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

Toxicity to reproduction

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Administrative data

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 October 2019 - 6 April 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

other: Analytical method

Cross-reference 2

Reason / purpose for cross-reference:

reference to other assay used for intermediate effect derivation

Reference

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

Based on ECHA decision No. TPE-D-2114456461-51-01/F.

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
- Premating exposure duration for parental (P0) animals: 10 weeks; 10 weeks premating exposure duration is required because there is no substance-specific information in the dossier supporting shorter premating exposure duration. Ten weeks exposure duration is supported also by the lipophilicity of the substance to ensure that the steady state in parental animals has been reached before mating.
- Basis for dose level selection: Within the repeated dose 90-day study (Charles River Study No. 511237) animals were dosed for 90 days at 0, 7.5, 25 and 75 mg/kg bw/day. At 75 mg/kg bw/day there were increases in the relative organ weight of the liver, kidney and adrenal weight. This was accompanied by minimal histopathological changes in the liver, spleen, thymus, thyroid, kidney and adrenals, the study concluded that these findings were considered not to be adverse. Additionally, there were small changes in albumin and creatinine in both sexes and urea in the females. It was concluded that the findings were considered not to be adverse. No toxicologically significant findings were noted in clinical appearance, functional observations, body weight, food consumption, ophthalmoscopy or macroscopic evaluations. Although none of the findings at 75 mg/kg bw/day in the 90-day toxicity study indicated a significant impairment of a vital organ function or indicated significant organ damage, based on the relative increase in several organ weights, the no observed adverse effect level (NOAEL) for 2-amylanthraquinone for the 90-day toxicity study was considered to be 25 mg/kg bw/day. Based on this study a high dose level of 75 mg/kg/day was considered to be suitable to induce moderate but not excessive effects of toxicity.
- Inclusion/exclusion of extension of Cohort 1B: not included, since the criteria lead down in column 2 of Section 8.7.3 (Annex X) are not met, i.e. there are no uses leading to significant exposure of consumers and professionals, the substance does not display any genotoxic effects in somatic cell mutagenicity tests in vivo, there are no toxicokinetic indications for a steady state of the internal dose for the active substance and/or its metabolites after extended exposure, there are no indications for an endocrine disrupting mode of action.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: not included, since there are no particular concerns for developmental neurotoxicity (criteria in column 2 of 8.7.3 (Annex X) are not met).
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: not included, since there are no particular concerns for developmental immunotoxicity (criteria in column 2 of Section 8.7.3 (Annex X) are not met).
- Route of administration: The oral route of administration was selected for this study as this route is considered the most appropriate route of administration for liquid substances such as the test substance.

Test material

Test material information

Constituent 1

Test material form:

solid

Remarks:

The test item was found to solidify at ambient temperature.

Details on test material:

- Appearance: Solid at room temperature. At temperatures of 37°C - 55°C the test item was a yellow/light brown liquid.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in an incubator set to maintain 55°C.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: determined in Charles River Study No. 511240 as well as during dose formulation analysis as part of the present study.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: determined in Charles River Study No. 511240.
- Reactivity of the test material with the incubation material used: glass vials were used.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

CRL:WI (Han)

Details on species / strain selection:

The Han Wistar rat was chosen as the animal model for this study as it is a rodent species
accepted by regulatory agencies for toxicity testing.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, United Kingdom.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 7-9 wks
- Weight at study initiation: (P) Males: 219-372 g (target 200-350 g); Females: 134-208 g (target: 125-225 g). The difference in the males' actual bodyweight and target bodyweight was considered not to impact the study as all animal were the correct age and were therefore considered to be suitable for use on this study.
- Fasting period before study: not applicable.
- Housing: Animals were initially housed 2 or 3 per cage by sex in appropriately sized suspended polycarbonate cages with stainless steel grid tops and solid bottoms. A few days prior to mating, males were transferred to individual cages with solid bottoms. Females were transferred to these cages for mating. Mated females were transferred to individual solid bottomed cages. White paper tissue was supplied as nesting material from GD 20. F0 females with litters were retained in this type of cage until termination. On a suitable day after completion of mating, the males were re-housed with their original cage mates. Cohort 1A and 1B animals were housed 2 or 3 by sex per cage in appropriately sized suspended polycarbonate cages with stainless steel grid tops and solid bottoms. Bedding material was sterilised white wood shavings.
- Animal enrichment: Animals were socially housed for psychological/environmental enrichment and were provided with items such as a device for hiding in and an object for chewing, except when interrupted by study procedures/activities.
- Diet: SDS VRF-1 breeder diet was provided ad libitum throughout the study, except during designated procedures.
- Water: water ad libitum from the public supply from water bottles which were changed as necessary throughout the course of the study.
- Acclimation period: The F0 animals were allowed to acclimate for a period of 17 days before the commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C to 23°C (target 19 to 23°C)
- Humidity (%): 19% to 62% (target 40 to 70%)
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 25 October 2019 To: 7 May 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

Polyethylene glycol 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
Test item dosing formulations were prepared based on a method established at the Test Site (Test Site Study No. 511240) at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least weekly, stored in a refrigerator set to maintain 4°C protected from light, and dispensed daily.
The control item, Polyethylene glycol 400, was used as supplied and dispensed at least weekly, stored in a refrigerator set to maintain 4°C and protected from light, and dispensed daily for administration to Control animals.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: On Day 71 of dosing, females were housed with their allocated co-group male partner. The pairing period for each pair of animals was a maximum of 14 nights.
- Proof of pregnancy: vaginal plug and/or sperm in vaginal lavage referred to as Day 0 of pregnancy
- If evidence of mating was not observed by the end of the pairing period, the female was separated from the male during the morning following the last night of pairing and treated as if mating had occurred during that night. Procedures for that female continued as if it had mated on the last night of pairing.
- After successful mating, females were transferred to individual solid bottomed cages. White paper tissue was supplied as nesting material from GD 20. F0 females with litters were retained in this type of cage until termination.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis on Day 1, in Week 14 and in Week 25. For concentration analysis, samples were taken from the control and test item formulations. For homogeneity analysis, samples were taken from the test item formulations only. The homogeneity results obtained from the top, middle and bottom for the low, mid and high dose test item formulations were averaged and utilised as the concentration results.
Duplicate top, middle and bottom (duplicate middle only for control) sets of samples (500 mg) for each sampling time point were collected into cryo vials; triplicate top, middle and bottom samples (triplicate middle only for control) were retained at the Test Facility as backup samples. All samples were stored at <-15˚C within the established stability period. For stability analysis, a sample of test item was sent at ambient temperature, protected from light, along with the Day 1 samples. Analyses were performed by ultra performance liquid chromatography (UPLC) using a validated analytical procedure (Test Site Study No. 511240).
Concentration results were considered acceptable if sample concentration results were within or equal to ± 10% of theoretical concentration. Each individual sample concentration result was considered acceptable if it was within or equal to ± 15%. After acceptance of the analytical results, backup samples were discarded. For homogeneity, the criteria for acceptability was a relative standard deviation (RSD) of concentrations of ≤ 10% for each group.
Stability analyses performed previously in conjunction Test Site Study No. 511240 demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Duration of treatment / exposure:

F0 males were treated daily for 10 weeks prior to mating until necropsy after the termination of the F0 females; total exposure 123-127 days.
F0 females were treated daily for 10 weeks prior to mating, then through mating, gestation and until at least LD 21 (termination between LD 22-24).
Cohort 1A animals were dosed daily from PND 21 until at least PND 90 (termination between PND 91 and 96).
Cohort 1B animals were dosed daily from PND 21 until at least PND 90 (termination between PND 98 and 100).

Frequency of treatment:

daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Low dose group

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Mid dose group

Doses / concentrations 3

Dose / conc.:

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

Remarks:

High dose group

No. of animals per sex per dose:

F0: 25 animals/sex/group
F1 weanlings - non-selected pups: 10 animals/sex/group
Cohort 1A: 20 animals/sex/group
Cohort 1B: 20 animals/sex/group, except for the high dose group, to which 21 males and 19 females were allocated. This was due mis-sexing, with a male pup inadvertently selected for the female group. This was noticed on Study Day 120. However, this animal was unable to be replaced as the dam and remaining pups from the litter had been sent for necropsy. This animal was kept on study so the same number of animals were dosed within each treatment group. There was considered to be a sufficient number of both sexes to be able to meet the objective of the study. This was therefore considered not to impact the study.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the repeated dose 90-day study (CRL Study No. 511237) a high dose level of 75 mg/kg bw/day was considered to be suitable to induce moderate but not excessive effects of toxicity.
- Rationale for animal assignment (if not random): F0: during the week before the commencement of dosing, individual body weights were checked to ensure that the mean body weights of each sex within each treatment group were within ± 5% of the mean weight of each sex. Cohort 1A and 1B: from each group, 40 males and 40 females were selected for post-weaning assessments, nominally by selecting up to 2 males and 2 females from each litter. Where fewer than 40 litters were weaned, the necessary additional animals were obtained by selecting an additional pup from appropriate litters; these appropriate litters were normally selected arbitrarily but attention was paid to the retention of as wide a genetic pool as possible. The selected pups were the median’th weight pups of that sex in the litter on LD 21. These pups were removed from their mother on LD 21, individually identified and housed in their new cage. Pups that were not selected for post-weaning assessments remained with their mother until sacrifice.
- Fasting period before blood sampling for clinical biochemistry: yes

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily throughout the study
- Cage side observations: observations for general health/mortality and moribundity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly starting in the week prior to the start of dosing (week -1).
- Animals were removed from the cage for examination.
- Observations included, but were not limited to, changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture, response to handling, as well as the presence of clonic or tonic movements, stereotypy or bizarre behaviour were also assessed.

PRE-/POST-DOSE OBSERVATIONS
Animals were observed regularly throughout the day on each day of dosing for signs of reaction to treatment, with particular attention being paid to the animals during and for the first hour after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males were weighed weekly beginning Week -1.
F0 females were weighed weekly from Week -1 until pairing for mating, and then on GD 0, 7, 14 and 20, and LD 1, 7, 14 and 21.
Litters were weighed en masse and by sex on LD 4, 7 and 14 and individually on LD 1 and 21.
F1 cohort 1A and 1B were weighed weekly from weaning, starting on a suitable day within one week of weaning for the majority of litters.
A weight was recorded on the day of scheduled necropsy for all animals.

FOOD CONSUMPTION (no feeding study):
Food consumption was measured as follows;
-for F0 animals, weekly from Week -1 for both sexes until pairing for mating.
-for F0 males weekly after mating and re-housing, and
-for mated F0 females over the periods GD 0-7, 7-14 and 14-20 and LD 1-7, 7-14 and 14-21.
-for F1 cohort 1A and 1B,weekly, starting on a suitable day within one week of weaning for the majority of litters.

WATER CONSUMPTION: Water consumption was monitored on a regular basis throughout the study by visual inspection of the water bottles. No inter-group differences were noted.

OBSERVATIONS OF FEMALES DURING PARTURITION AND LACTATION
The females were allowed to litter normally. Any observed difficulty or prolongation of parturition was recorded. The day of birth of the litter (day on which first pups were born) was designated LD 0. The duration of gestation in days was calculated.
Deficiencies in maternal care were recorded: inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups, or apparently inadequate lactation or feeding. White paper tissue was supplied to each mother for incorporation in the nest. This was replaced when it became soiled.

CLINICAL PATHOLOGY
-On the morning of necropsy, 10-13 F0 animals/sex/group were sampled for haematology, coagulation and clinical chemistry investigations. Animals were fasted prior to blood sampling. Blood was collected via the orbital sinus.
For haematology, K2EDTA was used as anticoagulant and the following parameters were analysed: Red blood cell count, Haemoglobin concentration, Haematocrit, Mean corpuscular volume, Red blood cell distribution width, Mean corpuscular haemoglobin concentration, Mean corpuscular haemoglobin, Reticulocyte count (absolute), Platelet count, White blood cell count, Neutrophil count (absolute), Lymphocyte count (absolute), Monocyte count (absolute), Eosinophil count (absolute), Basophil count (absolute) and Large unstained cells (absolute).
For coagulation, blood samples were collected using 3.2% (w/v) trisodium citrate as anticoagulant and processed for plasma, which was analysed for the following parameters: Activated partial thromboplastin time, Fibrinogen Prothrombin time and Sample quality.
For clinical chemistry, blood samples were collected using lithium heparin as anticoagulant and processed for plasma, which was analysed for the following parameters: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Gamma-glutamyltransferase, Creatine Kinase, Total bilirubin, Urea, Creatinine, Calcium, Phosphate, Total protein, Albumin, Globulin, Albumin/globulin ratio, Glucose, Cholesterol, Triglycerides, Sodium, Potassium, Chloride and Sample quality.

-In the last week of dosing, 10 animals/sex/group were housed in individual metabolism cages, with access to water only, over a period of 6 hours. Urine was collected for analysis of the following parameters: Colour, Appearance/Clarity, Specific gravity, Volume, pH, Protein, Glucose, Bilirubin, Ketones and Blood.

THYROID HORMONE ANALYSIS
On the morning of necropsy, blood samples were collected from 10 F0 animals/sex/group from the jugular vein. Animals were fasted prior to sampling. Samples were analysed for triiodothyronine (T3), thyroxine (T4), and thyroid stimulating hormone (TSH) using validated analytical procedures. T3 and T4 were measured by using solid phase, competitive chemiluminescent enzyme immunoassays. TSH was measured by using a solid phase enzyme immunometric assay.

Oestrous cyclicity (parental animals):

Estrous cycles were monitored daily for F0 females from Day 57 of dosing until day of detection of a copulatory plug in situ and/or of sperm in the lavage. Vaginal lavages were taken early each morning and the stages of estrous observed was recorded. Vaginal smears were also examined on the morning of necropsy to determine the stage of estrous cycle to allow correlation with histopathology of ovaries.

Sperm parameters (parental animals):

Parameters examined in F0 males:
- testis weight (both sides)
- epididymis weight (both sides)
- epidydimis (right cauda): assessment of percentage motile sperm, percentage progressive motile sperm and straightline velocity (VSL) and total sperm count expressed per cauda epididymis and per gram of cauda epididymis.
In addition, a sperm smear was prepared and stained. From the control and high dose animals (both generations), at least two hundred sperm per animal were evaluated for morphological abnormalities.
- one testis: determination of total spermatid count expressed per testis and per gram of testis.

Litter observations:

PARAMETERS EXAMINED IN F1 OFFSPRING
LD 0-LD 4
The number of live and dead pups born in each litter was recorded as soon as possible after
completion of parturition on LD 0. The live pups were counted, sexed and weighed individually on PND 1 and examined daily for the presence of milk in the stomach and for any externally visible abnormalities daily up to and including LD 4.
Assessment of ano-genital distance was measured for both sexes on PND 1.

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded. Where 4 males or 4 females were not available, extra pups of the opposite sex were retained to ensure a total number of 8 pups. When the total number of pups in a litter on LD 4 was ≤ 8 pups, no litter size adjustment occurred.

LD 5 - LD 21
From LD 5, the total live pups were counted daily, and were sexed and examined for abnormality again on LD 7, 14 and 21. These pups were weighed en masse, sexes separate, on LD 4, 7 and 14. On LD 1 and 21 all pups were weighed individually.
Nipple retention was assessed in males on PND 13.

SEXUAL MATURATION
Cohort 1A and 1B animals were examined for sexual maturation.
Commencing at 28 days of age, F1 females were examined daily for vaginal opening. The day on which the vagina became open was recorded, as was the body weight on that day.
F1 females had their estrous cycles monitored daily from the day after vaginal patency (circa PND 28) until 1 estrous cycle was confirmed, then daily from at least PND 75 up to and including the day of scheduled necropsy.
Commencing at 35 days of age, males were examined daily for balano-preputial separation. The day on which separation occurred was recorded, as was the body weight on that day.

SPERM PARAMETERS
Parameters examined in Cohort 1A males:
- testis weight (both sides)
- epididymis weight (both sides)
- epidydimis (right cauda): assessment of percentage motile sperm, percentage progressive motile sperm and straightline velocity (VSL) and total sperm count expressed per cauda epididymis and per gram of cauda epididymis.
In addition, a sperm smear was prepared and stained. From the control and high dose animals (both generations), at least two hundred sperm per animal were evaluated for morphological abnormalities.
- one testis: determination of total spermatid count expressed per testis and per gram of testis.

CLINICAL PATHOLOGY
-On the morning of necropsy, 10-14 animals/sex/group from Cohort 1A were sampled for haematology, coagulation and clinical chemistry investigations. Animals were fasted prior to blood sampling. Blood was collected via the orbital sinus. Sampling, processing and analyses were similar to those described for F0 animals.
-In the last week of dosing, 10 animals/sex/group from Cohort 1A were housed in individual metabolism cages, with access to water only, over a period of 6 hours. Urine was collected for urinanalysis. Parameters were identical to those described for F0 animals.

THYROID HORMONE ANALYSIS
The following blood samples were taken from the F1 litters:
-PND 4: culled offspring (up to 4 rats/litter), taken via cardiac puncture without anticoagulant. At least 1 mL of blood per litter (from 2 or more pups per litter, as necessary) was collected. Samples were collected from all litters at each dose level with sufficient numbers of culled pups.
-PND 22-24: unselected pups (10 rats/sex/group from 10 litters), taken from the jugular vein. At least 0.8 mL/pup was collected.
-Cohort 1A (10 rats/sex/group) on the morning of scheduled necropsy, taken from the jugular vein (1 mL). Procedures were similar as for F0 animals.
Samples were analysed for triiodothyronine (T3), thyroxine (T4), and thyroid stimulating hormone (TSH) using validated analytical procedures, identical to those described for the F0 samples.

GROSS EXAMINATION OF DEAD PUPS:
- Animals euthanised or found dead before LD 14: where practicable, these animals were sexed and then checked for the presence of milk in the stomach and for the presence of any externally visible abnormalities. Any externally abnormal pups were fixed in 10% formalin for optional further examination. Externally normal pups were discarded.
- Animals euthanised of found dead after LD 14: These animals were subject to a gross necropsy. An external examination was followed by an inspection of the cranial, thoracic and abdominal contents. Representative samples of any abnormal tissues were preserved.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed between Study Days 123-127, after termination of the F0 females.
- Maternal animals: All surviving animals were sacrificed between LD 22-24, after weaning of the F1 litters. Females which failed to produce a litter by their expected GD 24 were sent for necropsy.
- All adult animals were euthanized by exposure to carbon dioxide, followed by exsanguination.

GROSS NECROPSY
- Gross necropsy consisted of a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
The reproductive tracts of all F0 females were examined for signs of implantation, the number of any implantation sites were recorded. The total number of corpora lutea graviditatis was recorded for each female. The uteri of all non-pregnant females were fixed, stained and examined for implantation sites.

HISTOPATHOLOGY / ORGAN WEIGHTS
For all groups, the tissues indicated in Table 1 were weighed and prepared for microscopic examination. Organ weights were not recorded for animals euthanised in poor condition or in extremis. Paired organs were weighed together. Terminal body weights were used for organ weight analysis. Tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with haematoxylin and eosin.
For F0 animals, from each ovary, 6 step serial sections (sectioning a small amount into the ovary e.g. 25 µm before the next section was taken) were taken. One section was stained with haematoxylin and eosin and 5 sections were stained for IHC using PCNA marker for enumeration of primordial and primary follicles.
All tissue samples from all high dose and control group animals were examined for histology and histopathology. For the low and mid dose group animals, only gross lesions and the target tissues (identified as liver and kidney for males, and liver for females) were included for histology and histopathology.

Testes
A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted.

Ovaries
The examination of the ovaries included quantification of the primordial and growing oocytes and the confirmation of the presence or absence of corpora lutea.

Sperm evaluation
From all males from the F0 animals, the right cauda epididymis was placed in Medium 199 and the sperm were allowed to “swim out” into the medium. An appropriate dilution of the sperm suspension was prepared and examined using a Hamilton Thorne sperm motility analyser, to assess percentage motile sperm, percentage progressive motile sperm and straightline velocity (VSL).
The cauda epididymis was minced and suspended. Dilutions of this sperm suspension were counted using a haemocytometer to obtain a total sperm count which was expressed per cauda epididymis and per gram of cauda epididymis.
From all samples of the sperm suspension, a sperm smear was prepared and stained with eosin Y solution. From the high dose and control group 1 and 4 animals, at least two hundred sperm per animal were evaluated for morphological abnormalities.
One testis was decapsulated and homogenised. The homogenate was sonicated to reduce tissue debris etc, if required. The number of homogenisation resistant spermatids in dilutions of this suspension was counted using a haemocytometer to obtain a total spermatid count which was expressed per testis and per gram of testis.

Postmortem examinations (offspring):

SACRIFICE
Timing:
- Culled pups on PND 4
- Non-selected pups at weaning between PND 22-24 (at the same time as their mother)
- Cohort 1A animals between PND 91 and 96
- Cohort 1B animals between PND 98 and 100

- Animals less than 10 days old were killed by intraperitoneal injection of sodium pentobarbitone or cervical dislocation, followed by exsanguination. Animals 10 days of age or more were euthanised by exposure to carbon dioxide, followed by exsanguination.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Non-selected pups on PND 4: these animals were necropsied. This consisted of external examination followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Samples of any grossly abnormal tissues were preserved.
-Non-selected pups on PND 22-24: from each litter, 1 male and 1 female pup (where they were available) were necropsied from 10 litters/group. This consisted of external examination followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Samples of selected tissues and any grossly abnormal tissues were preserved.
The remaining pups in each litter were checked for externally visible abnormalities at the time of killing. Any found to have such an abnormality had a gross necropsy performed and any abnormalities were preserved.
- Cohort 1A (PND 91-96 ): Necropsy was performed as described for the F0 animals.
- Cohort 1B (PND 98-100 ): Necropsy was performed as described for the F0 animals.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table 1 (Cohort 1A), Table 2 (Non-selected pups on PND 22) and Table 3 (Cohort 1B) were weighed and prepared for microscopic examination.
From non-selected pups terminated on PND 22-24, full (limited) tissue histology and histopathology was performed for the high dose and control animals. For the low and mid dose animals, only gross lesions were included in histology and histopathology.
From Cohort 1A, all tissue samples from all high dose and control group animals were examined for histology and histopathology. For the low and mid dose group animals, only gross lesions and the target tissues (identified as liver and kidney for males, and liver for females) were included for histology and histopathology.
No histopathology was performed for samples from Cohort 1B. For control animals, all tissues were processed to paraffin blocks.

For Cohort 1 animals, from each ovary, 6 step serial sections (sectioning a small amount into the ovary e.g. 25 µm before the next section was taken) were taken. One section was stained with haematoxylin and eosin and 5 sections were stained for IHC using PCNA marker for enumeration of primordial and primary follicles.

Testes
A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted.

Ovaries
The examination of the ovaries included quantification of the primordial and growing oocytes and the confirmation of the presence or absence of corpora lutea.

Sperm evaluations
Sperm evaluations were performed for all Cohort 1A males, as described for the F0 males.

IMMUNOPHENOTYPING
Spleen samples (approximately half of the spleen) were taken from 10 rats per sex per group of the Cohort 1A animals at necropsy and were collected into tubes containing RPMI medium and stored immediately on wet ice, and were analysed using a validated flow cytometry-based analytical method.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two-sided tests and have been reported at the 1%, and 5% levels. The following variables were analysed according to sex and occasion (groups with les that 3 observations were excluded: body weight (gains), food consumption, haematology variables, coagulation variables, clinical chemistry variables, urinalysis variables, TSH, T3 and T4 variables, organ weights, organ weight relative to body weight, ovarian scoring (total number of oocytes per animal). Pair-wise comparisons of the low-, mid- or high dose group relative to the control group were made.
Levene’s test was used to assess the homogeneity of group variances. Groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively. Datasets with two groups were compared using a Dunnett’s test (equivalent to t-test in Nevis 2012 tables) or Dunn’s test (equivalent to Wilcoxon Rank-Sum test in Nevis 2012 tables).

Reproductive indices:

Female Mating Index = (Number of Females with Evidence of Mating (or no confirmed mating date and pregnant))/Number of Females Paired;
Female Fertility Index = Number of Pregnant Females / (Number of Females with Evidence of Mating (or no confirmed mating date and pregnant));
Female Pregnancy Index = Number of Pregnant Females/Number of Females Paired;
Male Mating Index = (Number of Males with Evidence of Mating (or female partner confirmed pregnant))/Number of Males Paired;
Male Fertility Index = Number of Males Impregnating a Female/(Number of Males with Evidence of Mating (or female partner confirmed pregnant));
Male Pregnancy Index = Number of Males Impregnating a Female/Number of Males Paired;
Gestation Length = Number of days from GD 0 to the day the first pup is observed;
Gestation Index (Percentage of pregnancies that resulted in birth of live litters) = Number of Animals with Live Offspring/Number of Animals Pregnant x 100;
Post-Implantation Loss/Litter = ((Number of Implants – Total Newborn Pups)/Number of Implants) x 100.

Offspring viability indices:

Live Birth Index (Percentage of pups born alive) = Number of Live Newborn Pups/Number of Newborn Pups x 100; Viability Index (Percentage of pups born that survived 4 days postpartum) = Number of Live Pups on Day 4 Postpartum/Number of Live Newborn Pups x 100;
Lactation Index (Percentage of pups that survived 21 days postpartum) = Number of Live Pups on Day 21 Postpartum/Number of Live Pups on Day 4 (postculling) Postpartum x 100;
Sex Ratio (% males) (Percentage of male pups per litter) = Number of Live Male Pups/Total Number of Live Pups x 100.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were clinical observations of salivation at 5, 20 or 75 mg/kg bw/day in the F0 males as well as at 20 or 75 mg/kg bw/day in the F0 females, whereas there were no observations in the control. There was also a higher incidence of ploughing at 5, 20 or 75 mg/kg bw/day in the F0 animals when compared to the control. Salivation and ploughing had a dose-dependent relationship as there was a higher number of incidences at increasingly higher dose levels. Both salivation and ploughing were predominantly observed immediately post-dose with only few occasions at the 0-1 hour timepoint after dosing, therefore it is considered that these findings are most likely due to the taste of the formulation so were considered not to be adverse.
All other clinical observations were considered to be incidental background findings commonly observed in this species.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths on the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no bodyweight or body weight gain effects considered to be related to the test item in the F0 animals at dose levels of 5, 20 or 75 mg/kg bw/day. Any statistical significance seen was due to individual variation within the groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no effects on food consumption considered to be related to the test item in the F0 animals at dose levels of 5, 20 or 75 mg/kg bw/day. Any statistical significance seen was due to individual variation within the groups.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Mild to moderate changes in haematology parameters related to the administration of 2-Amylanthraquinone were observed at 75 mg/kg bw/day in the F0 animals. The changes are summarized in Table 3.
At 75 mg/kg bw/day there were higher numbers of reticulocytes in F0 generation animals and this correlated with lower levels of red blood cells; an increase in red blood cell distribution width and correspondingly lower levels of haemoglobin and haematocrit. Additionally, in the F0 females there were high mean corpuscular volume and corpuscular haemoglobin which could be related to the lower levels of red blood cells. At 75 mg/kg bw/day there were higher levels of monocytes in the F0 males and lymphocytes in the F0 females. The changes in the F0 males corresponded with a higher level of white blood cells.
There were no effects in the haematology parameters considered to be related to the test item
at 5 or 20 mg/kg bw/day in the F0 animals. All other values were considered to be within normal biological variation when compared with the controls.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Mild to moderate changes in clinical chemistry parameters related to the administration of 2-Amylanthraquinone were observed at 75 mg/kg bw/day in the F0 males, and at 20 or 75 mg/kg bw/day in the F0 females. These changes are summarised in Table 3.
There were higher levels of albumin at 75 mg/kg bw/day in the F0 males as well as lower level of globulin at 75 mg/kg bw/day in the F0 animals when compared with the control. The changes in albumin and globin corresponded with higher albumin/globulin ratio at 75 mg/kg bw/day in the F0 animals. Additionally there were lower levels of triglycerides at 20 or 75 mg/kg bw/day in the F0 females when compared with the control.
There were no effects in the clinical chemistry parameters considered to be related to the test item at 5 or 20 mg/kg bw/day in the F0 males or at 5 mg/kg bw/day in the F0 females. All other values were considered to be within normal biological variation when compared with the controls.

Coagulation
At 75 mg/kg bw/day there were lower levels of fibrinogen in the F0 males with a 0.87-times lower fold difference respectively when compared with the control. There were no test item-related effects on coagulation parameters at 5, 20 or 75 mg/kg bw/day of 2-mylanthraquinone in the F0 females or at 5 or 20 mg/kg bw/day in the F0 males.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Changes in the thyroid hormones related to the administration of 2-Amylanthraquinone were observed at 75 mg/kg bw/day in the F0 animals. The levels of T3, T4 and TSH at 75 mg/kg bw/day in the F0 animals are summaries in Table 4.
At 75 mg/kg bw/day there were lower levels of T4 in the F0 males and T3 in the F0 males and females. There were also higher levels of TSH at 75 mg/kg bw/day in the F0 animals.
There were no test item related effects at 5 or 20 mg/kg bw/day in the in the F0 animals. All other values were considered to be within normal biological variation when compared with the controls.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no urinalysis findings considered related to the test item at 5, 20 or 75 mg/kg bw/day.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were test item-related histopathological findings at 75 mg/kg bw/day and at 20 mg/kg bw/day in the F0 males. These finding have been summarised in Table 6.
At 20 or 75 mg/kg bw/day the liver showed incidences of minimal focal hepatocellular degeneration in the F0 males, whereas there were no observations within the control. At 75 mg/kg bw/day there were were incidences of minimal hepatocellular hypertrophy in the liver in the F0 males as well as mild focal hepatocellular necrosis in the F0 males.
There were no findings considered to be related to the test item at 5, 20, or 75 mg/kg bw/day in the F0 females, or at 5 mg/kg/day in the F0 males. All other microscopic findings observed were of the nature commonly observed in this strain and age of rat, or occurred at a similar incidence in control and treated animals, and, therefore, were considered not to be test item related.

Ovarian follicle counts
There were no findings in the ovarian follicle counts that were considered to be related to the test item at 5, 20 or 75 mg/kg bw/day of 2-Amylanthraquinone in the F0 females.
There were no findings in the histopathology examination of the ovaries to indicate an increase in degenerating ovarian follicles. Therefore although there were mean ovarian follicles counts that were statistically significantly different from their respective controls, these differences were considered to be within normal biological variation for this species.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no effects on the estrous cycles considered to be related to the test item at 5, 20 or 75 mg/kg bw/day.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no findings in the sperm straight line velocity or the percentage of motile or progressive motile sperm considered to be related to the test item at 5, 20 or 75 mg/kg bw/day
2-Amylanthraquinone in the F0 males.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no effects on the maternal performance considered to be related to the test item at 5, 20 or 75 mg/kg bw/day. There were no effects on the duration of gestation, litter performance or survival considered to be related to the dosing of test item at 5, 20 or 75 mg/kg bw/day.

Details on results (P0)

The changes in the haematology, coagulation and clinical chemistry parameters are considered likely to be related to the changes in the organ weights and/or histopathology observed in the spleen, liver and kidney. These findings were considered not to be adverse as they were mild to moderate in severity and there were no associated adverse histopathological or toxicology effects.

Higher kidney weights in the F0 generation and Cohort 1A and 1B animals were considered not to be adverse as they were not associated with any adverse histopathological changes. In the liver there were test item-related mild/minimal histological findings of focal hepatocellular degeneration, hepatocellular hypertrophy and/or focal hepatocellular necrosis at ≥ 20 mg/kg bw/day in the F0 males. Hepatocyte hypertrophy in the liver often results from induction of microsomal metabolic enzymes, which is an adaptive response to xenobiotics (Maronpot et al 2010, Hall et al 2012). These histological findings were classified as either mild or minimal changes and so are considered not to be adverse. Additionally, the liver weight (as % of body weight) was 29.4 - 45.2 % higher at 75 mg/kg bw/day in the F0, Cohort 1A and 1B males and 15.1 - 34.7 % higher at 75 mg/kg bw/day in the F0, Cohort 1A and 1B females, these changes were not considered to be adverse as they were not associated with any adverse histopathological changes.
Test item-related higher spleen weights at 75 mg/kg bw/day and higher pituitary gland weights
at ≥ 20 mg/kg bw/day were not associated with any gross pathological or histological findings
therefore these organ weight changes were considered not to be adverse.
At 75 mg/kg bw/day there were lower levels of T4 and T3 as well as higher levels of TSH. These changes in the F0 males were associated with a higher thyroid weight. Given the trend of increased liver weights described above, the thyroid changes may be also related to these changes. The changes in the thyroid hormone levels were considered to be related to the test item however, under the conditions of this study no adverse effects could be linked to the changes in the thyroid hormones therefore this change was not taken into account when determining the parental NOAEL.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of effect on reproduction parameters.

Target system / organ toxicity (P0)

1

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:

not specified

2

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:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

PUP AND LITTER (LD 1- LD 21): There were no pup or litter observations considered to be related to the dosing of the test item at 5, 20 or 75 mg/kg bw/day.
COHORT 1A and 1B: There were clinical observations of salivation at 20 or 75 mg/kg bw/day in the Cohort 1A animals and Cohort 1B animals, whereas there were no observations in the control. There was also a higher incidence of ploughing at 5, 20 or 75 mg/kg bw/day in the Cohort 1A and 1B animals when compared to the control. Salivation and ploughing had a dose-dependent relationship as there was a higher number of incidences at increasingly higher dose levels. Both salivation and ploughing were predominantly observed immediately post-dose with only few occasions at the 0-1 hour timepoint after dosing, therefore it is considered that these findings are most likely due to the taste of the formulation so were considered not to be adverse.
All other clinical observations were considered to be incidental background findings commonly observed in this species.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no unscheduled deaths on the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

PUPS AND LITTER (LD 1 - 21): There were no effects on the litter weights or individual pup weights considered to be related to the test item at 5, 20 or 75 mg/kg bw/day.
COHORT 1A and 1B: There were no bodyweight or body weight gain effects considered to be related to the test item in the Cohort 1A or 1B animals at dose levels of 5, 20 or 75 mg/kg bw/day. Any statistical significance seen was due to individual variation within the groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no effects on food consumption considered to be related to the test item in the Cohort 1A or 1B animals at dose levels of 5, 20 or 75 mg/kg bw/day. Any statistical significance seen was due to individual variation within the groups.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Mild to moderate changes in haematology parameters related to the administration of 2-Amylanthraquinone were observed at 75 mg/kg bw/day in the Cohort 1A animals. These changes are summarised in Table 2.
At 75 mg/kg bw/day there were higher numbers of reticulocytes in Cohort 1A males and this correlated with lower levels of red blood cells; an increase in red blood cell distribution width and correspondingly lower levels of haemoglobin and haematocrit. At 75 mg/kg bw/day there were higher levels of platelets in the Cohort 1A males.
There were no effects in the haematology parameters considered to be related to the test item at 5 or 20 mg/kg bw/day in the Cohort 1A animals. All other values were considered to be within normal biological variation when compared with the controls.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Mild to moderate changes in clinical chemistry parameters related to the administration of 2-Amylanthraquinone were observed at 75 mg/kg bw/day in the Cohort 1A males and at 5, 20 or 75 mg/kg bw/day in the Cohort 1A females. These changes are summarised in Table 3.
There were higher levels of albumin at 5, 20 or 75 mg/kg bw/day in the Cohort 1A females as well as lower level of globulin at 75 mg/kg bw/day in the Cohort 1A females, when compared with the control. The changes in albumin and globin corresponded with higher albumin/globulin ratio at 75 mg/kg bw/day in the Cohort 1A females, as well as higher total protein levels at 5, 20 or 75 mg/kg bw/day in the Cohort 1A females. Additionally there were lower levels of triglycerides at 75 mg/kg bw/day in the Cohort 1A males when compared with the control.
There were no effects in the clinical chemistry parameters considered to be related to the test item at 5 or 20 mg/kg bw/day in the Cohort 1A males. All other values were considered to be within normal biological variation when compared with the controls.

Coagulation
At 75 mg/kg bw/day there were lower levels of fibrinogen in the Cohort 1A males with a 0.89 times lower fold difference respectively when compared with the control.
There were no test item-related effects on coagulation parameters at 5, 20 or 75 mg/kg bw/day of 2-Amylanthraquinone in the Cohort 1A females or at 5 or 20 mg/kg bw/day in the Cohort 1A males. All other values were considered to be within normal biological variation when compared with the controls.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no urinalysis findings considered related to the test item at 5, 20 or 75 mg/kg bw/day.

Sexual maturation:

no effects observed

Description (incidence and severity):

There were no findings related to sexual maturation (age and weight at vaginal opening or preputial separation) and no difference in time to first estrous cycle in Cohorts 1A and 1B considered to be related to the test item at 5, 20 or 75 mg/kg bw/day.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There were no findings in anogenital distance considered to be related to the test item at 5, 20 or 75 mg bw/kg/day.

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Changes in the absolute and relative to body weights related to the administration of 2-Amylanthraquinone were observed at 20 or 75 mg/kg bw/day in the Cohort 1A and Cohort 1B animals. Test item-related organ weight differences are summarised in Table 5.
At 75 mg/kg bw/day there were higher kidney and liver weights in the Cohort 1A and 1B animals, as well as higher spleen weights in the Cohort 1B animals and Cohort 1A females, when compared with the control. At 20 mg/kg bw/day there were higher kidney weights in the Cohort 1B males and higher liver weights in the Cohort 1B males. when compared with the control.
The liver weight changes at 75 mg/kg bw/day in the Cohort 1A animals correlated with hepatocellular hypertrophy.
There no findings considered to be related to the test item at 20 mg/kg bw/day in the Cohort 1A animals or Cohort 1B females or at 5 mg/kg bw/day in the Cohort 1A or Cohort 1B animals. There were other individual organ weight values that were different from their respective controls, however, there were no patterns or correlating data (taking into account differences in sexual maturity) to suggest these values were test item-related.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross findings in the culled F1 pups on PND4, unselected F1 animals on PND22-24, Cohort 1A animals or Cohort 1B animals. Gross findings observed were of the nature commonly observed in this strain and age of rat, or occurred at a similar incidence in control and treated animals, and, therefore, were considered not to be test item-related.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were test item-related histopathological findings at 75 mg/kg/day in the Cohort 1A animals. These findings have been summarised in Table 6.
At 75 mg/kg bw/day in the liver, there were incidences of minimal hepatocellular hypertrophy in the Cohort 1A males and females. At 75 mg/kg bw/day in the kidney there were higher incidences of minimal focal mineralization and minimal/mild accumulation of hyaline droplets in the Cohort 1A males and minimal/mild tubular basophilia in the Cohort 1A males and females.
There were no findings considered to be related to the test item at 5, 20, or 75 mg/kg bw/day in the unselected F1 pups on PND22-24, or at 5 or 20 mg/kg bw/day in the Cohort 1A males and females. All other microscopic findings observed were of the nature commonly observed in this strain and age of rat, or occurred at a similar incidence in control and treated animals, and, therefore, were considered not to be test item related.

Ovarian follicle counts
There were no findings in the ovarian follicle counts that were considered to be related to the test item at 5, 20 or 75 mg/kg bw/day of 2-Amylanthraquinone in the Cohort 1A females. Additionally, there were no findings in the histopathology examination of the ovaries to indicate an increase in degenerating ovarian follicles. Therefore although there were mean ovarian follicles counts that were statistically significantly different from their respective controls, these differences were considered to be within normal biological variation for this species.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

THYROID HORMONE ANALYSIS F1
Changes in the thyroid hormones related to the administration of 2-Amylanthraquinone were observed at 75 mg/kg bw/day in the Cohort 1A males. The levels of T3, T4 and TSH at 75 mg/kg/day in the Cohort 1A animals are summaries in Table 4.
At 75 mg/kg bw/day there were higher levels of TSH in the Cohort 1A males. There were no test item related effects at 5, 20 or 75 mg/kg bw/day of 2-Amylanthraquinone in the F1 pups culled on PND 4 or the unselected F1 pups on PND 22-24 or the Cohort 1A females. There were also no test item related effects at 5 or 20 mg/kg bw/day in the in the Cohort 1A males. All other values were considered to be within normal biological variation when compared with the controls.

SPERM MEASURES
There were no findings in the sperm straight line velocity or the percentage of motile or progressive motile sperm considered to be related to the test item at 5, 20 or 75 mg/kg bw/day
2-Amylanthraquinone in the Cohort 1A males. There were no effects on sperm morphology, sperm counts or spermatid counts considered to be related to the test item at 5, 20 or 75 mg/kg bw/day 2-Amylanthraquinone in the Cohort 1A males.

IMMUNOPHENOTYPING
There were no test item, dose dependent or sex related effects observed for any of the immune cell populations analysed within the rat spleens at 5, 20 or 75 mg/kg bw/day (Cohort 1A).

Details on results (F1)

See also Details on results (P0)
At 75 mg/kg bw/day there were test-item-related mild/minimal findings in the kidney of focal
mineralization in the Cohort 1A animals as well as accumulation of hyaline droplets and
tubular basophilia in the Cohort 1A males. In the kidney, hyaline droplet accumulation can be
induced by xenobiotics (including some hydrocarbons) that disrupt the lysosomal catabolism
of alpha-2u-globulin in the renal proximal tubules (Alden 1986, Frazier et al 2012). Because
this globulin is synthesised in especially large amounts by the male rat liver and is not present
in humans, the finding is not considered relevant to humans. These findings were therefore
considered not to be adverse as they were mild/minimal in severity and were not associated
any degenerative changes. The accumulation of hyaline droplet could be associated with the
higher kidney weights in the Cohort 1A males.

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of effects on developmental parameters.

Target system / organ toxicity (F1)

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:

not specified

2

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:

not specified

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

DOSE FORMULATION ANALYSES

A summary of the analysis of the dose formulation samples taken in study week 1, 14 and 25 is given in Table 1 below.

No test item was detected in the formulations for the control group (0 mg/mL).

 

Table 1: Results of dose formulation analysis

Dose concentration (mg/mL)	Accuracy range (%)	Mean Accuracy (%)	Homogeneity (coefficient of variation [%])
Week 1
0	n.a.	n.a.	n.a.
1	95-98	97	1.2
4	101-105	104	1.5
25	89-98	94	3.3
Week 14
0	n.a.	n.a.	n.a.
1	91-97	95	2.1
4	86-94	91	3.3
25	84-93	90	4.3
Week 25
0	n.a.	n.a	n.a.
1	101-102	102	0.59
4	91-98	96	3.1
25	95-98	97	1.0

In conclusion, all formulations were within the acceptance criteria (mean concentration within 15%, with a relative standard deviation <10%). No test item was detected in the control groups formulations. Dose levels were thus adequate.

 

HAEMATOLOGY  

Table 2 Haematology Findings in the F0 and Cohort 1A animals

	F0		Cohort 1A
	Males	Females	Males	Females
Dose Level (mg/kg/day	75	75	75	75
Haemoglobin (g/L)	x0.92	-	x0.92	-
Haematocrit (L/L)	x0.93	-	x0.94	-
Red Blood Cells (x10^12/L)	x0.93	x0.89	x0.92	-
White Blood Cells (x109/L)	x1.13	-	-	-
Monocytes (x10^9/L)	x1.30	-	-	-
Reticulocyte (x10^9/L)	x1.40	x1.57	-	-
Red Blood Cell Distribution Width (%)^a	x1.14	-	x1.16	-
Platelets (10^9/L)	-	-	x1.10	-
Reticulocyte count (10^9/L)	-	-	x1.27	x1.15
Lymphocytes (x10^9/L)	-	x1.14	-	-
Mean Corpuscular Volume (fL)	-	x1.05	-	-
Mean Corpuscular Heamoglobin (pg)	-	x1.06	-	-
A dash (-) indicates absence of a test item related change. Numerical value indicates fold difference of group values relative to vehicle control group value. Bolded values were statistically significant from the control group (P ≤ 0.05). a – describes the variation in the size of the red blood cells

 

 

 

CLINICAL CHEMISTRY

Table 3 Clinical Chemistry Findings F0 and Cohort 1A animals

	F0			Cohort 1A
	Males	Females		Males	Females
Dose Level (mg/kg/day)	75	20	75	75	5	20	75
Triglycerides (mmol/L)	-	x0.59	x0.53	x0.73	-	-	-
Albumin (g/L)	x1.10	-	-	-	x1.05	x1.10	x1.14
Globulin (g/L)	x0.82	-	x0.83	-	-	-	x0.96
Albumin/Globulin (ratio)	x1.34	-	x1.28	-	-	x1.09	x1.19
Total Protein (g/L)	-	-	-	-	x1.06	x1.08	x1.08
A dash (-) indicates absence of test item related change. Numerical value indicates fold difference of group values relative to vehicle control group value. Bolded values were statistically significant from the control group (P ≤ 0.05).

 

THYROID HORMONE ANALYSIS

Table 4: TSH, T3 and T4 Findings F0 and Cohort 1A animals

	F0		Cohort 1A
	Males	Females	Males	Females
Dose Level (mg/kg/day)	75	75	75	75
T3 (ng/mL)	x0.79	x0.80	x0.94	x1.36
TSH (ng/mL)	x2.37	x1.84	x2.53	x1.23
T4 (ng/mL)	x0.78	x0.90	x0.92	x0.97
Bolded values were statistically significant from the control group (P ≤ 0.05).

 

ORGAN WEIGHTS

Table 5: Summary Group Mean Organ Weight Data – F0, Cohort 1A and Cohort 1B animals.

	F0				Cohort 1A		Cohort 1B
	Male		Female		Male	Female	Male		Female
Dose Level (mg/kg/day)	20	75	20	75	75	75	20	75	75
Kidney
Absolute Value	-	19.7	-	10.9	12.7	9.29	-	16.1	12.7
% of body weight	9.29	25.7	-	9.83	13.2	7.62	8.92	20.8	8.69
Liver
Absolute Value	-	33.7	-	15.8	27.7	36.5	-	40.0	27.9
% of body weight	-	40.2	-	15.1	29.4	34.7	10.0	45.2	30.1
Spleen
Absolute Value	-	10.2	-	18.4	-	18.4	-	-	18.1
% of body weight	-	15.9	-	17.0	-	16.1	-	19.1	19.7
Thyroid
Absolute Value	-	30.3	-	-	-	-	-	-	-
% of body weight	20.4	36.9	-	-	-	-	-	-	-
Pituitary Gland
Absolute Value	-	-	17.3	17.1	-	-	-	-	-
% of body weight	-	-	15.6	15.2	-	-	-	-	-
A dash (-) indicates absence of test item related change. Numerical value indicates a percentage difference (%) of group values relative to vehicle control group value. Bolded values were statistically significant from the control group (P ≤ 0.05).

 

HISTOPATHOLOGY

Table 6: Summary Test Item-related Microscopic Findings – F0 and Cohort 1A animals.

	F0			Cohort 1A
	Males*			Males		Female
Dose Level (mg/kg/day)	0	20	75	0	75	0	75
Liver (No. Examined)	(25)	(25)	(25)	(20)	(20)	(20)	(20)
Minimal focal hepatocellular degeneration	0	1	2	-	-	-	-
Minimal hepatocellular hypertrophy	0	-	9	0	5	0	8
Mild focal hepatocellular necrosis	0	-	1	-	-	-	-
Kidney (No. Examined)	(25)	(25)	(25)	(20)	(20)	(20)	(20)
minimal focal mineralization	-	-	-	2	10	-	-
minimal/mild tubular basophilia	-	-	-	4	16	4	7
minimal/mild accumulation of hyaline droplets	-	-	-	11	20	-	-
A dash (-) indicated the absence of test item related change. Where a test item related change was observed the number of incidence in the control was also included in the table to display the difference in the number of incidences in the control compared to the test item group. *In F0 females, like for F0 males no notable changes in histopathology findings  relative to the control animals were done.

 

Applicant's summary and conclusion

Conclusions:

Based on the results of an extended one generation reproduction toxicity study with 2-Amylanthraquinone in rats performed according to OECD guideline 443 and following GLP principles, the NOAEL for systemic toxicity for the F0 and the F1 generation was 20 mg/kg bw/day. The NOAEL for reproduction toxicity was 75 mg/kg bw/day. The NOAEL for F1 developmental toxicity was at least 75 mg/kg bw/day.

Executive summary:

An extended one generation study was performed in rats with 2-Amylanthraquinone according to OECD guideline 443 and under GLP principle. Male and female rats from the F0 and F1 generation were exposed to the test substance by oral gavage at dose levels of 5, 20 or 75 mg/kg bw/day. The dose levels were established based on a 90-day repeated dose toxicity study. Dose formulation analyses were performed and demonstrated accurate dosing.

F0 males were treated daily for 10 weeks prior to mating until necropsy after the termination of the F0 females. F0 females were treated daily for 10 weeks prior to mating, then through mating, gestation and until at least Lactation Day (LD) 21. Cohort 1A and 1B animals were then dosed daily from Postnatal Day (PND) 21 until at least PND 90.

The following parameters and end points were evaluated in this study: clinical observations, body weights, food consumption, estrous cycling, mating performance, observations of females and litters during lactation, pre-weaning physical development of F1 animals, clinical pathology parameters (haematology, coagulation, clinical chemistry, urinalysis, thyroid stimulating hormone, T3 and T4), gross necropsy findings, organ weights, sperm evaluations and histopathological examinations.

There was a dose dependent increase in the incidence of ploughing and/or salivation at ≥ 5 mg/kg bw/day in the F0, Cohort 1A and 1B animals. These findings were predominantly observed immediately post-dose.

At 75 mg/kg bw/day there were higher numbers of reticulocytes in F0 generation animals and Cohort 1A males and this correlated with lower levels of red blood cells; an increase in red blood cell distribution width and correspondingly lower levels of haemoglobin and haematocrit. Additionally, in the F0 generation females there were high mean corpuscular volume and corpuscular haemoglobin which could be related to the lower levels of red blood cells. At 75 mg/kg bw/day there were higher levels of monocytes in the F0 males, platelets in the Cohort 1A males and lymphocytes in the F0 females the changes, in the F0 males corresponded with a higher level of white blood cells.
At 75 mg/kg bw/day there were lower levels of fibrinogen in the F0 and Cohort 1A males. There were higher levels of albumin at 75 mg/kg bw/day in the F0 males and at ≥ 5 mg/kg bw/day in the Cohort 1A females as well as lower level of globulin at 75 mg/kg bw/day in the F0 animals and Cohort 1A females. The changes in albumin and globulin corresponded with a higher albumin/globulin ratio at 75 mg/kg bw/day in the F0 animals and Cohort 1A females as well as a higher total protein levels at ≥ 5 mg/kg bw/day in the Cohort 1A females. Additionally, there were lower levels of triglycerides at ≥ 20 mg/kg/day in the F0 females and 75 mg/kg bw/day in the Cohort 1A males.
The changes in the haematology, coagulation and clinical chemistry parameters were considered not to be adverse as they were mild to moderate in severity and there were no associated adverse histopathological or toxicology effects.

At 75 mg/kg bw/day there were test item-related histological findings in the kidney of focal mineralization and accumulation of hyaline droplets the Cohort 1A males and tubular basophilia in the Cohort 1A males and females. There were also higher kidney weights in the F0, Cohort 1A and 1B animals at 75 mg/kg bw/day and at 20 mg/kg bw/day in F0 and Cohort 1B males. These changes were considered not to be adverse as they were not associated with any adverse histopathological changes.
In the liver at ≥ 20 mg/kg bw/day test item-related histological findings of focal hepatocellular degeneration in the F0 males. Additionally, at 75 mg/kg bw/day there were test item-related histological findings in the liver of hepatocellular hypertrophy in the F0 males and Cohort 1A animals as well as focal hepatocellular necrosis in the F0 males. The histological findings in the liver were considered to be mild or minimal changes therefore considered not to be adverse. Additionally, there were also higher liver weights in the F0, Cohort 1A and 1B animals at 75 mg/kg bw/day and in the Cohort 1B males at 20 mg/kg bw/day. These changes in the weights of the liver were not considered to be adverse as they were not associated with any adverse histopathological changes.
There were higher spleen weights at 75 mg/kg bw/day in the F0 animals, Cohort 1B animals and Cohort 1A females and higher pituitary gland weights at ≥ 20 mg/kg bw/day in the F0 females. There were no associated gross pathological or histological findings with these organ weight changes therefore they were considered not to be adverse.

At 75 mg/kg bw/day there were lower levels of T4 in the F0 males and T3 in the F0 animals. There were also higher levels of TSH at 75 mg/kg bw/day in the F0 animals and Cohort 1A males. The changes in the levels of the thyroid hormones was associated with a higher thyroid weight at 75 mg/kg bw/day in the F0 males.

Given the trend of increased liver weights described above, the thyroid changes may be also related to these changes. The changes in the thyroid hormone levels were considered to be related to the test item however, under the conditions of this study no adverse effects could be linked to the changes in the thyroid hormones therefore this change was not taken into account when determining the parental NOAEL.
There were no treatment related findings related to bodyweights or food consumption in the F0 generation or Cohort 1A and 1B animals. There were no treatment related effects seen estrous cycling, mating performance, observations of females and litters during lactation, preweaning physical development of F1 animals or sexual maturation of the F1 animals. There were also no effects on the number of ovarian follicles in the ovaries of the F0 or Cohort 1A females.

In conclusion, the administration of 2-Amylanthraquinone by once daily oral gavage was considered to have a systemic No Observed Adverse Effect Level (NOAEL) of 20 mg/kg bw/day for the F0 and F1 generation, due to the higher organ weights of the kidney and liver and associated minimal to mild histopathology  findings in both liver and kidney, as well as mild to moderate changes in the haematology and clinical pathology parameters at 75 mg/kg bw/day. Findings in the kidney were minimal mineralisation and minimal to mild tubular basophilia; findings in the liver were minimal hepatocellular hypertrophy and degeneration, especially in males. Although individually, these findings were considered not to be adverse and did not indicate significant organ damage or impaired function, the combined changes within these parameters were considered to be adverse at 75 mg/kg bw/day.
The NOAEL for reproduction toxicity was considered to be at least 75 mg/kg bw/day as there was no adverse effect on any of the reproductive endpoints examined within this study. The NOAEL for F1 developmental toxicity was considered to be at least 75 mg/kg bw/day as there was no adverse effect on the developmental endpoints examined within the study.