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

Endpoint summary

Description of key information

In a reliable 28-days study 2-amylanthraquinone administered at 3.75, 15 and 60 mg/kg bw/day by oral gavage to male and female rats caused a significant prolongation in the prothrombin time in male rats at 15 and 60 mg/kg bw/day. Also, in the 60 mg/kg bw/day males, a significant increase in the absolute and relative weight of the liver, accompanied by centrolobular hypertrophy of hepatocytes, was observed. From the above results, the NOAEL for male rats is 3.75 mg/kg bw/day, while the NOAEL for female rats is 15 mg/kg bw/day.

In a reliable guideline 90-day oral gavage study with rats, 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 in the high-dose animals, the NOAEL for risk asessment purposes was set at 25 mg/kg bw/day. Considering that the 90-day study was conducted under GLP and according to the OECD guideline, it is considered to be reliable without restrictions. As the effects observed in the 28 -day study were either not observed, or occurred at higher dose levels in the reliable study with a longer exposure duration, the NOAEL from the 90 -day study shall be taken forward for risk assesment and DNEL derivation.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed according to OECD guidelines and GLP principles; study is in Japanese, with a translation in English. There are no individual data available. Only 5 animals per sex per dose.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

no individual data available; study in Japanese, article in Engllish.

Principles of method if other than guideline:

14-day recovery period

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan Inc
- Age at study initiation: 5-week-old
- Weight at study initiation: average in all male dose groups 163.9-165.8 g, female dose groups 135.6-139.6 g
- Fasting period before study: not stated
- Housing: metal wire mesh floor cages, one rat per cage
- Diet: ad libitum solid food (CE-2, CLEA Japan, Inc.)
- Water: ad libitum tap water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ~ 25.0
- Humidity (%): 40.0 to 75.0
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The oral dose samples were prepared by first weighing out the dose and using a graduated cylinder to add the medium of corn oil (NACALAI TESQUE, INC.) and make a 1.2 w/v% solution. The 1.2 w/v% solution was diluted in steps to prepare 0.3 to 0.075 w/v% solutions. The prepared samples were then placed in glass bottles and put in refrigerated storage. Also, the stability of the 0.05 and 5 w/v% sample preparations, after 8 days of cold storage, was confirmed using HPLC. The administration of the doses was done within 8 days of the preparation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Both before and after the animal testing, the test substance stability (incl. its stability over the course of the test) was confirmed using HPLC.

Duration of treatment / exposure:

28 days (recovery period was 14 days)

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
0, 3.75, 15.0, 60 mg/kg 2-pentylanthraquinone
Basis:
other: in corn oil

No. of animals per sex per dose:

10 in both the control and high dose group, of which 5 were used for the recovery
5 for the low and medium dose group.

Control animals:

yes

Details on study design:

- Dose selection rationale: Dose administered for this test was based on the results of a preliminary test conducted prior to this test. At the lowest dose tested 62.5 mg/kg some effects were observed after 7 days of dosing to 3 animals/sexe/dose. Therefore 60 mg/kg was chosen as it could be predicted that this dose would show an effect after 28 days of dosing.

Positive control:

no

Observations and examinations performed and frequency:

General Condition
The general condition (during the administration at least once before and after administration and during recovery once a day) of the rats was observed including checking for any deaths.

Detailed Observation of Symptoms and Function Examination
On a weekly basis, using the scoring method, detailed observation of symptoms was conducted for three hours (13:00→16:00). First, after observation (postural position, spontaneous movement, vocalizations, tremor, convulsions) from outside the cage, the animal was taken out of the cage and observed for easy retrieval, handling ease, heartbeat, body temperature, fur, skin color, visible mucosa, tearing, bulging eyes, pupil diameter and salivation. Then, on the bench, postural stance, seeking behavior, grooming, vocalizations, tail lift, walking, normal behavior, strange behavior, tremors, convulsions, respiratory rate, piloerection, eye tears, frequency of urination and bowel movements, response to removal, response to handling and response to aural stimulation were observed. This was done under blind conditions with the dosage information and the number of the animal unknown to the researcher. Also, at the fourth week of dosage administration and the second week of recovery, during the detailed examination of symptoms a function examination was also performed. The function examination was for auditory response to a stimulus (startle response), visual response to a stimulus (visual orientation, pupillary response) and response to specific stimuli (righting reflex).

Weight and Feed Intake
The weight of the rats was measured three times the first week of administration and thereafter at a frequency of twice weekly. Weight was also measured on the dosage administration period end day (the 28th day) the end of the test recovery period (the 14th day) and on the day of the autopsy. Feed intake for the day was measured once every week.

Urine Test
The animals were placed in a metabolism cage in the fourth week (after the start of dose administration) and in the second week (of recovery) and urine collected after the expiration of four hours. The urine sample was visually inspected for color and turbidity. pH, occult blood, protein, glucose and ketone bodies, urobilinogen and bilirubin were examined using the test strip method. Microscopical examination of urine sediment was performed.

Blood Sample
The animals were not fed for a period of 18 to 21 hours from the day of the end of the dosing or recovery period until the autopsy the next day. Then, under pentobarbital sodium anesthesia, from the posterior major abdominal vein, blood samples were taken using sodium citrate as the anti-coagulant for prothrombin time activity and partial thromboplastin time measurement, EDTA-2K as the anticoagulant for other hematological tests and heparin as the anticoagulant for blood biochemistry testing. As much as possible, blood samples were taken in the order of control, high, medium and low dose groups with one rat from each group starting with the lowest number animal selected.

Hematological Examination
With an automated blood analyzer (CELL-DYN3500, Abbott Laboratories) red blood cell count (RBC), mean cell volume (MCV), and platelet count were measured using the electrical resistance method. Using the flow cytometry technique with laser light scattering/electrical resistance method white blood cell count (WBC) was measured. The flow cytometry technique with laser light scattering method was used for measuring white blood cell categories and hemoglobin amount. Using these measurements as the basis, the hematocrit, mean corpuscular hemoglobin (MCH) and the mean corpuscular hemoglobin concentration (MCHC) was calculated. The fully automated blood coagulation analyzer (CA-1000, Toa Medical Electronics), using the light scattering detection method was used for measuring prothrombin time (PT) and activated partial thromboplastin time (APTT). Moreover, using the Brecher method with an optical microscope, the ratio of reticulocytes was calculated.

Blood Biochemistry Examination
An automatic biochemical analyzer (COBAS MIRA plus, Roche Diagnostics) was used and the albumin/globulin (A/G) ratio was calculated after measurement of total protein concentration (biuret method), albumin concentration (BCG method), total cholesterol level (cholesterol-oxidase/HDAOS method), triglyceride concentration (GPO-HDAOS glycerol blanking method), glucose level (hexokinase/G-6-PDH method), urea nitrogen concentration (BUN) levels (urease GIDH method) , creatinine concentration (Jaffe method), total bilirubin concentration (azobilirubin method), alkaline phosphatase (ALP) activity (GSCC method), aspartate aminotransferase (AST) activity, alanine aminotransferase (ALT) activity and gamma-glutamyl transpeptidase (γ-GTP) activity (IFCC method), calcium concentration (OCPC method), and inorganic phosphate concentration (direct molybdate method). Also, a fully automated electrolyte analyzer (EA05, A&T Corporation) was used to measure sodium ion concentration, potassium ion concentration and chlorine ion concentration (ion electrode method).

Sacrifice and pathology:

After taking the blood sample, if necessary, the axillary artery was cut and the animal killed and the organs and tissue were subject to visual observation. Also, the weight (actual weight) of each animal's liver, kidneys, adrenal glands and testes, epididymis, ovaries, thymus, spleen, brain, heart and thyroid was measured. In addition, the relative weight value for each organ (the weight of each organ at the autopsy divided by body weight) was calculated. Visual observation was continued to see if there were any gross examples of lesions of the brain, spinal cord, stomach, small intestine (the duodenum, the jejunum, the ileum), the large intestine (colon, rectum), liver, kidney, adrenal gland, spleen, heart, thymus, thyroid, trachea, lung (bronchi), gonads (testes and ovaries), accessory reproductive organ (epididymis, prostate, uterus, seminal vesicle and vagina), bladder, lymph nodes (mesenteric lymph nodes, lymph nodes under the jaw), sciatic nerve (including calf), thigh bone and bone marrow, the aorta, tongue, esophagus, pancreas, submandibular gland, the sublingual gland, the pituitary gland, parathyroid eyeballs and then the organs were fixed in a 0.1 M phosphate buffer fixed 10% formalin solution. However, the testis and epididymis were fixed in a BUAN solution. Next, the organs and tissues of the high-dose group and control group fixed at the end of the testing (however, not including the duodenum, the jejunum, rectum, seminal vesicle, vagina, thigh bone, the aorta, tongue, esophagus and parathyroid) were embedded in paraffin and sliced into thin sections with hematoxylin eosin staining. Then, using an optical microscope, histopathological examinations were performed. Because the test results indicated the possibility that the test substance administration caused changes in the liver, a histological examination of the livers of the rats in all groups, including the control group, was performed.

Statistics:

For each group an average value as well as the standard deviation value was sought for weight and feed intake, hematology and blood chemistry test values and the values of each organ weight measurement. All four groups (including the control group) were subjected to the statistical analysis using the Bartlett’s balancing method for uniformity and one-way analysis of variance and the Dunnett method or Kruskal-Wallis or Dunnett type verification for multiple comparisons. For two of the groups, including the control group, the F-test along with Student's t-test or Aspin-Welch's t-test was also conducted.
The test-strip paper-based test results of urine tests, urine color and turbidity (4th week of administration) were used and a χ2-test with the cumulative column or separate tables was performed and a Dunnett type test method used for multiple comparisons. All the test results for the 2nd week of the recovery period were obtained and subjected to a Wilcoxon rank-sum test.
The histopathological findings grade separated data was subjected to a Mann-Whitney U-test or a Fisher’s one-sided exact test on the total value of the positive grade was conducted.
In addition, in either case, the significant difference level was 5 percent.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the administration, from the third week in the 60 mg/kg group, temporary salivation immediately after dose administration was found here and there in three males and four females. In addition, in the 15 mg/kg group, on the day of the autopsy there was one instance of bleeding from the nail area noted due to, it was thought, getting the nail caught on one of the wires of the wire cage.
During the recovery test period, there were no changes in the general condition of any of the groups.

Mortality:

no mortality observed

Description (incidence):

There were no examples of death or the appearance of imminent death in any of the groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Both during the dose administration period and recovery period no significant difference in body weight or body weight increase was found for any of the test groups in comparison to the control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Both during the dose administration period and recovery period no significant difference in feed intake was found for any of the test groups in comparison to the control group.

Food efficiency:

no effects observed

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 the examination at the end of the dosing period, a significant extension of the prothrombin time was seen in the male rats of the 15 and 60 mg/kg dosage groups. In addition, a lowering trend for red blood cell count was seen in the male rats of the 60 mg/kg dosage group. Also, a significant difference was noted in the reduction of the neutrophil ratio and lymphocyte ratio increase for male rats in the 15 mg/kg dosage group and an increase in the monocytes ratio for females in the 15 mg/kg dosage group. Expressing the white blood cell count ratio in terms of absolute number, no significant difference was found between any of the test groups and the control group. Therefore, it was judged that the changes in the white blood cell count percentage analysis ratio were not caused by the administered dosages.
At the end of the recovery period, a significant difference in the number of red blood cells and hemoglobin amount was seen for male rats in the 60 mg/kg dose administration group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the dosing period, significant decreases were seen in alkaline phosphatase activity for males in the 60 mg/kg group. In addition, a significant decrease was found in the total protein concentration for males in the 15 mg/kg group. However, albumin concentration and A/G ratio changes were not seen and there were no significant difference for the 60 mg/kg dose group. Therefore, it was judged that this effect was not caused by the administered dosages.
At the test at the end of the recovery period, a significant decrease in glucose concentration levels for the females in the 60 mg/kg group was 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 the autopsy examination at the end of the dosing period, a significant increase in the actual and relative weights of the liver for male rats in 60 mg/kg dosage group was seen. Also, a significant increase in the actual weight of the thyroid for males in the 60 mg/kg group and a significant increase in the relative weight of the thyroid for males in the 15 mg/kg dosage group were found.
Besides, a significant reduction in the actual weight of the epididymis in male rats of the 60 mg/kg dose group was seen. However, the relative weight did not change and no weight or histological changes of the epididymis were found at the autopsy at the end of the dosage administration period. Therefore, it was judged that this was not due to the administered dosages.

Gross pathological findings:

no effects observed

Description (incidence and severity):

In the autopsy examination at the end of the dosing period, an example of thickening of the mucous lining of the forestomach was observed in a male rat of the 60 mg/kg dosed group. In the autopsy examination at the end of the recovery period, visual observation did not show any abnormality findings.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Both during the test substance administration period and the recovery period the groups did not show any appreciable changes to note and there were no observations to suggest a neurotoxic effect. Also, no abnormal condition in any of the rats was demonstrated during the function tests conducted during the fourth week of dose administration and the second week of recovery.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Minor centrolobular hypertrophy of hepatocytes in four male rats in the high dose group was seen. The incidence of htis findings as compared to the control group, was significantly higher. All other changes were comparable to the control group.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

3.75 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: Increased prothrombin time at mid dose, at high dose increased liver weight and increased incidence in minor centrolobular hypertrophy of hepatocytes.

Critical effects observed:

not specified

Conclusions:

In the 28-day oral gavage study with rats, the test substance 2-amylanthraquinone was administered to male and female rats at 3.75, 15 and 60 mg/kg bw/day. Based on the observed effects (prolongation in prothrombin time in mid- and high-dose males, increased liver weight and centolobular hepatocellular hypertrophy ) the NOAEL was set at 3.5 mg/kg bw/day for male and 15 mg/kg bw/day for female rats.

Executive summary:

The 28-days of repeated oral administration with a recovery period of 14-day was performed with 2-pentylanthraquinone administered in 3.75, 15 and 60 mg/kg doses to male and female Sprague-Dawley rats. There were no cases of death or the appearance of imminent death in any of the groups. The results show  a change in salivation immediately after dosage administration to the 60 mg/kg rat group. In hematological tests, after the end of dosing, a significant prolongation in the prothrombin time was observed in the 15 and 60 mg/kg administered dose male rats.  Also, in the 60 mg/kg dose group of male rats, a significant increase in the actual and relative weight of the liver was observed.  In the histological examination centrolobular hypertrophy of hepatocytes was observed.

No effect from the dosing was shown in the weight and feed intake and urine testing.  Also, a detailed observation of symptoms and function tests did not show any abnormalities.

From the above results,  the test shows that the 2-pentylanthraquinone NOAEL for male rats is 3.75 mg/kg/day and 15 mg/kg/day for female rats.

Reference 2

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

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

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.

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

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

Dose / conc.:

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

Remarks:

Vehicle controls

Dose / conc.:

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

Dose / conc.:

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

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

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.

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.

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

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

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

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

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).

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Reliable without restrictions

System:

hepatobiliary

Organ:

adrenal glands

kidney

liver

spleen

thymus

thyroid gland

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

Mode of Action Analysis / Human Relevance Framework

No information on the mode of action of the test substance is available. However, based on the results of the 90 -day repeated dose toxicity study, the main target organs appear to be liver and kidneys. An increased incidence and severity of hyaline droplet accumulation in kidneys observed in high-dose male rats in the repeated 90 -day toxicity study 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, and is thus not relevant for human risk assessment.

The relevance of other observed effects for humans cannot be excluded.

Additional information

A reliable 28 -day study with rats with a 14 -day recovery period and a reliable 90 -day toxicity study by oral route of administration are available.

A 28-days study with repeated oral administration with a recovery period of 14-day was performed with 2-pentylanthraquinone administered by gavage at 3.75, 15 and 60 mg/kg bw/day to male and female Sprague-Dawley rats. There were no cases of death or the appearance of imminent death in any of the groups. The results show a change in salivation immediately after dosage administration to the 60 mg/kg bw/day rat group. In hematological tests, after the end of dosing, a significant prolongation in the prothrombin time was observed in the 15 and 60 mg/kg bw/day dose group male rats. Also, in the 60 mg/kg bw/day dose group of male rats, a significant increase in the actual and relative weight of the liver was observed. In the histological examination centrolobular hypertrophy of hepatocytes was observed. No effect from the dosing was shown in the weight and feed intake and urine testing. Also, a detailed observation of symptoms and function tests did not show any abnormalities. From the above results, the test shows the 2-pentylanthraquinone NOAEL for male rats is 3.75 mg/kg bw/day and 15 mg/kg bw/day for female rats.

In a GLP-compliant OECD Guideline 408 study, the test substance 2 -amylanthrazuinone 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 bw/day (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 levels in both sexes and urea concentration in females only. Based on these effects, the NOAEL was set at 25 mg/kg bw/day.

Considering that the 90-day study was conducted under GLP and according to the OECD guideline, it is considered to be reliable without restrictions. As the effects observed in the 28 -day study were either not observed, or occurred at higher dose levels in the reliable study with a longer exposure duration, the NOAEL from the 90 -day study shall be taken forward for risk assesment and DNEL derivation.

Justification for classification or non-classification

In a reliable 90 -day study with 2-amylanthraquinone adverse effects were observed at a dose level of 75 mg/kg bw/day. These included increased relative organ weights (liver, spleen and kidneys in both sexes, thyroids in males, adrenals in females), accompanied by minimal histopathological changes (centrilobular hepatocellular hypertrophy and follicular cells hypertrophy in the thyroids, slightly inreased incidence and severity of hemosiderin pigmentation and diffuse congestion in the spleen, increased lymphocytolysis and lymphoid depletion in the thymus, hyaline droplets accumulation in kidneys of male rats). Also small changes in haematological and biochemical parameters were evidenced. However, none of these effects are considered to indicate a significant impairment of a vital organ function or indicate a significant organ damage. Based on this, classification of the substance for specific target organ toxicity by repeated exposure is considered to be not warranted in accordance with Regulation 1272/2008/EC.