Sciatic nerve
Salivary glands - mandibular, sublingual
Seminal vesicles
(Skeletal muscle)
Skin
Spinal cord -cervical, midthoracic, lumbar
Spleen
Sternum with bone marrow
Stomach
Testes
Thymus
Thyroid including parathyroid [if detectable]
(Tongue)
Trachea
Urinary bladder
Uterus
Vagina
All gross lesions
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from all Group 1 and 4 animals.
- all gross lesions.
- Thymus, liver and spleen of all males and females of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4.
- Thyroid gland and kidneys of all males of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4.
- Adrenal glands of all females of Groups 2 and 3, based on (possible) treatment-related changes in this organ in Group 4.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No toxicologically relevant clinical signs during daily observations or during weekly arena observations were noted during the study period.
Salivation noted after dosing for all animals, including vehicle controls, was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to dosing procedure and vehicle rather than a sign of systemic toxicity.
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated animals remained in the same range as controls over the study period.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in food consumption before or after correction for body weight were recorded.
Statistically significant decrease in absolute food consumption during Week 9-10 in males were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment and the control value was high compared to historical control data. No statistically significant changes in relative food consumption were observed.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmology findings were noted that were considered to be related to treatment.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The following (statistically significant) changes in haematology parameters distinguished treated from control animals:
- Lower red blood cell counts for both sexes at 75 mg/kg bw/day (males: 8.50 x 10e12/L vs. 9.12 x 10e12/L in controls, p = 0.05 (Dunnett test), females: 7.82 x 10e12/L vs 8.37 x 10e12 in controls, p = 0.01 (Dunnett test)))
- Higher reticulocytes for both sexes at 75 mg/kg bw/day (males: 3.5% vs 2.4% in controls, p = 0.01 (Dunnett test), females: 3.8% vs. 2.5% in controls, p = 0.01 (Dunnett test)) and males at 25 mg/kg bw/day (2.7% vs. 2.4% in controls, p = 0.01 (Dunnett test))
- Higher red cell distribution width for males at 75 mg/kg bw/day (13.6% vs 12.4% in controls, p = 0.01 (Dunnett test))
- Lower haemaglobin for both sexes at 75 mg/kg bw/day (males: 9.3 mmol/L vs 9.9 mmol/L in controls, p = 0.05 (Dunnett test), females: 9.2 mmol/L vs. 9.6 mmol/L in controls, p = 0.01 (Dunnett test))
- Higher mean corpuscular haemoglobin (MCH) and mean corpuscular volume (MCV) for females at 75 mg/kg bw/day (MCH: 1.18 fmol vs 1.15 in controls, p = 0.05 (Dunnett test); MCHC: 20.63 mmol/L vs 21.23 mmol/L in controls, p = 0.01 (Dunnett test)).
Any other statistically significant changes in haematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following (statistically significant) changes in clinical biochemistry parameters distinguished treated from control animals:
- Higher albumin for both sexes at 25 mg/kg bw/day (males: 33.9 g/L vs. 32.6 g/L in controls, p = 0.01 (Dunnett test); females: 37.9 g/L vs/ 36.1 g/L in controls; p = 0.05 (Dunnett test) and 75 mg/kg bw/day (males: 33.8 g/L vs. 32.6 g/L in controls, p = 0.05 (Dunnett test); females: 38.8 g/L vs. 36.1 g/L; p = 0.01 (Dunnett test)). No dose-response was evident in males.
- Higher urea for females at 25 and 75 mg/kg bw/day (9.4 and 10.3 mmol/L vs. 8.0 in controls, p = 0.05 and 0.01, respectively (Dunnett test)).
- Higher creatinine for both sexes at 25 and 75 mg/kg bw/day (males: 41.4 µmol/L and 42.8 µmol/L vs. 37.8 µmol/L in controls, p = 0.01 in both cases (Dunnett test); females: 47.8 and 48.3 µmol/L vs. 45.3 µmol/L in controls, not statistically significant).
Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals. All groups showed a similar motor activity habituation profile with a decreasing trend in
activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant test item-related organ weight changes were observed:
- Higher thyroid gland weights were noted in males at 75 mg/kg bw/day (absolute and relative to body weight) (+ 25% compared to controls, p < 0.01).
- Higher liver weights were noted at 25 mg/kg bw/day (relative to body weight, males: +12% (p < 0.01); females +11% (p < 0.05)) and at 75 mg/kg bw/day (absolute and relative to body weight, males: + 31% and +36% compared to controls, respectively (p < 0.01); females +27% and +26% compared to controls, respectively (p < 0.01)).
- Higher spleen weights were noted in both sexes at 75 mg/kg bw/day (absolute and relative to body weight) (males: +24% and +29% relative to controls, p < 0.01, females: +33% and +31% relative to controls; p < 0.01)
- Higher kidney weights were noted in both sexes at 75 mg/kg bw/day (absolute and relative to body weight) (males: +13% and +19%, p < 0.01; females: +19% and +19%, p < 0.01)
- Higher adrenal gland weights were noted in females at 75 mg/kg bw/day (absolute and relative to body weights) (+16% and +19%, p < 0.05)

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in thymus, liver and spleen of both sexes, thyroid gland and kidney of males and adrenal glands of females.
Thymus: A minor increased incidence and severity of lymphoid depletion was recorded in males at 75 mg/kg bw/day (2/10 males minimal-slight). Minimal increased lymphocytolysis was recorded in 5/10 females at 75 mg/kg bw/day. The minimal increased lymphocytolysis in a single female at 7.5 and 25 mg/kg bw/day is considered to be within background pathology of female rats of this age and strain.
Liver: Minimal centrilobular hepatocellular hypertrophy of the liver was recorded in 6/10 males and 4/10 females at 75 mg/kg bw/day.
Spleen: A slightly increased incidence and severity of pigmentation, hemosiderin compared to the control groups was recorded in females at 25 and 75 mg/kg bw/day and in males at 75 mg/kg bw/day. Diffuse congestion was recorded in 3/10 males and 5/10 females at 75 mg/kg bw/day(minimal-slight)
Thyroid gland (males): A slightly increased incidence and severity of follicular cell hypertrophy was recorded in males at 75 mg/kg (5 minimal, 2 slight) compared to a minimal degree of some males in the remaining dose groups.
Kidney (males): A slightly increased severity of hyaline droplet accumulation was recorded in males at 75 mg/kg bw/day (3 minimal, 6 slight, 1 moderate) compared to a minimal or slight degree in most males of the remaining dose groups.
Adrenal gland (females): An increased incidence and severity of vacuolation of the zona glomerulosa was recorded in females at 75 mg/kg bw/day (7/10 females, minimal-slight). The minimal vacuolation of the zona glomerulosa in a single female at 7.5 and 25 mg/kg bw/day is considered to be within background pathology of female rats of this age and strain.
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

No toxicologically significant changes were noted in clinical appearance, functional observations, body weight, food consumption, ophthalmoscopy and macroscopic examination.
Microscopic examination revealed test item related changes in the liver, spleen, thymus, thyroid, kidney and adrenals.
Minimal centrilobular hepatocellular hypertrophy of the liver was noted in high dose males and females accompanied with higher organ weight. In the absence of any degenerative findings is considered to be a non-adverse adaptive response.
In the spleen a slightly increased pigmentation and low grades of congestion was noted, accompanied with a higher spleen weight. In haematology test item related changes were found in red blood cell parameters in both sexes. At the recorded incidences and severities of histopathological findings and in absence of any other indicators of toxicity these findings are considered to be non-adverse.
In the thymus, lymphocytolysis and lymphoid depletion can be seen as a spontaneous finding at low incidence and severity. The slightly increased incidence of minimal lymphocytolyisis in some high dose females and the minimal or slight lymphoid depletion in a few high dose males are regarded to be a non-adverse microscopic findings.
Hypertrophy of follicular cells of the thyroid gland in rats is usually an adaptive response to induction of hepatic enzymes. This results in increase in the hepatic/biliary clearance of T3/T4 leading to increase in TSH and compensatory follicular cell hypertrophy and/or hyperplasia. The slightly increased incidence and severities of follicular cell hypertrophy as recorded in the high dose males in the current study, accompanied with a higher thyroid weight, is regarded to be an adaptive response and considered to be nonadverse.
In the kidney, an increased incidence and severity of hyaline droplet accumulation as recorded for high dose likely represents alpha2uglobulin, a normal protein in male rats which undergoes re-absorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation. The slightly increased hyaline droplet accumulation recorded in males was not accompanied by any degenerative change and was therefore considered to be a non-adverse finding. This male rat specific protein is not present in female rats nor in higher mammals, including man. The higher kidney weight is suggested to
be related to hyaline droplet accumulation and therefore not considered adverse. For females no microscopic correlate was found for the higher organ weight.
Low grades of vacuolation of the zona glomerulosa were noted in the adrenal gland of high dose females, accompanied with higher adrenal weight. This can be seen as a spontaneous finding at low incidence and severity. Therefore, the increased incidence in the high dose females in the absence of any degenerative changes was considered to be non-adverse.
In clinical biochemistry test item related changes were found in albumin and creatinine in both sexes and urea in females only. Since these findings were slight and not accompanied by degenerative changes these findings were not considered adverse.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

haematopoietic

Organ:

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

3

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Accuracy and homogeinity
The formulations were analysed for accuracy of preparation and homogeneity in week 1, 3, 6 and 13.

The concentrations analyzed in the formulations of Group 3 and Group 4 were in agreement with target concentrations (week 1: accuracy 99% (n = 2) and 101% (n = 6); week 3: 97% (n = 2) and 97% (n = 6); week 6: 96% (n = 2) and 101% (n = 6); week 13: 94% (n = 2) and 94% (n = 2), for Group 3 and Group 4, respectively).

For the formulation of Group 2 prepared for use in Week 1, the mean accuracy was below the target concentration (i.e. 72% of target, n = 6). Based on these results, additional analyses were performed with new prepared Group 2 samples. These results were in agreement with target concentrations (i.e. 100%, n = 6). For the formulation of Group 2 prepared for use in Week 6, the mean accuracy was slightly below the target concentration (i.e. 88% of target, n = 6). The analysed concentrations in the formulations of Group 2 in the other weeks were in agreement with target concentrations (week 3: 102%, week 13: 97%, n = 6).

The formulations of Group 2 and Group 4 were homogeneous (coefficients of variation: week 1: 2.9% and 2.6%, week 3: 0.94% and 1.1%; week 6: 0.48% and 1.6%; week 13: 1.1% and 1.5%, n = 6, for Group 2 and Group 4, respectively).

Applicant's summary and conclusion

Conclusions:

In the 90-day oral gavage study with rats, the test substance 2-amylanthraquinone was administered to male and female rats at 7.5, 25 and 75 mg/kg bw/day. Based on the observed effects (organ weight changes, slight histopathological changes in the liver, thyroid, spleen, adrenals, thymus) and slight changes in haematological parameters and clinical biochemistry, the NOAEL was set at 25 mg/kg bw/day.

Executive summary:

In a GLP-compliant OECD Guideline 408 study, the test substance 2 -amylanthraquinone was administered to male and female Wistar rats at 7.5, 25 and 75 mg/kg bw/day once daily by oral gavage for 13 weeks. No toxicologically significant changes were noted in clinical appearance, functional observations, body weight, food consumption, ophthalmoscopy and macroscopic examination. Microscopic examination revealed test item related changes in the liver, spleen, thymus, thyroid, kidney and adrenals. Minimal centrilobular hepatocellular hypertrophy of the liver was noted in high dose males and females accompanied with higher organ weight (+36% and +26% relative to vehicle controls in males and females, respectively). In the spleen a slightly increased pigmentation and low grades of congestion was noted, accompanied with a higher spleen weight (+29% and +31% relative to vehicle controls in males and females, respectively). In haematology test item related changes were found in red blood cell parameters in both sexes. A minor increased incidence and severity of lymphoid depletion was recorded in males at 75 mg/kg (2/10 males minimal-slight). Minimal increased lymphocytolysis was recorded in 5/10 females at 75 mg/kg bw/day. In the kidney, an increased incidence and severity of hyaline droplet accumulation as recorded for high dose likely represents alpha2uglobulin, a normal protein in male rats which undergoes re-absorption in the proximal cortical tubules. This male rat specific protein is not present in female rats nor in higher mammals, including man. The relative kidney weights were increased for both sexes at the highest dose level (+19% compared to vehicle controls). Low grades of vacuolation of the zona glomerulosa were noted in the adrenal gland of high dose females, accompanied with higher adrenal weight. In clinical biochemistry test item related changes were found in albumin and creatinine in both sexes and urea in females only. Based on these effects, the NOAEL was set at 25 mg/kg bw/day.