Reaction mass of Amines, C10-14-branched and linear alkyl, [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]chromate(1-) and Amines, C10-14-branched and linear alkyl, bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)]chromate(1-) and Amines, C10-14-branched and linear alkyl, bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]chromate(1-)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 May 2017 -

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 May 2017 -

Reliability:

1 (reliable without restriction)

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Identification: Orasol Black X51
- Appearance: Black solid
- EC Number: 939-191-9
- Batch: 001-150606
- Test Facility Test Item Number: 208107/A
- Purity/Composition: Approx. 95%
- Test item storage: At room temperature
- Stable under storage conditions until: 26 May 2020 (expiry date)

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: At initiation of dosing, males were 11 weeks and females were 12 weeks old.
- Weight at study initiation: At initiation of dosing, males weighed between 268 g and 305 g and females weighed between 194 g and 248 g.
- Housing: On arrival and following the pre-test (females only) and pre-mating period, Main animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). Recovery males and females were housed as such during the entire study period. During the mating phase, Main males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, Main males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Main Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). During the lactation phase, Main females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not be left without their dam or a bottle filled with warm water for longer than 30-40 minutes. 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. The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water: Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours.
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for 7 days prior to start of the pre-test period (females) or 7 days before the commencement of dosing (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 21
- Humidity (%): 50 to 76
- Air changes (per hr): 10 or greater with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 5 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.

VEHICLE
- Concentration in vehicle: 1, 3 and 10 mg/mL dosing solutions correspond to dose levels of 5, 15 and 50 mg/kg bw/day respectively
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected during week 1 of the pre-mating period. Concentrations were determined for all groups, homogenicity for groups 2 and 4 (The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 preparations were averaged and utilized as the concentration results). All samples were stored on dry ice immediately after sampling and shipped. Upon receipt at the analytical laboratory, the samples were stored in the ultra-low freezer ≤ -70°C until analysis. Analyses were performed by using a validated analytical procedure.
- Concentration analysis: Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to 15% for suspensions of target concentration.
- Homogeneity analysis: Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.
- Stability analyses performed previously in conjunction with the method development and validation study 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:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days. Main males and Recovery males were treated for 29 days, including at least 2 weeks of treatment prior to mating and during the mating period for Main males. For both Main and Recovery males treatment ended one day before scheduled necropsy of Main males. Main females that delivered were treated for 42 to 56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Recovery females were treated during the same period as Main females, until one day before the first scheduled necropsy of Main Group 4 females. Females which failed to deliver or had a total litter loss were treated for 42 to 45 days.

Frequency of treatment:

once daily

Dose / conc.:

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

Remarks:

Group 2

Dose / conc.:

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

Remarks:

Group 3

Dose / conc.:

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

Remarks:

Group 4

No. of animals per sex per dose:

10 animals per dose per sex (main test) or 5 animals per dose per sex (additional recovery test groups: group 1 and group 4)

Control animals:

yes, concurrent vehicle

Details on study design:

Basis for dose level selection: The dose levels were selected based on the results of a 14-day dose range finder with oral exposure in rat.
For the control group (group 1) and high dose group (group 4) 5 recovery animals (of each sex) were used to study potential reversibility of possible toxic effects. The recovery period was 15 days.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from their cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS:
Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing and recovery periods up to the day prior to necropsy. During the dosing period, these observations were performed 1 hour (± 30 min) post-dose, based on the peak effect of occurrence of clinical signs observed in the dose range finder. The time of onset, grade and duration of any observed sign was 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. Clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment and recovery. These observations were initiated 1 hour (± 30 min) post-dose.

BODY WEIGHT:
Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated Main females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION
Food consumption was quantitatively measured weekly, except for Main males and Main females which were housed together for mating and for Main females without evidence of mating. Food consumption of mated Main females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

FUNCTIONAL TESTS
Functional tests were performed on the selected 5 Main males and all Recovery males during Week 4 of treatment, on the selected 5 Main Group 1-3 females during the last week of lactation (i.e. PND 8-12), on the selected 5 Main Group 4 females during Week 6 of treatment, and on all Recovery females on the first day a Main female was tested. These tests were initiated 1 hour (± 30 min) post-dose, after completion of clinical observations (including arena observation, if applicable). The following tests were performed: Hearing ability, Pupillary reflex, Static righting reflex, Fore- and hind-limb grip strength and Locomotor activity.

HAEMATOLOGY
- Time schedule for collection of blood: Blood was collected on the day of scheduled necropsy.
- Anaesthetic used for blood collection: Yes (isoflurane
- Animals fasted: Yes
- How many animals: 5 animals per sex of the main group, all animals of the recovery group
- Blood samples were analyzed for the following parameters: White blood cells (WBC), Red Blood Cell Distribution Width (RDW), Neutrophil (absolute), Haemoglobin, Lymphocyte (absolute), Haematocrit, Monocyte (absolute), Mean corpuscular volume (MCV), Eosinophil (absolute), Mean corpuscular haemoglobin (MCH), Basophil (absolute), Mean corpuscular haemoglobin concentration (MCHC), Red blood cells, Platelets and Reticulocyte (absolute).
- In addition the following coagulation parameters were assessed: Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT)

CLINICAL CHEMISTRY
- Time schedule for collection of blood: Blood was collected on the day of scheduled necropsy.
- Animals fasted: Yes
- How many animals: 5 animals per sex of the main group, all animals of the recovery group
- Blood samples were processed for plasma or serum (bile acids), which was analyzed for the following parameters: Alanine aminotransferase (ALAT), Creatinine, Aspartate aminotransferase (ASAT), Glucose, Alkaline Phosphatase (ALP), Cholesterol, Gamma glutamyl transpeptidase (GGT), Sodium, Total protein, Potassium, Albumin, Chloride, Total Bilirubin, Calcium, Bile Acids, Inorganic Phosphate (Inorg. Phos), Urea.

THYROID HORMONE
- Time schedule for collection of blood: Blood was collected on the day of scheduled necropsy.
- Animals fasted: Yes
- How many animals: all animals main group, all animals of the recovery group
- Blood samples were processed for serum, and serum was analyzed for Thyroxine (T4) and Thyroid Stimulating Hormone (TSH) by using the IMMULITE® 1000 immunoassay-system.

Sacrifice and pathology:

SACRIFICE
Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. Scheduled necropsies were conducted on the following days:
- Main Males (which sired or failed to sire): Following completion of the mating period (a minimum of 28 days of administration).
- Recovery males: After the recovery period of 15 days, which was 15 days after the scheduled necropsy of Main males.
- Main females which delivered: PND 14 or 16.
- Main females which failed to deliver but showed evidence of mating: Post-coitum Days 25-26.
- Main female with total litter loss (one female in group 3): Dam with no surviving pups was euthanized within 24 hours after the last pup was found dead or missing.
- Recovery females: At least 14 days after the first dose-free day of the Recovery females.
Except for the female with total litter loss, all animals surviving to scheduled necropsy were fasted (overnight with a maximum of 24 hours) before their scheduled necropsy.

GROSS NECROPSY
- A necropsy was conducted for animals that died on study, and specified tissues were saved.
- All animals (both Main and Recovery) were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
- The numbers of former implantation sites were recorded for all paired Main females. In case no macroscopically visible implantation sites were present in Main females, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

TISSUE COLLECTION
The following tissues and organs were collected for all selected Main animals, all Recovery animals, and all animals that died spontaneously: Artery aorta, Body cavity nasopharynx, Bone marrow (femur), Bone sternum, Brain (seven levels), Cervix, Epididymis, Esophagus, Eye, Gland adrenal, Gland coagulation, Gland harderian, Gland lacrimal, Gland mammary, Gland parathyroid, Gland pituitary, Gland prostate, Gland salivary (mandibular and sublingual), Gland seminal vesicle, Gland thyroid, Gross lesions/masses, Gut-associated lymphoid tissue, Heart, Kidney, Large intestine cecum, Large intestine colon, Large intestine rectum, Larynx, Liver, Lung, Lymph node (axillary and mesenteric site), Muscle skeletal, Nerve optic, Nerve sciatic, Nose (nasal cavity), Ovaries, Oviducts, Pancreas, Pharynx, Small intestine duodenum, Small intestine ileum, Small intestine, jejunum, Spinal cord, Spleen, Stomach (forestomach and glandular stomach), Testes, Thymus, Tongue, Trachea, Urinary bladder, Uterus and Vagina.

ORGAN WEIGHTS
- The following organs were weighted at necropsy for all scheduled euthanasia animals:
Cervixa (Weighed together with the uterus), Epididymides, Gland coagulation (Weighed together with the seminal vesicles, Paired organ weight.), Gland parathyroid (Weighed together with the thyroid), Gland prostate, Gland seminal vesicle, Gland thyroid, Ovaries (Paired organ weight), Testes (Paired organ weight), Uterus.
- Organs Weighed at Necropsy for al selected main animals and recovery animals sacrificed on schedule: Brain, Gland adrenal (Paired organ weight), Heart, Kidneys, Liver, Spleen and Thymus.

HISTOLOGY
The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
- Selected Main animals, all Recovery animals and unscheduled deaths (found dead): Tissues identified under tissue collection (except animal aorta, esophagus, harderian gland, lacrimal gland, nasopharynx, mammary gland (female), salivary gland, larynx, optic nerve, pancreas, tongue).
- Males that failed to sire (except for males which were selected), females that failed to deliver pups, and the female with total litter loss: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina.
- Female with total litter loss: Mammary gland.
- Remaining animals: Gross lesions/masses.

HISTOPATHOLOGY
- Reproductive organs (cervix, coagulation gland, epididymides, ovaries, prostate gland, seminal vesicles, testes, uterus, and vagina): All Main animals of the control and high dose group, all males that failed to sire to examine staging of spermatogenesis and histopathology of interstitial cell structure, all females that failed to deliver pups.
- For ovaries, a qualitative evaluation of 1 section from each ovary was made.
- For testes, a detailed qualitative examination 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 stagespecificity of testicular findings were noted.
- Remaining organs: First five males and lactating females of the control and high dose group, gross lesions for all Main and Recovery animals.

Statistics:

- All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
- Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations.
- The following pairwise comparisons were made: Group 2 vs. Group 1, Group 3 vs. Group 1, roup 4 vs. Group 1
- Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
- Non-Parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. The Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group.
- Incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- At 15 and 50 mg/kg, piloerection was recorded for females, starting during the mating phase, with a dose-related increase in incidence. At 15 mg/kg bw/day, this finding resolved prior to initiation of the recovery phase, while at 50 mg/kg bw/day this resolved in the first week of the recovery period.
- Black discoloration of faeces and/or a general blue discoloration of the skin was noted in both sexes at 15 and 50 mg/kg bw/day. The onset of these findings showed a dose-related trend and lasted until the end of the recovery phase at 50 mg/kg bw/day.
- Blue discolouration of the eyes was recorded for both sexes only at 15 mg/kg bw/day during three consecutive days of the premating period. This limited occurrence at 15 mg/kg bw/day may be due to an easier detection of discolouration of eyes compared to other body parts, while at 50 mg/kg bw/day generalized discolouration was more pronounced/sudden without being first detected in the eyes. This discoloration is considered to be related to the black color of the test item and not to represent a sign of toxicity.
- No findings were noted during the weekly arena observations in this study.
- No clinical signs were noted at 5 mg/kg bw/day. Incidental findings that were noted in other dose groups 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. At the incidence observed, these were not considered to be signs of toxicological relevance.

Mortality:

mortality observed, treatment-related

Description (incidence):

- At 50 mg/kg bw/day, a total of 3 Main group females were found dead. One female was found dead on Day 36 prior to dosing (i.e. after 35 days of treatment). No relevant signs were noted prior to the spontaneous death for this animal. Apart from autolysis and many black foci noted on the thymus, main macroscopic and microscopic findings were comparable to the remaining animals of this treatment group and consisted of black discoloration of all organs. No clear cause of death could be established histopathologically for this animal.
- In addition, two Main females at 50 mg/kg bw/day died at terminal blood sampling (prior to scheduled necropsy). No relevant signs were noted for these animals and no cause of death could be established histopathologically. Although it is conceivable that these deaths could be related to the blood sampling procedure/anaesthesia, given that in total 3 high dose Main females were found dead in this study, a possible treatment-related effect could not be ruled out.
- One male at 15 mg/kg bw/day was found dead on the last day of treatment (on Day 29). No relevant signs were noted prior to the spontaneous death for this animal. Apart from autolysis, main macroscopic and microscopic findings were comparable to the remaining animals of this treatment group and consisted of black discoloration of all organs. In addition, an enlarged liver and thoracic cavity filled with black, watery-clear fluid was observed. The latter finding may point to a gavage-related incident although histopathologically a cause of death for this animal could not be confirmed. This death was regarded incidental and unrelated to the treatment with the test item.
- One Main female at 15 mg/kg bw/day was sacrificed due to total litter loss on PND 5; two of her pups were found dead on Day 2 and the other two pups were sacrificed in extremis on Day 5.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- At 50 mg/kg bw/day, a statistically significantly lower body weight and body weight gain was recorded for females from Day 4 or 7 of the post-coitum period onwards, respectively. Slight body weight loss (on average approximately 6% based on mean body weight) was recorded for all these females between Days 17 and 20 of the post-coitum period (along with a more pronounced decrease in food intake). These effects on body weight and food intake were considered to have occurred secondary to the hampered fetal development (none of the females at this dose delivered offspring).
- At 15 mg/kg bw/day, body weight and body weight gain of females was lower than controls from the second week of the post-coitum period onwards, achieving a level of statistical significance on most occasions.
- The statistically significantly lower mean body weights and body weight gains of males at 5 and 50 mg/kg bw/day during the premating and repro period occurred in the absence of a dose-related trend; the mean body weight of the 5 and 50 mg/kg bw/day dose groups at the end of treatment were similar, and the mean body weight and body weight gain at the middose of 15 mg/kg bw/day was similar to the control mean. As such, these variations were not considered to be related to treatment. The subsequent lower starting body weight on Day 1 of the recovery phase was considered to have resulted in statistically significantly lower body weights and body weight gain for males during the recovery period. Body weight gain over Days 1-15 of the recovery period for males at 50 mg/kg bw/day based on Day 1 of the recovery period was the same as for control males (i.e. 13%).
- Occasional statistically significantly lower mean body weight gain recorded during the treatment and recovery period for females at 50 mg/kg bw/day was not consistently seen with continuation of treatment. Also, mean body weight at the end of recovery was similar to the control mean body weight. Therefore, these variations were not considered to represent an effect of treatment.
- see table 1 and 2

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- At 50 mg/kg bw/day, a lower food consumption (before and after correction for body weight) was recorded for females throughout the post-coitum period, achieving a level of statistical significance on most occasions. The difference in food consumption to the control mean became more pronounced over Days 17-20 of the post-coitum period.
- At 15 mg/kg bw/day, food consumption of females before and after correction for body weight was statistically significantly lower than controls during the last week of lactation.
- Variations in food consumption across male dose groups were not considered to represent an effect of treatment since these occurred in the absence of a dose-related trend and since these variations were of a minor degree.
- Any other (statistically significant) variations in food consumption were not considered to be related to treatment as these occurred in the absence of a dose-related trend, were of a minor degree and/or were not consistently recorded with continuation of treatment.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant, non-adverse changes in haematological parameters at 50 mg/kg bw/day distinguished treated from control animals at the end of treatment:
- Lower red blood cell counts in males (0.94x of control).
- Higher reticulocyte counts in males and Recovery group females (1.5x and 1.65x of control for males and females, respectively).
- Higher red blood cell distribution width (RDW) in males and Recovery group females (1.12x of control for males and females).
- Higher mean corpuscular haemoglobin (MCH) in males (1.05x of control.
- Higher mean corpuscular haemoglobin concentration (MCHC) in males (1.03x of control).
- Higher lymphocyte counts in females (1.35x of control).

At the end of recovery, mean corpuscular volume (MCV; 1.08x of controls) and mean corpuscular haemoglobin (1.06x of control) were (still) higher in males at 50 mg/kg bw/day, while affected parameters in Recovery group females had returned to control values.
The statistically significantly lower or higher lymphocyte counts in Main females at 15 and 50 mg/kg bw/day respectively at the end of treatment were not considered to be related to treatment as they did not show a dose-related trend (Groups 1-3, i.e. lactating females), were of a minor degree and were attributed to physiological differences due to pregnancy status between Main control females (lactating) and Main females at 50 mg/kg bw/day (implantation sites only).

Coagulation parameters of treated rats were not considered to have been affected by treatment.
The statistically significantly lower prothrombin time (PT) in males at 50 mg/kg bw/day at the end of recovery was absent at the end of the treatment period, and not considered toxicologically relevant given the direction of change (i.e. a more pronounced and opposite effect would be expected in case of target organ toxicity). As such, this change was not considered to be related to treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- The following (statistically significant, non-adverse) changes in clinical biochemistry parameters distinguished treated from control animals at the end of treatment:
1. Lower total protein in males and Recovery females at 50 mg/kg bw/day (0.92x and 0.95x of control for males and females, respectively)
2. Lower chloride in females at 15 mg/kg bw/day (0.95x of control).
3. Higher inorganic phosphate in non-lactating females at 50 mg/kg bw/day.

- At the end of the recovery phase, total protein remained lower for Recovery group females of the 50 mg/kg bw/day group (0.95x of control).
- Grey/black discoloration of blood samples was considered to have affected total bilirubin and cholesterol measurements. This resulted in a statistically significantly lower total bilirubin level in Main females at 5 mg/kg bw/day at the end of treatment (0.37x of control), and in Recovery males (0.45x of control) and Recovery females (0.58x of control) at 50 mg/kg bw/day at the end of recovery. Total bilirubin could not be determined at dose levels of 15 and 50 mg/kg bw/day at the end of treatment, and bile acids could not be determined at 50 mg/kg bw/day in most animals at the end of treatment. Blood sample discolouration was also considered to have resulted in lower cholesterol in males (0.38x of control) and non-lactating females (0.33x of controls) at 50 mg/kg bw/day at the end of treatment.
- Lower alanine aminotransferase (ALAT), aspartate aminotransferase activity (ASAT) and/or alkaline phosphatase activity (ALP) in males and/or females at 50 mg/kg bw/day at end of treatment or end of recovery were not considered to be toxicologically relevant since the opposite effect would be expected in case of target organ toxicity.
- 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 or were only recorded at the end of recovery (higher albumin and creatinine in males at 15 mg/kg bw/day at end of treatment, lower chloride in females at 15 mg/kg bw/day at end of treatment, and lower inorganic phosphate and calcium in Recovery females at 50 mg/kg bw/day at end of recovery).

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- At 50 mg/kg bw/day, a statistically significantly, non-adverse lower foreleg grip strength was recorded for Main females at the end of treatment (0.60x of control). It should be noted that these high dose females had implantation sites only, while selected females of Groups 1, 2 and 3 were all lactating at the time point of functional tests. Therefore values of Group 4 were compared with historical data from non-mated rats of this age and strain. Mean value for foreleg grip strength was at the lower end of the historical control range. However, the lower foreleg grip strength was considered not to be toxicologically relevant as fore leg grip strength values in nulliparous 50 mg/kg/day/day females (Recovery animals) were within the normal range for female rats of this strain and age. Moreover, there were no corroborating changes in hind limb grip strength or other end points in the neuromuscular domain.
- At 15 and 50 mg/kg bw/day (males) and 50 mg/kg (Recovery females and non-lactating Main females combined), a statistically significantly lower motor activity (total movements) was recorded at the end of treatment (0.70x and 0.72x of control for males at 15 and 50 mg/kg bw/day, respectively, and 0.91x of control for females at 50 mg/kg bw/day). Mean counts for ambulations also appeared lower than controls for males at 15 and 50 mg/kg bw/day (0.67x of control for both dose levels), but were not statistically significant. No toxicological relevance was attached to these findings because of the following reasons: all groups showed a similar motor activity habituation profile (with high activity in the first interval that decreased over the duration of the test period); relatively high values were observed for the control group; no clear dose-related trend was recorded for males, and for females the difference to the control mean was slight; means for both treated males and females remained within the range considered normal for rats of this age and strain.
- Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- A statistically significant test-item-related, non-adverse increase in liver weight (relative to body weight) was noted in 15 mg/kg bw/day and 50 mg/kg bw/day Main males. This was not present after the 15-day recovery period. There was no microscopic correlate. see table 3
- Any differences in absolute and relative organ weights of Main females at 50 mg/kg bw/day compared to control means were attributed to differences in pregnancy status and/or resulting lower body weights, and were therefore not considered to be related to the test-item.
- Any other differences, including those that reached statistical significance were considered not to treatment related due to absence of a similar change at the end of treatment and/or general overlap and/or variability in individual values.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were non-adverse, test item-related macroscopic findings starting in females at 5 mg/kg bw/day and starting in males at 15 mg/kg bw/day, consisting of:
- Liver, bluish discoloration in 3/10 females of the 5 mg/kg bw/day group, without microscopic correlate.
- General observation, black or bluish discoloration of all organs in both sexes of the 15 and 50 mg/kg bw/day group (including the premature decedents and accidental deaths). This finding was still present in all Recovery rats of the 50 mg/kg bw/day group.
- Preputial glands, several black foci in 2/5 Recovery males of the 50 mg/kg bw/day group.
- Lung, several/many black foci in 4/7 scheduled sacrificed Main females and in 1/5 Recovery males of the 50 mg/kg bw/day group.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related, non-adverse microscopic findings after treatment were noted in the lung, spleen, testes and uterus:
- In the lungs, an increased incidence and severity of alveolar macrophage aggregations (up to moderate degree) was present in both sexes at 15 and 50 mg/kg bw/day. The single incidence at minimal degree at 5 mg/kg bw/day was regarded to be within background range. In general, at 50 mg/kg bw/day the macrophages had a foamier (finely vacuolated) appearance, without black pigment. This finding was still present up to moderate degree in males and up to slight degree in females after a 15-day treatment free period.
- In the spleen, an increased incidence and severity of extramedullary hematopoiesis (up to slight) was present in males of the 50 mg/kg bw/day group. After a 15-day treatment free period, incidences and severities were within background range.
- In th testes, black-brown pigmented macrophages in the interstitium of the testes (up to slight) were present at 50 mg/kg bw/day. This finding was still present up to slight degree in males after a 15-day treatment free period
- In the Uterus, black-brown pigmented macrophages at the implantation sites (slight degree) were present in females at 50 mg/kg bw/day.
All the above findings were considered as non-adverse, based on their mild characters and absence of any other test-item related changes indicative of tissue/organ damage.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.
- see table 4

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

THYROID HORMONE ANALYSIS
- At end of treatment, significantly higher serum levels of total T4 were noted in males at 15 and 50 mg/kg bw/day (1.3x and 1.2x of control, respectively), and in non-lactating females at 50 mg/kg bw/day (2.4x of control). Concurrently, there was a trend towards lower values of TSH in treated males as compared to concurrent controls (not statistically significant), but not in treated females. It should be noted that the variation in TSH level between animals within the same group was relatively high.
-Therefore, and since TSH levels were comparable in offspring at PND 13-15, an effect of the test compound on TSH levels in parental males is considered unlikely.
-At the end of the recovery period, serum levels of T4 and TSH in males and females at 50 mg/kg bw/day were comparable in all groups.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: whole body

Organ:

other: mortality

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Dose Formulation Analyses

- No test item was detected in the Group 1 (control group) formulation.

- The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with the target concentrations (i.e. mean accuracies between 85% and 115%).

- The formulations of Group 2 and Group 4 prepared were homogeneous (i.e. coefficient of variation ≤ 10%).

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Identification: Orasol Black X51
- Appearance: Black solid
- EC Number: 939-191-9
- Batch: 001-150606
- Test Facility Test Item Number: 208107/A
- Purity/Composition: Approx. 95%
- Test item storage: At room temperature
- Stable under storage conditions until: 26 May 2020 (expiry date)

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: At initiation of dosing, males were 11 weeks and females were 12 weeks old.
- Weight at study initiation: At initiation of dosing, males weighed between 268 g and 305 g and females weighed between 194 g and 248 g.
- Housing: On arrival and following the pre-test (females only) and pre-mating period, Main animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). Recovery males and females were housed as such during the entire study period. During the mating phase, Main males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, Main males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Main Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). During the lactation phase, Main females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not be left without their dam or a bottle filled with warm water for longer than 30-40 minutes. 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. The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water: Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours.
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for 7 days prior to start of the pre-test period (females) or 7 days before the commencement of dosing (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 21
- Humidity (%): 50 to 76
- Air changes (per hr): 10 or greater with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 5 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.

VEHICLE
- Concentration in vehicle: 1, 3 and 10 mg/mL dosing solutions correspond to dose levels of 5, 15 and 50 mg/kg bw/day respectively
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected during week 1 of the pre-mating period. Concentrations were determined for all groups, homogenicity for groups 2 and 4 (The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 preparations were averaged and utilized as the concentration results). All samples were stored on dry ice immediately after sampling and shipped. Upon receipt at the analytical laboratory, the samples were stored in the ultra-low freezer ≤ -70°C until analysis. Analyses were performed by using a validated analytical procedure.
- Concentration analysis: Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to 15% for suspensions of target concentration.
- Homogeneity analysis: Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.
- Stability analyses performed previously in conjunction with the method development and validation study 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.

Details on mating procedure:

- M/F ratio per cage:1:1 within the same treatment group, avoiding sibling mating.
- Length of cohabitation: Once mating had occurred, the males and females were separated.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- After successful mating each pregnant female was caged: Main Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days. Main males and Recovery males were treated for 29 days, including at least 2 weeks of treatment prior to mating and during the mating period for Main males. For both Main and Recovery males treatment ended one day before scheduled necropsy of Main males. Main females that delivered were treated for 42 to 56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Recovery females were treated during the same period as Main females, until one day before the first scheduled necropsy of Main Group 4 females. Females which failed to deliver or had a total litter loss were treated for 42 to 45 days.

Frequency of treatment:

Once

Duration of test:

28 - 45 days

Dose / conc.:

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

Remarks:

Group 2

Dose / conc.:

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

Remarks:

Group 3

Dose / conc.:

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

Remarks:

Group 4

No. of animals per sex per dose:

10 animals per dose (main test) or 5 animals (additional recovery test groups: group 1 and group 4)

Control animals:

yes, concurrent vehicle

Details on study design:

Basis for dose level selection: The dose levels were selected based on the results of a 14-day dose range finder with oral exposure in rat.
For the control group (group 1) and high dose group (group 4) 5 recovery animals (of each sex) were used to study potential reversibility of possible toxic effects. The recovery period was 15 days.

Maternal examinations:

CAGE SIDE OBSERVATIONS:
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from their cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS:
Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing and recovery periods up to the day prior to necropsy. During the dosing period, these observations were performed 1 hour (± 30 min) post-dose, based on the peak effect of occurrence of clinical signs observed in the dose range finder. The time of onset, grade and duration of any observed sign was 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. Clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment and recovery. These observations were initiated 1 hour (± 30 min) post-dose.

BODY WEIGHT:
Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated Main females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION
Food consumption was quantitatively measured weekly, except for Main females which were housed together for mating and for Main females without evidence of mating. Food consumption of mated Main females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

FUNCTIONAL TESTS
Functional tests were performed on the selected 5 Main Group 1-3 females during the last week of lactation (i.e. PND 8-12), on the selected 5 Main Group 4 females during Week 6 of treatment, and on all Recovery females on the first day a Main female was tested. These tests were initiated 1 hour (± 30 min) post-dose, after completion of clinical observations (including arena observation, if applicable). The following tests were performed: Hearing ability, Pupillary reflex, Static righting reflex, Fore- and hind-limb grip strength and Locomotor activity.

HAEMATOLOGY
- Time schedule for collection of blood: Blood was collected on the day of scheduled necropsy.
- Anaesthetic used for blood collection: Yes (isoflurane
- Animals fasted: Yes
- How many animals: 5 animals per sex of the main group, all animals of the recovery group
- Blood samples were analyzed for the following parameters: White blood cells (WBC), Red Blood Cell Distribution Width (RDW), Neutrophil (absolute), Haemoglobin, Lymphocyte (absolute), Haematocrit, Monocyte (absolute), Mean corpuscular volume (MCV), Eosinophil (absolute), Mean corpuscular haemoglobin (MCH), Basophil (absolute), Mean corpuscular haemoglobin concentration (MCHC), Red blood cells, Platelets and Reticulocyte (absolute).
- In addition the following coagulation parameters were assessed: Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT)

CLINICAL CHEMISTRY
- Time schedule for collection of blood: Blood was collected on the day of scheduled necropsy.
- Animals fasted: Yes
- How many animals: 5 animals per sex of the main group, all animals of the recovery group
- Blood samples were processed for plasma or serum (bile acids), which was analyzed for the following parameters: Alanine aminotransferase (ALAT), Creatinine, Aspartate aminotransferase (ASAT), Glucose, Alkaline Phosphatase (ALP), Cholesterol, Gamma glutamyl transpeptidase (GGT), Sodium, Total protein, Potassium, Albumin, Chloride, Total Bilirubin, Calcium, Bile Acids, Inorganic Phosphate (Inorg. Phos), Urea.

THYROID HORMONE
- Time schedule for collection of blood: Blood was collected on the day of scheduled necropsy.
- Animals fasted: Yes
- How many animals: all animals main group, all animals of the recovery group
- Blood samples were processed for serum, and serum was analyzed for Thyroxine (T4) and Thyroid Stimulating Hormone (TSH) by using the IMMULITE® 1000 immunoassay-system.

GENERAL REPRODUCTION DATA
From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day. Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed. Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.

POST MORTUM EXAMINATIONS
SACRIFICE
Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. Scheduled necropsies were conducted on the following days:
- Main females which delivered: PND 14 or 16.
- Main females which failed to deliver but showed evidence of mating: Post-coitum Days 25-26.
- Main female with total litter loss (one female in group 3): Dam with no surviving pups was euthanized within 24 hours after the last pup was found dead or missing.
- Recovery females: At least 14 days after the first dose-free day of the Recovery females.
Except for the female with total litter loss, all animals surviving to scheduled necropsy were fasted (overnight with a maximum of 24 hours) before their scheduled necropsy.

GROSS NECROPSY
- A necropsy was conducted for animals that died on study, and specified tissues were saved.
- All animals (both Main and Recovery) were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
- The numbers of former implantation sites were recorded for all paired Main females. In case no macroscopically visible implantation sites were present in Main females, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition

TISSUE COLLECTION
The following tissues and organs were collected for all selected Main animals, all Recovery animals, and all animals that died spontaneously: Artery aorta, Body cavity nasopharynx, Bone marrow (femur), Bone sternum, Brain (seven levels), Cervix, Esophagus, Eye, Gland adrenal, Gland harderian, Gland lacrimal, Gland mammary, Gland parathyroid, Gland pituitary, Gland salivary (mandibular and sublingual), Gland thyroid, Gross lesions/masses, Gut-associated lymphoid tissue, Heart, Kidney, Large intestine cecum, Large intestine colon, Large intestine rectum, Larynx, Liver, Lung, Lymph node (axillary and mesenteric site), Muscle skeletal, Nerve optic, Nerve sciatic, Nose (nasal cavity), Ovaries, Oviducts, Pancreas, Pharynx, Small intestine duodenum, Small intestine ileum, Small intestine, jejunum, Spinal cord, Spleen, Stomach (forestomach and glandular stomach), Thymus, Tongue, Trachea, Urinary bladder, Uterus and Vagina.

ORGAN WEIGHTS
- The following organs were weighted at necropsy for all scheduled euthanasia animals:
Cervixa (Weighed together with the uterus), Gland parathyroid (Weighed together with the thyroid), Gland thyroid, Ovaries (Paired organ weight), Uterus.
- Organs Weighed at Necropsy for al selected main animals and recovery animals sacrificed on schedule: Brain, Gland adrenal (Paired organ weight), Heart, Kidneys, Liver, Spleen and Thymus.

HISTOLOGY
The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
- Selected Main animals, all Recovery animals and unscheduled deaths (found dead): Tissues identified under tissue collection (except animal aorta, esophagus, harderian gland, lacrimal gland, nasopharynx, mammary gland (female), salivary gland, larynx, optic nerve, pancreas, tongue).
- Females that failed to deliver pups, and the female with total litter loss: Cervix, ovaries, uterus and vagina.
- Female with total litter loss: Mammary gland.
- Remaining animals: Gross lesions/masses.

HISTOPATHOLOGY
- Reproductive organs (cervix, ovaries, uterus, and vagina): All Main animals of the control and high dose group, all females that failed to deliver pups.
- For ovaries, a qualitative evaluation of 1 section from each ovary was made.
- Remaining organs: Lactating females of the control and high dose group, gross lesions for all Main and Recovery animals.

Ovaries and uterine content:

- The numbers of former implantation sites were recorded for all paired Main females. In case no macroscopically visible implantation sites were present in Main females, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

Fetal examinations:

STANDARDISATION OF LITTERS
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

MORTALITY/MORIBUNDITY
Pups were observed daily for general health/mortality. The number of live and dead pups were determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings. Pups showing pain, distress or discomfort which was considered not transient in nature or is likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/2000/7). The circumstances of any death were recorded in detail.

PARAMETERS EXAMINED
- Clinical observations were performed at least once daily for all pups.
- Live pups were weighed individually on PND 1, 4, 7 and 13.
- Sex was externally determined for all pups on PND 1 and 4.
- Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
- All male pups in each litter were examined for the number of areola/nipples on PND 13.
- Blood samples were processed for serum, and serum was analyzed for Thyroxine (T4) for PND 4 and 13-15 pups. TSH was examined for PND 13-15 pups. Blood was collected from 2 pups/litter.
- Histopathological examination of the thyroid gland was performed.

GROSS EXAMINATION OF DEAD PUPS:
Pups that died before scheduled termination were examined externally and sexed (both externally and internally). Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally). The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Statistics:

- All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
- Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations.
- The following pairwise comparisons were made: Group 2 vs. Group 1, Group 3 vs. Group 1, roup 4 vs. Group 1
- Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
- Non-Parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. The Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group.
- Incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Indices:

- Mating (%) = number of females mated / number of females paired x 100
- Precoital time = number of days between initiation of cohabitation and conformation of mating
- Fertility index (%) = number of pregnant females / number of females mated x 100
- Gestation index (%) = number of females with living pups on Day 1 / Number of pregnant females x 100
- Duration of gestation = number of days between confirmation of mating and the beginning of parturition
- Post-implantation survival index (%) = total number of offspring born / total number of uterine implantation sites x 100
- Live birth index (%) = number of live offspring on Day 1 after littering / total number of offspring born
- Percentage live males at First Litter Check (%) = number of live male pups at First Litter Check / number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check (%) = number of live female pups at First Litter Check / number of live pups at First Litter Check x 100
- Viability index (%) = number of live offspring on Day 4 before culling / number of live offspring on Day 1 after littering x 100
- Lactation index (%) = number of live offspring on Day 13 after littering / number of live offspring on Day 4 (after culling) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- At 15 and 50 mg/kg, piloerection was recorded for females, starting during the mating phase, with a dose-related increase in incidence. At 15 mg/kg bw/day, this finding resolved prior to initiation of the recovery phase, while at 50 mg/kg bw/day this resolved in the first week of the recovery period.
- Black discoloration of faeces and/or a general blue discoloration of the skin was noted at 15 and 50 mg/kg bw/day. The onset of these findings showed a dose-related trend and lasted until the end of the recovery phase at 50 mg/kg bw/day.
- Blue discolouration of the eyes was recorded only at 15 mg/kg bw/day during three consecutive days of the premating period. This limited occurrence at 15 mg/kg bw/day may be due to an easier detection of discolouration of eyes compared to other body parts, while at 50 mg/kg bw/day generalized discolouration was more pronounced/sudden without being first detected in the eyes. This discoloration is considered to be related to the black color of the test item and not to represent a sign of toxicity.
- No findings were noted during the weekly arena observations in this study.
- No clinical signs were noted at 5 mg/kg bw/day. Incidental findings that were noted in other dose groups 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. At the incidence observed, these were not considered to be signs of toxicological relevance.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

- At 50 mg/kg bw/day, a total of 3 Main group females were found dead. One female was found dead on Day 36 prior to dosing (i.e. after 35 days of treatment). No relevant signs were noted prior to the spontaneous death for this animal. Apart from autolysis and many black foci noted on the thymus, main macroscopic and microscopic findings were comparable
to the remaining animals of this treatment group and consisted of black discoloration of all organs. No clear cause of death could be established histopathologically for this animal.
- In addition, two Main females at 50 mg/kg bw/day died at terminal blood sampling (prior to scheduled necropsy). No relevant signs were noted for these animals and no cause of death could be established histopathologically. Although it is conceivable that these deaths could be related to the blood sampling procedure/anaesthesia, given that in total 3 high dose Main females were found dead in this study, a possible treatment-related effect could not be ruled out.
- One Main female at 15 mg/kg bw/day was sacrificed due to total litter loss on PND 5; two of her pups were found dead on Day 2 and the other two pups were sacrificed in extremis on Day 5.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- At 50 mg/kg bw/day, a statistically significantly lower body weight and body weight gain was recorded for females from Day 4 or 7 of the post-coitum period onwards, respectively. Slight body weight loss (on average approximately 6% based on mean body weight) was recorded for all these females between Days 17 and 20 of the post-coitum period.
- At 15 mg/kg bw/day, body weight and body weight gain of females was lower than controls from the second week of the post-coitum period onwards, achieving a level of statistical significance on most occasions.
- Occasional statistically significantly lower mean body weight gain recorded during the treatment and recovery period for females at 50 mg/kg bw/day was not consistently seen with continuation of treatment. Also, mean body weight at the end of recovery was similar to the control mean body weight. Therefore, these variations were not considered to represent an effect of treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- At 50 mg/kg bw/day, a lower food consumption (before and after correction for body weight) was recorded for females throughout the post-coitum period, achieving a level of statistical significance on most occasions. The difference in food consumption to the control mean became more pronounced over Days 17-20 of the post-coitum period.
- At 15 mg/kg bw/day, food consumption of females before and after correction for body weight was statistically significantly lower than controls during the last week of lactation.
- Any other (statistically significant) variations in food consumption were not considered to be related to treatment as these occurred in the absence of a dose-related trend, were of a minor degree and/or were not consistently recorded with continuation of treatment.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes in haematological parameters at 50 mg/kg bw/day distinguished treated from control animals at the end of treatment:
- Higher reticulocyte counts in Recovery group females (1.65x of control).
- Higher red blood cell distribution width (RDW) in Recovery group females (1.12x of control).
- Higher lymphocyte counts in females (1.35x of control).

At the end of recovery the affected parameters in Recovery group females had returned to control values.
The statistically significantly lower or higher lymphocyte counts in Main females at 15 and 50 mg/kg bw/day respectively at the end of treatment were not considered to be related to treatment as they did not show a dose-related trend (Groups 1-3, i.e. lactating females), were of a minor degree and were attributed to physiological differences due to pregnancy status between Main control females (lactating) and Main females at 50 mg/kg bw/day (implantation sites only).

Coagulation parameters of treated rats were not considered to have been affected by treatment.

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 at the end of treatment:
1. Lower total protein in Recovery females at 50 mg/kg bw/day (0.95x of control)
2. Lower chloride in females at 15 mg/kg bw/day (0.95x of control).
3. Higher inorganic phosphate in non-lactating females at 50 mg/kg bw/day.

- At the end of the recovery phase, total protein remained lower for Recovery group females of the 50 mg/kg bw/day group (0.95x of control).
- Grey/black discoloration of blood samples was considered to have affected total bilirubin and cholesterol measurements. This resulted in a statistically significantly lower total bilirubin level in Main females at 5 mg/kg bw/day at the end of treatment (0.37x of control), and in Recovery females (0.58x of control) at 50 mg/kg bw/day at the end of recovery. Total bilirubin could not be determined at dose levels of 15 and 50 mg/kg bw/day at the end of treatment, and bile acids could not be determined at 50 mg/kg bw/day in most animals at the end of treatment. Blood sample discolouration was also considered to have resulted in lower cholesterol in non-lactating females (0.33x of controls) at 50 mg/kg bw/day at the end of treatment.
- Lower alanine aminotransferase (ALAT), aspartate aminotransferase activity (ASAT) and/or alkaline phosphatase activity (ALP) in females at 50 mg/kg bw/day at end of treatment or end of recovery were not considered to be toxicologically relevant since the opposite effect would be expected in case of target organ toxicity.
- 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 or were only recorded at the end of recovery (lower chloride in females at 15 mg/kg bw/day at end of treatment, and lower inorganic phosphate and calcium in Recovery females at 50 mg/kg bw/day at end of recovery).

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- At 50 mg/kg bw/day, a statistically significantly lower foreleg grip strength was recorded for Main females at the end of treatment (0.60x of control). It should be noted that these high dose females had implantation sites only, while selected females of Groups 1, 2 and 3 were all lactating at the time point of functional tests. Therefore values of Group 4 were compared with historical data from non-mated rats of this age and strain. Mean value for foreleg grip strength was at the lower end of the historical control range. However, the lower foreleg grip strength was considered not to be toxicologically relevant as fore limb grip strength values in nulliparous 50 mg/kg/day/day females (Recovery animals) were within the normal range for female rats of this strain and age. Moreover, there were no corroborating changes in hind limb grip strength or other end points in the neuromuscular domain.
- At 50 mg/kg (Recovery females and non-lactating Main females combined), a statistically significantly lower motor activity (total movements) was recorded at the end of treatment (0.91x of control for females at 50 mg/kg bw/day). No toxicological relevance was attached to these findings because of the following reasons: all groups showed a similar motor activity habituation profile (with high activity in the first interval that decreased over the duration of the test period); relatively high values were observed for the control group, and for females the difference to the control mean was slight; means remained within the range considered normal for rats of this age and strain.
- Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- Any differences in absolute and relative organ weights of Main females at 50 mg/kg bw/day compared to control means were attributed to differences in pregnancy status and/or resulting lower body weights, and were therefore not considered to be related to the test-item.
- Any other differences, including those that reached statistical significance were considered not to treatment related due to the direction of the change, lack of dose-related pattern, absence of a similar change at the end of treatment and/or general overlap and/or variability in individual values.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were test item-related macroscopic findings starting in females at 5 mg/kg bw/day, consisting of:
- Liver, bluish discoloration in 3/10 females of the 5 mg/kg bw/day group, without microscopic correlate.
- General observation, black or bluish discoloration of all organs of the 15 and 50 mg/kg bw/day group (including the premature decedents and accidental deaths). This finding was still present in all Recovery rats of the 50 mg/kg bw/day group.
- Lung, several/many black foci in 4/7 scheduled sacrificed Main females of the 50 mg/kg bw/day group.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment were noted in the lung, spleen and uterus:
- In the Lungs, an increased incidence and severity of alveolar macrophage aggregations (up to moderate degree) was present at 15 and 50 mg/kg bw/day. The single incidence at minimal degree at 5 mg/kg bw/day was regarded to be within background range. In general, at 50 mg/kg bw/day the macrophages had a foamier (finely vacuolated) appearance, without black pigment. This finding was still present up to slight degree in females after a 15-day treatment free period.
- In the uterus, black-brown pigmented macrophages at the implantation sites (slight degree) were present in females at 50 mg/kg bw/day.
All the above findings were considered as non-adverse, based on their mild characters and absence of any other test-item related changes indicative of tissue/organ damage.
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:

effects observed, treatment-related

Description (incidence and severity):

THYROID HORMONE ANALYSIS
Significantly higher serum levels of total T4 were recorded in non-lactating females at 50 mg/kg bw/day (2.4x of control) at end of treatment. This increase occurred in the absence of any corroborating changes in serum level of TSH or findings at the organ level for the thyroid gland (organ weight, morphology). However, given its magnitude (2.4x of control) the observed increase in total T4 was considered to represent a potentially n adverse, compound-related effect. At the end of the recovery period, serum levels of T4 and TSH in males and non-lactating females at 50 mg/kg bw/day were within the normal range of biological variation.

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

For two females at 50 mg/kg bw/day, red fluid was noted in the cage on post-coitum Day 19 or Day 21. Additionally, one of these females showed red discharge (presumably vaginal) on post-coitum Day 20. None of these females delivered live offspring, and it was considered that this observation was indicative of fetal loss. No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of other pregnant females revealed no signs of fetal loss or premature birth. No deficiencies in maternal care were observed.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

- Females at 50 mg/kg bw/day showed evidence of former pregnancy in the form of implantation sites in the uterus. Total litter loss was observed in one female of the 15 mg/kg bw/day group. No abnormalities were seen in the reproductive organs, which could account for the lack of offspring or total litter loss. There were no morphological findings in the reproductive organs.
- Post-implantation survival index: At 50 mg/kg bw/day, none of the implantations developed into offspring. At 15 mg/kg bw/day, the total number of offspring born compared to the total number of uterine implantations was slightly lower than the control group (87% vs. 97% in the control group, and 92% at 5 mg/kg bw/day). The mean post-implantation survival index at this dose remained at the lower end of the range considered normal for rats of this age and strain. Post-implantation survival index at 5 mg/kg bw/day was not considered to be affected by treatment.

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Gestation index and duration of gestation at dose levels up to 15 mg/kg bw/day were not considered to be affected by treatment.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Fertility index was not affected by treatment. Fertility index was 100 % for the control and 15 mg/kg bw/day group and 90% for the 5 and 15 mg/kg bw/day group. One female at 5 mg/kg bw/day and one female at 15 mg/kg bw/day were not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was not considered to be related to treatment.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Parturition/Maternal Care: For two females at 50 mg/kg bw/day, red fluid was noted in the cage on post-coitum Day 19 or Day 21. Additionally, one of these females showed red discharge (presumably vaginal) on post-coitum Day 20. None of these females delivered live offspring, and it was considered that this observation was indicative of fetal loss. No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of other pregnant females revealed no signs of fetal loss or premature birth. No deficiencies in maternal care were observed.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

mortality

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Pup body weights were not affected by treatment. At 15 mg/kg bw/day, mean body weights of male and female pups combined appeared slightly lower than controls on Day 13 of lactation (approximately 5% lower than controls). However, this difference was slight and not statistically significant, and mean body weights of male and female pups remained within the range considered normal for rats of this age and strain. Therefore, this was not considered to represent a treatment-related difference (table 2).

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Live birth index: The number of live offspring on Day 1 after littering compared to the total number of offspring born was not affected by treatment. Live birth index was 99% in the control group and at 15 mg/kg bw/day, and 100% at 5 mg/kg bw/day. One pup of the control group and one pup at 15 mg/kg bw/day were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age (table 1).

Changes in sex ratio:

effects observed, treatment-related

Description (incidence and severity):

At 15 mg/kg bw/day, sex ratio was statistically significantly lower (35/65 vs. 55/45 in the control group). Although this finding may have occurred by chance due to a lower mean litter size and subsequent higher relative contribution of a higher number of females in these litters, it could not be excluded that this was a treatment-related effect. Sex ratio at 5 mg/kg bw/day was not affected by treatment (table 5).

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Litter size: At 15 mg/kg bw/day, a statistically significantly lower mean number of living pups was recorded (9.6 vs. 12.5 in the control group and 12.3 at 5 mg/kg bw/day). Although the mean remained within the range considered normal for rats of this age and strain, this was considered to be a treatment–related effect (table 5).

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

- Viability index: At 15 mg/kg bw/day, the number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was lower than the control mean (90% vs. 98% in the control group). ). This mean was at the low end of the range considered normal for pups of this age and strain. At this dose, a total of 8 pups were found dead or missing between PND 2 and 4 Pups missing were most likely cannibalised. One control pup and one pup at 5 mg/kg bw/day were found dead on Days 4 and 3, respectively. This mortality incidence remained well within the range considered normal for pups of this age and was therefore not considered to be related to treatment (table 1).
- Lactation index: At 15 mg/kg bw/day, the number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was lower than the control mean (94% vs. 100% in the control group). This mean exceeded the range considered normal for pups of this age and strain3. At this dose, a total of 4 pups died before scheduled sacrifice and one pup was sacrificed in extremis on Day 5, and one pup of litter was found missing on Day 7 and was most likely cannibalized. One pup at 5 mg/kg bw/day was found missing on Day 6 and was most likely cannibalized. This single mortality incidence remained well within the range considered normal for pups of this age and was therefore not considered to be related to treatment (table 1).

External malformations:

effects observed, treatment-related

Description (incidence and severity):

Blue discolouration of the whole body was recorded for a few pups at 5 mg/kg bw/day and for most pups at 15 mg/kg bw/day.

Skeletal malformations:

not examined

Visceral malformations:

no effects observed

Description (incidence and severity):

Microscopic findings recorded for the thyroid gland of the F1-pups were within the range of background pathology encountered in rats of this age and strain.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

THYROID HORMONES (table 4.1-4.3)
Note: No pups were available in the 50 mg/kg bw/day group.
In pups from PND 4, a slight higher serum level of total T4 was noted at 15 mg/kg bw/day (both sexes combined, if applicable). This difference of 19% did not reach statistical significance. No measurement of TSH was performed in PND 4 pups (not applicable).
In pups from PND 13-15 (both sexes), a dose dependent increase in serum levels of total T4 was noted at 5 and 15 mg/kg bw/day. When compared to the concurrent controls, T4-values were increased by a factor of 1.4x and 1.6x, respectively, in male pups and a factor of 1.3x and 1.4x, respectively, in female pups. These changes occurred without any relation to TSH.
Microscopic findings recorded for the thyroid gland of the F1-pups were within the range of background pathology encountered in rats of this age and strain.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in postnatal survival

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1 DEVELOPMENTAL DATA

	GROUP 1 CONTROL	GROUP 2 5 MG/KG	GROUP 3 15 MG/KG	GROUP 4 50 MG/KG
Total number of offspring born	126	111	87	0
Total number of uterine implantation sites	130	121	104	112
Number of live offspring on Day 1 after littering	125	111	86	0
Number of live offspring on Day 4 (before culling)	124	110	78	0
Number of live offspring on Day 4 (after culling)	80	72	66	0
Number of live offspring on Day 13 after littering	80	71	62	0
Post-implantation survival index (%)	97	92	84	0
(Total number of offspring born/Total number of uterine implantation sites) * 100
Live birth index (%)	99	100	99	-
(Number of live offspring on Day 1 after littering/Total number of offspring born) * 100
Viability index (%)	99	99	91	-
(Number of live offspring on Day 4 (before culling)/Number of live offspring on Day 1 after littering)*100
Lactation index (%) (Number of live offspring on Day 13 after littering/Number of live offspring on Day 4 (after culling)) * 100	100	99	94	-

Table 2 BODY WEIGHTS OF PUPS (GRAM) F0-GENERATION - LACTATION

DAY	SEX		GROUP 1 CONTROL	GROUP 2 5 MG/KG	GROUP 3 15 MG/KG
1	M	MEAN	6.3	6.4	6.2
		ST.DEV.	0.5	0.4	0.4
		N	10	9	9
	F	MEAN	6.0	6.1	5.9
		ST.DEV.	0.4	0.4	0.3
		N	10	9	9
	M+F	MEAN	6.1	6.2	6.0
		ST.DEV.	0.5	0.4	0.3
		N	10	9	9
4	M	MEAN	9.0	9.1	9.1
		ST.DEV.	0.8	0.7	0.5
		N	10	9	8
	F	MEAN	8.6	8.7	8.4
		ST.DEV.	0.8	0.7	1.3
		N	10	9	9
	M+F	MEAN	8.8	8.9	8.4
		ST.DEV.	0.8	0.7	1.3
		N	10	9	9
7	M	MEAN	14.9	15.1	14.7
		ST.DEV.	1.3	1.2	1.0
		N	10	9	8
	F	MEAN	14.3	14.7	14.1
		ST.DEV.	1.3	1.4	1.1
		N	10	9	8
	M+F	MEAN	14.6	14.9	14.3
		ST.DEV.	1.3	1.3	1.1
		N	10	9	8
13	M	MEAN	28.7	29.3	27.4
		ST.DEV.	2.0	2.9	1.6
		N	10	9	8
	F	MEAN	27.9	28.4	26.7
		ST.DEV.	1.9	3.1	1.7
		N	10	9	8
	M+F	MEAN	28.3	28.9	26.9
		ST.DEV.	2.0	3.0	1.7
		N	10	9	8

 */** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 3.1 ANOGENITAL DISTANCE AND NIPPLE RETENTION PER GROUP F0-GENERATION - LACTATION

  

		GROUP 1 CONTROL	GROUP 2 5 MG/KG	GROUP 3 15 MG/KG
anogenital dist M mm	MEAN	2.67	2.67	2.72
	ST. DEV.	0.17	0.11	0.31
	N	10	9	9
anogenital dist F mm	MEAN	0.99	0.94	1.00
	ST. DEV.	0.08	0.03	0.08
	N	10	9	9
Number of nipples	MEAN	0.00	0.00	0.00
	MEDIAN (+)	0.00	0.00	0.00
	N	10	9	8

 +/++ Steel-test significant at 5% (+) or 1% (++) level

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

Table 3.2 CORRECTED ANOGENITAL DISTANCE 

PND 1		GROUP 1 CONTROL	GROUP 2 5 MG/KG	GROUP 3 15 MG/KG
norm anog dist M	MEAN	1.45	1.44	1.49
mm	ST.DEV	0.10	0.05	0.14
	N	10	9	9
norm anog dist F	MEAN	0.55	0.52	0.56
mm	ST.DEV	0.05	0.02	0.05
	N	10	9	9

+/++ Steel-test significant at 5% (+) or 1% (++) level

Table 4.1 T4 level pups PND 4 Summary

PND 4 PUPS		GROUP 1 CONTROL	GROUP 2 5 MG/KG	GROUP 3 15 MG/KG
Total T4	MEAN	1.54	1.51	1.84
ug/dL	ST.DEV	0.31	0.31	0.14
	N	9	8	3

 */** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 4.2 T4 level pups PND 13 -15 Males

		GROUP 1 CONTROL	GROUP 2 5 MG/KG	GROUP 3 15 MG/KG
PND 13-15 TSH	MEAN	0.124	0.132	0.121
uIU/mL	ST.DEV	0.089	0.105	0.062
	N	10	9	8
Total T4	MEAN	4.16	5.77 **	6.49 **
ug/dL	ST.DEV	0.71	0.85	1.34
	N	10	9	8

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 4.3 T4 level pups PND 13 -15 females 

		GROUP 1 CONTROL	GROUP 2 5 MG/KG	GROUP 3 15 MG/KG
PND 13-15 TSH	MEAN	0.132	0.142	0.128
uIU/mL	ST.DEV	0.083	0.072	0.113
	N	10	9	8
Total T4	MEAN	4.25	5.47 **	5.85 **
ug/dL	ST.DEV	0.53	0.82	1.04
	N	10	9	8

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 5 DEVELOPMENTAL DATA F0-GENERATION - LACTATION 

	Group 1 Control	Group 2 5 mg/kg bw		Group 3 15 mg/kg bw
LITTERS TOTAL	10	9		9
DURATION OF GESTATION
MEAN (+)	21.3		21.2		21.7
ST.DEV.	0.5		0.4		0.5
N	10		9		9
DEAD PUPS AT FIRST LITTER CHECK
LITTERS AFFECTED (#)	1		0		1
TOTAL	1		0		1
MEAN (+)	0.1		0.0		0.1
ST.DEV.	0.3		0.0		0.3
N	10		9		9
LIVING PUPS AT FIRST LITTER CHECK
% OF MALES / FEMALES (#)	55 / 45		46 / 54		35 / 65 ##
TOTAL	125		111		86
MEAN (+)	12.5		12.3		9.6 ++
ST.DEV.	1.5		2.5		2.4
N	10		9		9
POSTNATAL LOSS
% OF LIVING PUPS	0.8		0.9		9.3
LITTERS AFFECTED (#)	1		1		4
TOTAL (#)	1		1		8 ##
MEAN (+)	0.1		0.1		0.9
ST.DEV.	0.3		0.3		1.2
N	10		9		9
CULLED PUPS
TOTAL	44		38		12
LIVING PUPS DAY 4 P.P.
TOTAL	80		72		66
MEAN (+)	8.0		8.0		7.3
ST.DEV.	0.0		0.0		2.0
N	10		9		9
BREEDING LOSS DAYS 5 - 13 P.P. % OF LIVING PUPS AT DAY 4 P.P.	0.0		1.4		6.1
LITTERS AFFECTED (#)	0		1		3
TOTAL (#)	0		1		4 #
MEAN (+)	0.0		0.1		0.4
ST.DEV.	0.0		0.3		0.7
N	10		9		9
LIVING PUPS DAY 13 P.P.
% OF MALES / FEMALES (#)	53 / 48		51 / 49		35 / 65
TOTAL	80		71		62
MEAN (+)	8.0		7.9		6.9
ST.DEV.	0.0		0.3		2.6
N	10		9		9

+/++ Steel-test significant at 5% (+) or 1% (++) level

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

Cross-reference 3

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

14-day dose range finder

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

07 Apr 2017 to 07 jun 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The objective of this dose range finder was to select dose levels for a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening. Animals were dosed by oral gavage at dose levels of 50, 100, 200 and 1000 mg/kg bw/ day for 14 days The following parameters and end points were evaluated in this study: clinical signs, body weights, food consumption, clinical pathology parameters (hematology, coagulation and clinical chemistry), gross necropsy findings and organ weights.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Identification: Orasol Black X51
- Appearance: Black solid
- Batch: 001-150606
- Purity/Composition: Approx. 95% can be assumed
- Test item storage: At room temperature
- Stable under storage conditions until: 26 May 2020 (expiry date)

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on species / strain selection:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 12 weeks (Groups 1-4) and 14 weeks (Group 5)
- Weight at study initiation: between 329 g and 377 g (males Groups 1-3), 192 g and 232 g (females Groups 1-4), and 244 g and 258 g (females Group 5).
- Housing: On arrival and following randomization, animals were group housed (up to 4 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon type IV, height 18 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Animals were separated during designated procedures/activities. For psychological/environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom), except when interrupted by study procedures/activities.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study.
- Water: Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: at least 5 days before the commencement of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8 to 20.7
- Humidity (%): 46.53 to 61.62
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Details on route of administration:

The oral route of exposure was selected as this is a possible route of human exposure during manufacture, handling or use of the test item.

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) was homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations are prepared daily and dosed within 5 hours after adding the vehicle to the test item. Test item dosing formulations are kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle are continuously stirred until and during dosing.

VEHICLE
- Concentration in vehicle: 10, 20, 40 and 100 mg/mL correspond to dose levels of 50, 100, 200 and 1000 mg/kg bw/day, respectively
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed by LC-DAD using a validated analytical procedure. Stability of the test item under test conditions was demonstrated in an analytical method development and validation study:
- Concentration Analysis: Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration.
- Homogeneity Analysis: Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.
- Stability Analysis: Stability analyses performed previously 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:

14 days

Frequency of treatment:

once daily

Dose / conc.:

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

Remarks:

Group 4

Dose / conc.:

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

Remarks:

Group 5

Dose / conc.:

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

Remarks:

Group 3

Dose / conc.:

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

Remarks:

Group 2

No. of animals per sex per dose:

Group 1-4: 4 males and 4 females
Group 5: 4 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The first dose level of 1000 mg/kg bw/day (Group 2) was selected based on single dose toxicity data with the test compound, which yielded a LD50 > 10000 mg/kg bw. Subsequent dose levels for Groups 3, 4 and 5 were selected based on the results of the previous group(s).

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from their cage during observation, unless necessary for identification or confirmation of possible findings.
At least twice daily, animals showing pain, distress or discomfort which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7).

DETAILED CLINICAL OBSERVATIONS:
Clinical observations were performed once daily, beginning at the first administration of the test item and lasting throughout the dosing period. During the dosing period, these observations were performed in all animals at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing. The time of onset, grade and duration of any observed sign was 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:
Animals were weighed individually, starting on Day 1 prior to dosing and on Days 4, 7, 10 and 14. On the day of necropsy, a non-fasted weight was recorded for all live Group 5 animals, and a fasted weight was recorded for all live Group 2 animals.

FOOD CONSUMPTION:
Food consumption was quantitatively measured over Days 1-4, 4-7, 7-10, 10-14.

WATER CONSUMPTION:
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

HAEMATOLOGY:
- Blood of Group 4 animals (except for one male which was found dead) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) in the animal facility. Additional blood samples were obtained from Group 4 animal nos. 26 and 30 (due to clotting of non-serum samples) in the necropsy room. After collection, samples were transferred to the appropriate laboratory for processing. Animals were fasted overnight with a maximum of approximately 25.25 hours before blood sampling, but water was available.
- Blood samples were analyzed for the following parameters: White blood cells (WBC), Red Blood Cell Distribution Width (RDW), Neutrophils (absolute), Haemoglobin, Lymphocytes (absolute), Haematocrit, Monocytes (absolute), Mean corpuscular volume (MCV), Eosinophils (absolute), Mean corpuscular haemoglobin (MCH), Basophils (absolute), Mean corpuscular haemoglobin concentration (MCHC), Red blood cells, Platelets and Reticulocyte (absolute). In addition, the following coagulation parameters were analyzed: Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT)

CLINICAL CHEMISTRY:
- The same blood as collected for haematology was used for clinical chemistry.
- Blood samples were processed for serum, and the serum was analyzed for the following parameters: Alanine aminotransferase (ALAT), Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate (Inorg. Phos), Aspartate aminotransferase (ASAT), Alkaline phosphatase (ALP), Gamma glutamyl transferase (GGT), Total protein, Albumin, Total bilirubin, Bile acids and Urea.

Sacrifice and pathology:

GROSS PATHOLOGY AND TERMINAL PROCEDURES:
All live animals were deeply anaesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to an external, thoracic and abdominal examination on Day 15 (scheduled necropsy) or sooner (decedents). Group 4 animals (except for one male which was found dead) were deprived of food overnight (with a maximum of approximately 25.25 hours) prior to blood sampling on Day 15 (scheduled necropsy).
Terminal body weight, kidney, adrenal glands, spleen and liver weight were determined for all Group 4 animals (fasted) that survived until necropsy on Day 15 and for all Group 2 animals (non-fasted) euthanized in extremis on Day 4.

HISTOPATHOLOGY
From all animals in this study, kidney, adrenal glands, spleen and liver were collected and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), but no histopathological examination was performed.

Statistics:

Due to the complex design of this dose range finder (i.e. reallocation of control animals to treatment Group 3, addition of treatment Groups 4 and 5), no statistical analysis was applied on the available data.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Prior to the unscheduled deaths at 100, 200 and 1000 mg/kg bw/day, piloerection (both sexes) and hunched posture (females only) were noted. In addition, one female at 200 mg/kg bw/day was observed with orange discoloration of her mouth 3 hours after dosing on Day 2. The general observation of black discoloration of the faeces and blue discoloration of different parts of the body was related to the black color of the test item and thus no sign of toxicity. Alopecia noted for a single female at 100 mg/kg bw/day was not considered to be treatment-related as it 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.
- In the 50 mg/kg bw/day dose group piloerection (both sexes) and hunched posture (females only) were noted on one or more days. In addition, rales and salivation (both at slight degree) were observed incidentally (both sexes). Taking into account the nature and minor severity of these latter two findings, the route of test item administration (oral gavage), the time point of occurrence (i.e. immediate after dosing), and the fact that rales and salivation occurred on isolated days and were not observed at the higher doses tested, no toxicological relevance was attached to these findings. The general observation of black discoloration of the faeces and blue discoloration of different parts of the body was most likely related to the black color of the test item and thus no sign of toxicity.

Mortality:

mortality observed, treatment-related

Description (incidence):

Treatment at 100, 200 and 1000 mg/kg bw/day was not tolerated and resulted in preterm deaths in these groups. The survival period decreased with higher dose:
- 1000 mg/kg bw/day: In the morning of Day 3, two females were found dead and treatment of all remaining animals at this dose level was stopped. In the morning of Day 4, the remaining two females were found dead. As treatment at 1000 mg/kg bw/day seemed to be tolerated by the males, it was decided in consultation with the Sponsor to continue treatment of males at this dose level on Day 4. In the morning of Day 5, all males were found dead.
- 200 mg/kg bw/day: One female was found dead in the morning of Day 4. All remaining animals in this group were euthanized for humane reasons.
- 100 mg/kg bw/day: Three out of the four females were found dead in the morning of Day 9; the last female in this dose group was found dead in the morning of Day 11.
- 50 mg/kg bw/ day: One male in the 50 mg/kg bw/day group was found dead after blood collection on Day 15. His death was considered to be related to complications during anaesthesia and not to treatment with the test item. All other animals in the 50 mg/kg bw/day group survived until scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- As animals at 100, 200 and 1000 mg/kg bw/day died very early in the study, data on body weight were very limited. In general, body weight loss together with reduced food consumption (absolute and relative to body weight) were seen at either dose level. Severity increased with higher dose, and females seemed to be more sensitive to the treatment than males.
- For the 50 mg/kg bw/ day dose group no treatment-related effects on body weight/body weight gain (absolute and relative to body weight) were noted during the observation period. All values were considered to be within the normal range of biological variation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- As animals at 100, 200 and 1000 mg/kg bw/day died very early in the study, data on food consumption were very limited. In general, reduced food consumption was seen at either dose level. Severity increased with higher dose, and females seemed to be more sensitive to the treatment than males.
- For the 50 mg/kg bw/ day dose group no treatment-related effects on food consumption were noted during the observation period. All values were considered to be within the normal range of biological variation.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the 14-days treatment period in the 50 mg/kg bw/day group the changes observed in haematology parameters (including clotting) were considered to be unrelated to treatment as changes were relatively slight, occurred in the absence of changes in correlating parameters, and/or no corroborative findings in the opposite sex were noted.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the 14-days treatment period in the 50 mg/kg bw/day group, blood levels of cholesterol were markedly decreased in both sexes. Ranges of 0.75-1.08 mmol/L (males) and 0.28-0.55 mmol/L (females) were measured. In addition, higher levels of bile acids were recorded in one male (105.8 μmol/L) and all females (84.0-96.0 μmol/L). All other changes in clinical biochemistry were considered to be unrelated to treatment as changes were relatively slight, occurred in the absence of changes in correlating parameters, and/or no corroborative findings in the opposite sex were noted.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- For animals that were euthanized in extremis (Group 3: 200 mg/kg bw/day), terminal body weight together with organ weights of liver, kidneys, adrenals and spleen were determined at necropsy. Higher organ weights of liver and kidneys were recorded for one male (absolute weights only) and one female (absolute and relative to body weight). In addition, one male had a relatively high adrenal weight (absolute and relative). All remaining weights were considered to be within the normal range of biological variation.
- There were no general effects on organ weights in the 50 mg/kg bw/ day group. Changes in single animals included higher absolute and relative weights of liver (1 male), kidneys (1 female) and adrenals
(2 female).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- At necropsy, two females at 1000 mg/kg bw/day had several to many, purple foci on their thymus. In addition, all animals were noted with general bluish discoloration of their bodies, including internal tissues/organs, which most likely was related to the black color of the test item. Furthermore, depending on the time span between the animal had died and discovery of its death, beginning or advanced autolysis was observed for some animals.
- One dead male at 100 mg/kg bw/day was found partially cannibalized with his spleen missing. Both, the dark discoloration of the tissues and state of autolysis made it difficult to identify macroscopic abnormalities. Therefore, it cannot be excluded that possible changes in tissues/organs have been missed.
- At necropsy of the 50 mg/kg bw group, there were no macroscopic findings apart from the general bluish discoloration of the body, including internal tissues/organs, which most likely was caused by the black test item. It cannot be excluded that certain macroscopic abnormalities like changes in color or pattern of tissues have been obscured by this dark discoloration.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Dose formulation analysis

- No test item was detected in the Group 1 (control group) formulation.

- The concentrations analyzed in the formulations of Groups 2 and 3 were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%). The formulations of Group 2 and Group 3 prepared were homogeneous (i.e. coefficient of variation ≤ 10%).

- Note: In consultation with the Sponsor, no dose formulation analysis was performed for the two extra groups (Groups 4 and 5) that were added during the course of the study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

- Route of administration: The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Amines, C10-14-branched and linear alkyl, [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]chromate(1-) and Amines, C10-14-branched and linear alkyl, bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)]chromate(1-) and Amines, C10-14-branched and linear alkyl, bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]chromate(1-)

EC Number:

939-191-9

Molecular formula:

C32H18CrN6O8.C10-15H21-31NH2

IUPAC Name:

Reaction mass of Amines, C10-14-branched and linear alkyl, [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]chromate(1-) and Amines, C10-14-branched and linear alkyl, bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)]chromate(1-) and Amines, C10-14-branched and linear alkyl, bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]chromate(1-)

Specific details on test material used for the study:

- Identification: Orasol Black X51
- Appearance: Black solid
- EC Number: 939-191-9
- Batch: 001-150606
- Test Facility Test Item Number: 208107/A
- Purity/Composition: Approx. 95%
- Test item storage: At room temperature
- Stable under storage conditions until: 26 May 2020 (expiry date)

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: At initiation of dosing, males were 11 weeks and females were 12 weeks old.
- Weight at study initiation: At initiation of dosing, males weighed between 268 g and 305 g and females weighed between 194 g and 248 g.
- Housing: On arrival and following the pre-test (females only) and pre-mating period, Main animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). Recovery males and females were housed as such during the entire study period. During the mating phase, Main males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, Main males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Main Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). During the lactation phase, Main females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not be left without their dam or a bottle filled with warm water for longer than 30-40 minutes. 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. The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water: Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours.
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for 7 days prior to start of the pre-test period (females) or 7 days before the commencement of dosing (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 21
- Humidity (%): 50 to 76
- Air changes (per hr): 10 or greater with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 5 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.

VEHICLE
- Concentration in vehicle: 1, 3 and 10 mg/mL dosing solutions correspond to dose levels of 5, 15 and 50 mg/kg bw/day respectively
- Amount of vehicle (if gavage): 5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage:1:1 within the same treatment group, avoiding sibling mating.
- Length of cohabitation: Once mating had occurred, the males and females were separated.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- After successful mating each pregnant female was caged: Main Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected during week 1 of the pre-mating period. Concentrations were determined for all groups, homogenicity for groups 2 and 4 (The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 preparations were averaged and utilized as the concentration results). All samples were stored on dry ice immediately after sampling and shipped. Upon receipt at the analytical laboratory, the samples were stored in the ultra-low freezer ≤ -70°C until analysis. Analyses were performed by using a validated analytical procedure.
- Concentration analysis: Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to 15% for suspensions of target concentration.
- Homogeneity analysis: Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.
- Stability analyses performed previously in conjunction with the method development and validation study 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:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days. Main males and Recovery males were treated for 29 days, including at least 2 weeks of treatment prior to mating and during the mating period for Main males. For both Main and Recovery males treatment ended one day before scheduled necropsy of Main males. Main females that delivered were treated for 42 to 56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Recovery females were treated during the same period as Main females, until one day before the first scheduled necropsy of Main Group 4 females. Females which failed to deliver or had a total litter loss were treated for 42 to 45 days.

Frequency of treatment:

once daily

Details on study schedule:

For the control group (group 1) and high dose group (group 4) 5 recovery animals (of each sex) were used to study potential reversibility of possible toxic effects. These animals were not mated and consequently were not used for the assessment of reproduction/developmental toxicity. The recovery period was 15 days.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Group 2

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Group 3

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Group 4

No. of animals per sex per dose:

10 animals per dose per sex (main test) or 5 animals per dose per sex (additional recovery test groups: group 1 and group 4)

Control animals:

yes, concurrent vehicle

Details on study design:

Basis for dose level selection: The dose levels were selected based on the results of a 14-day dose range finder with oral exposure in rat.

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from their cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS:
Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing and recovery periods up to the day prior to necropsy. During the dosing period, these observations were performed 1 hour (± 30 min) post-dose, based on the peak effect of occurrence of clinical signs observed in the dose range finder. The time of onset, grade and duration of any observed sign was 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. Clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment and recovery. These observations were initiated 1 hour (± 30 min) post-dose.

BODY WEIGHT:
Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated Main females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION
Food consumption was quantitatively measured weekly, except for Main males and Main females which were housed together for mating and for Main females without evidence of mating. Food consumption of mated Main females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

FUNCTIONAL TESTS
Functional tests were performed on the selected 5 Main males and all Recovery males during Week 4 of treatment, on the selected 5 Main Group 1-3 females during the last week of lactation (i.e. PND 8-12), on the selected 5 Main Group 4 females during Week 6 of treatment, and on all Recovery females on the first day a Main female was tested. These tests were initiated 1 hour (± 30 min) post-dose, after completion of clinical observations (including arena observation, if applicable). The following tests were performed: Hearing ability, Pupillary reflex, Static righting reflex, Fore- and hind-limb grip strength and Locomotor activity.

HAEMATOLOGY
- Time schedule for collection of blood: Blood was collected on the day of scheduled necropsy.
- Anaesthetic used for blood collection: Yes (isoflurane
- Animals fasted: Yes
- How many animals: 5 animals per sex of the main group, all animals of the recovery group
- Blood samples were analyzed for the following parameters: White blood cells (WBC), Red Blood Cell Distribution Width (RDW), Neutrophil (absolute), Haemoglobin, Lymphocyte (absolute), Haematocrit, Monocyte (absolute), Mean corpuscular volume (MCV), Eosinophil (absolute), Mean corpuscular haemoglobin (MCH), Basophil (absolute), Mean corpuscular haemoglobin concentration (MCHC), Red blood cells, Platelets and Reticulocyte (absolute).
- In addition the following coagulation parameters were assessed: Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT)

CLINICAL CHEMISTRY
- Time schedule for collection of blood: Blood was collected on the day of scheduled necropsy.
- Animals fasted: Yes
- How many animals: 5 animals per sex of the main group, all animals of the recovery group
- Blood samples were processed for plasma or serum (bile acids), which was analyzed for the following parameters: Alanine aminotransferase (ALAT), Creatinine, Aspartate aminotransferase (ASAT), Glucose, Alkaline Phosphatase (ALP), Cholesterol, Gamma glutamyl transpeptidase (GGT), Sodium, Total protein, Potassium, Albumin, Chloride, Total Bilirubin, Calcium, Bile Acids, Inorganic Phosphate (Inorg. Phos), Urea.

THYROID HORMONE
- Time schedule for collection of blood: Blood was collected on the day of scheduled necropsy.
- Animals fasted: Yes
- How many animals: all animals main group, all animals of the recovery group
- Blood samples were processed for serum, and serum was analyzed for Thyroxine (T4) and Thyroid Stimulating Hormone (TSH) by using the IMMULITE® 1000 immunoassay-system.

GENERAL REPRODUCTION DATA
From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day. Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed. Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females (Main and Recovery) during 14 days prior to treatment (pre-test period) and the first 14 days of treatment. For Main females, daily vaginal lavage was continued during mating until evidence of copulation was observed. On the day of necropsy, a vaginal lavage was also taken from Main and Recovery females to determine the stage of estrus. This was done for all females, except for females that died spontaneously.

Litter observations:

STANDARDISATION OF LITTERS
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

MORTALITY/MORIBUNDITY
Pups were observed daily for general health/mortality. The number of live and dead pups were determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings. Pups showing pain, distress or discomfort which was considered not transient in nature or is likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/2000/7). The circumstances of any death were recorded in detail.

PARAMETERS EXAMINED
- Clinical observations were performed at least once daily for all pups.
- Live pups were weighed individually on PND 1, 4, 7 and 13.
- Sex was externally determined for all pups on PND 1 and 4.
- Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
- All male pups in each litter were examined for the number of areola/nipples on PND 13.
- Blood samples were processed for serum, and serum was analyzed for Thyroxine (T4) for PND 4 and 13-15 pups. TSH was analyzed for PND 13-15 pups. Blood was collected from 2 pups/litter.
- Histopathological examination of the thyroid gland was performed.

GROSS EXAMINATION OF DEAD PUPS:
Pups that died before scheduled termination were examined externally and sexed (both externally and internally). Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally). The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Postmortem examinations (parental animals):

SACRIFICE
Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. Scheduled necropsies were conducted on the following days:
- Main Males (which sired or failed to sire): Following completion of the mating period (a minimum of 28 days of administration).
- Recovery males: After the recovery period of 15 days, which was 15 days after the scheduled necropsy of Main males.
- Main females which delivered: PND 14 or 16.
- Main females which failed to deliver but showed evidence of mating: Post-coitum Days 25-26.
- Main female with total litter loss (one female in group 3): Dam with no surviving pups was euthanized within 24 hours after the last pup was found dead or missing.
- Recovery females: At least 14 days after the first dose-free day of the Recovery females.
Except for the female with total litter loss, all animals surviving to scheduled necropsy were fasted (overnight with a maximum of 24 hours) before their scheduled necropsy.

GROSS NECROPSY
- A necropsy was conducted for animals that died on study, and specified tissues were saved.
- All animals (both Main and Recovery) were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
- The numbers of former implantation sites were recorded for all paired Main females. In case no macroscopically visible implantation sites were present in Main females, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

TISSUE COLLECTION
The following tissues and organs were collected for all selected Main animals, all Recovery animals, and all animals that died spontaneously: Artery aorta, Body cavity nasopharynx, Bone marrow (femur), Bone sternum, Brain (seven levels), Cervix, Epididymis, Esophagus, Eye, Gland adrenal, Gland coagulation, Gland harderian, Gland lacrimal, Gland mammary, Gland parathyroid, Gland pituitary, Gland prostate, Gland salivary (mandibular and sublingual), Gland seminal vesicle, Gland thyroid, Gross lesions/masses, Gut-associated lymphoid tissue, Heart, Kidney, Large intestine cecum, Large intestine colon, Large intestine rectum, Larynx, Liver, Lung, Lymph node (axillary and mesenteric site), Muscle skeletal, Nerve optic, Nerve sciatic, Nose (nasal cavity), Ovaries, Oviducts, Pancreas, Pharynx, Small intestine duodenum, Small intestine ileum, Small intestine, jejunum, Spinal cord, Spleen, Stomach (forestomach and glandular stomach), Testes, Thymus, Tongue, Trachea, Urinary bladder, Uterus and Vagina.

ORGAN WEIGHTS
- The following organs were weighted at necropsy for all scheduled euthanasia animals:
Cervixa (Weighed together with the uterus), Epididymides, Gland coagulation (Weighed together with the seminal vesicles, Paired organ weight.), Gland parathyroid (Weighed together with the thyroid), Gland prostate, Gland seminal vesicle, Gland thyroid, Ovaries (Paired organ weight), Testes (Paired organ weight), Uterus.
- Organs Weighed at Necropsy for al selected main animals and recovery animals sacrificed on schedule: Brain, Gland adrenal (Paired organ weight), Heart, Kidneys, Liver, Spleen and Thymus.

HISTOLOGY
The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
- Selected Main animals, all Recovery animals and unscheduled deaths (found dead): Tissues identified under tissue collection (except animal aorta, esophagus, harderian gland, lacrimal gland, nasopharynx, mammary gland (female), salivary gland, larynx, optic nerve, pancreas, tongue).
- Males that failed to sire (except for males which were selected), females that failed to deliver pups, and the female with total litter loss: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina.
- Female with total litter loss: Mammary gland.
- Remaining animals: Gross lesions/masses.

HISTOPATHOLOGY
- Reproductive organs (cervix, coagulation gland, epididymides, ovaries, prostate gland, seminal vesicles, testes, uterus, and vagina): All Main animals of the control and high dose group, all males that failed to sire to examine staging of spermatogenesis and histopathology of interstitial cell structure, all females that failed to deliver pups.
- For ovaries, a qualitative evaluation of 1 section from each ovary was made.
- For testes, a detailed qualitative examination 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 stagespecificity of testicular findings were noted.
- Remaining organs: First five males and lactating females of the control and high dose group, gross lesions for all Main and Recovery animals.

Postmortem examinations (offspring):

SACRIFICE
- Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND 13 to 15), except for the two pups per litter selected for blood collection, were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%). The pups selected for blood collection on PND 13 to 15 were anesthetized using isoflurane followed by exsanguination.
- Pups that died or were euthanized before scheduled necropsy date were examined externally and sexed. The stomach of these pups was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

GROSS NECROPSY
On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible. All remaining pups were euthanized on PND 13 or 15. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (one male and one female pup) was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation. Histopathological examination of the thyroid gland was performed.

Statistics:

- All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
- Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations.
- The following pairwise comparisons were made: Group 2 vs. Group 1, Group 3 vs. Group 1, roup 4 vs. Group 1
- Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
- Non-Parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. The Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group.
- Incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

- Mating (%) = number of females mated / number of females paired x 100
- Precoital time = number of days between initiation of cohabitation and conformation of mating
- Fertility index (%) = number of pregnant females / number of females mated x 100
- Gestation index (%) = number of females with living pups on Day 1 / Number of pregnant females x 100
- Duration of gestation = number of days between confirmation of mating and the beginning of parturition
- Post-implantation survival index (%) = total number of offspring born / total number of uterine implantation sites x 100

Offspring viability indices:

- Live birth index (%) = number of live offspring on Day 1 after littering / total number of offspring born
- Percentage live males at First Litter Check (%) = number of live male pups at First Litter Check / number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check (%) = number of live female pups at First Litter Check / number of live pups at First Litter Check x 100
- Viability index (%) = number of live offspring on Day 4 before culling / number of live offspring on Day 1 after littering x 100
- Lactation index (%) = number of live offspring on Day 13 after littering / number of live offspring on Day 4 (after culling) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- At 15 and 50 mg/kg, piloerection was recorded for females, starting during the mating phase, with a dose-related increase in incidence. At 15 mg/kg bw/day, this finding resolved prior to initiation of the recovery phase, while at 50 mg/kg bw/day this resolved in the first week of the recovery period.
- Black discoloration of faeces and/or a general blue discoloration of the skin was noted in both sexes at 15 and 50 mg/kg bw/day. The onset of these findings showed a dose-related trend and lasted until the end of the recovery phase at 50 mg/kg bw/day.
- Blue discolouration of the eyes was recorded for both sexes only at 15 mg/kg bw/day during three consecutive days of the premating period. This limited occurrence at 15 mg/kg bw/day may be due to an easier detection of discolouration of eyes compared to other body parts, while at 50 mg/kg bw/day generalized discolouration was more pronounced/sudden without being first detected in the eyes. This discoloration is considered to be related to the black color of the test item and not to represent a sign of toxicity.
- No findings were noted during the weekly arena observations in this study.
- No clinical signs were noted at 5 mg/kg bw/day. Incidental findings that were noted in other dose groups 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. At the incidence observed, these were not considered to be signs of toxicological relevance.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

- At 50 mg/kg bw/day, a total of 3 Main group females were found dead. One female was found dead on Day 36 prior to dosing (i.e. after 35 days of treatment). No relevant signs were noted prior to the spontaneous death for this animal. Apart from autolysis and many black foci noted on the thymus, main macroscopic and microscopic findings were comparable to the remaining animals of this treatment group and consisted of black discoloration of all organs. No clear cause of death could be established histopathologically for this animal.
- In addition, two Main females at 50 mg/kg bw/day died at terminal blood sampling (prior to scheduled necropsy). No relevant signs were noted for these animals and no cause of death could be established histopathologically. Although it is conceivable that these deaths could be related to the blood sampling procedure/anaesthesia, given that in total 3 high dose Main females were found dead in this study, a possible treatment-related effect could not be ruled out.
- One male at 15 mg/kg bw/day was found dead on the last day of treatment (on Day 29). No relevant signs were noted prior to the spontaneous death for this animal. Apart from autolysis, main macroscopic and microscopic findings were comparable to the remaining animals of this treatment group and consisted of black discoloration of all organs. In addition, an enlarged liver and thoracic cavity filled with black, watery-clear fluid was observed. The latter finding may point to a gavage-related incident although histopathologically a cause of death for this animal could not be confirmed. This death was regarded incidental and unrelated to the treatment with the test item.
- One Main female at 15 mg/kg bw/day was sacrificed due to total litter loss on PND 5; two of her pups were found dead on Day 2 and the other two pups were sacrificed in extremis on Day 5.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- At 50 mg/kg bw/day, a statistically significantly lower body weight and body weight gain was recorded for females from Day 4 or 7 of the post-coitum period onwards, respectively. Slight body weight loss (on average approximately 6% based on mean body weight) was recorded for all these females between Days 17 and 20 of the post-coitum period along with a more pronounced decrease in food intake. These effects on body weight and food intake were considered to have occurred secondary to the hampered fetal development (none of the females at this dose delivered offspring).
- At 15 mg/kg bw/day, body weight and body weight gain of females was lower than controls from the second week of the post-coitum period onwards, achieving a level of statistical significance on most occasions.
- The statistically significantly lower mean body weights and body weight gains of males at 5 and 50 mg/kg bw/day during the premating and repro period occurred in the absence of a dose-related trend; the mean body weight of the 5 and 50 mg/kg bw/day dose groups at the end of treatment were similar, and the mean body weight and body weight gain at the middose of 15 mg/kg bw/day was similar to the control mean. As such, these variations were not considered to be related to treatment. The subsequent lower starting body weight on Day 1 of the recovery phase was considered to have resulted in statistically significantly lower body weights and body weight gain for males during the recovery period. Body weight gain over Days 1-15 of the recovery period for males at 50 mg/kg bw/day based on Day 1 of the recovery period was the same as for control males (i.e. 13%).
- Occasional statistically significantly lower mean body weight gain recorded during the treatment and recovery period for females at 50 mg/kg bw/day was not consistently seen with continuation of treatment. Also, mean body weight at the end of recovery was similar to the control mean body weight. Therefore, these variations were not considered to represent an effect of treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- At 50 mg/kg bw/day, a lower food consumption (before and after correction for body weight) was recorded for females throughout the post-coitum period, achieving a level of statistical significance on most occasions. The difference in food consumption to the control mean became more pronounced over Days 17-20 of the post-coitum period.
- At 15 mg/kg bw/day, food consumption of females before and after correction for body weight was statistically significantly lower than controls during the last week of lactation.
- Variations in food consumption across male dose groups were not considered to represent an effect of treatment since these occurred in the absence of a dose-related trend and since these variations were of a minor degree.
- Any other (statistically significant) variations in food consumption were not considered to be related to treatment as these occurred in the absence of a dose-related trend, were of a minor degree and/or were not consistently recorded with continuation of treatment.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant, non-adverse changes in haematological parameters at 50 mg/kg bw/day distinguished treated from control animals at the end of treatment:
- Lower red blood cell counts in males (0.94x of control).
- Higher reticulocyte counts in males and Recovery group females (1.5x and 1.65x of control for males and females, respectively).
- Higher red blood cell distribution width (RDW) in males and Recovery group females (1.12x of control for males and females).
- Higher mean corpuscular haemoglobin (MCH) in males (1.05x of control.
- Higher mean corpuscular haemoglobin concentration (MCHC) in males (1.03x of control).
- Higher lymphocyte counts in females (1.35x of control).

At the end of recovery, mean corpuscular volume (MCV; 1.08x of controls) and mean corpuscular haemoglobin (1.06x of control) were (still) higher in males at 50 mg/kg bw/day, while affected parameters in Recovery group females had returned to control values.
The statistically significantly lower or higher lymphocyte counts in Main females at 15 and 50 mg/kg bw/day respectively at the end of treatment were not considered to be related to treatment as they did not show a dose-related trend (Groups 1-3, i.e. lactating females), were of a minor degree and were attributed to physiological differences due to pregnancy status between Main control females (lactating) and Main females at 50 mg/kg bw/day (implantation sites only).

Coagulation parameters of treated rats were not considered to have been affected by treatment.
The statistically significantly lower prothrombin time (PT) in males at 50 mg/kg bw/day at the end of recovery was absent at the end of the treatment period, and not considered toxicologically relevant given the direction of change (i.e. a more pronounced and opposite effect would be expected in case of target organ toxicity). As such, this change was not considered to be related to treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- The following (statistically significant, non-adverse) changes in clinical biochemistry parameters distinguished treated from control animals at the end of treatment:
1. Lower total protein in males and Recovery females at 50 mg/kg bw/day (0.92x and 0.95x of control for males and females, respectively)
2. Lower chloride in females at 15 mg/kg bw/day (0.95x of control).
3. Higher inorganic phosphate in non-lactating females at 50 mg/kg bw/day.

- At the end of the recovery phase, total protein remained lower for Recovery group females of the 50 mg/kg bw/day group (0.95x of control).
- Grey/black discoloration of blood samples was considered to have affected total bilirubin and cholesterol measurements. This resulted in a statistically significantly lower total bilirubin level in Main females at 5 mg/kg bw/day at the end of treatment (0.37x of control), and in Recovery males (0.45x of control) and Recovery females (0.58x of control) at 50 mg/kg bw/day at the end of recovery. Total bilirubin could not be determined at dose levels of 15 and 50 mg/kg bw/day at the end of treatment, and bile acids could not be determined at 50 mg/kg bw/day in most animals at the end of treatment. Blood sample discolouration was also considered to have resulted in lower cholesterol in males (0.38x of control) and non-lactating females (0.33x of controls) at 50 mg/kg bw/day at the end of treatment.
- Lower alanine aminotransferase (ALAT), aspartate aminotransferase activity (ASAT) and/or alkaline phosphatase activity (ALP) in males and/or females at 50 mg/kg bw/day at end of treatment or end of recovery were not considered to be toxicologically relevant since the opposite effect would be expected in case of target organ toxicity.
- 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 or were only recorded at the end of recovery (higher albumin and creatinine in males at 15 mg/kg bw/day at end of treatment, lower chloride in females at 15 mg/kg bw/day at end of treatment, and lower inorganic phosphate and calcium in Recovery females at 50 mg/kg bw/day at end of recovery).

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- At 50 mg/kg bw/day, a statistically significantly, non-adverse lower foreleg grip strength was recorded for Main females at the end of treatment (0.60x of control). It should be noted that these high dose females had implantation sites only, while selected females of Groups 1, 2 and 3 were all lactating at the time point of functional tests. Therefore values of Group 4 were compared with historical data from non-mated rats of this age and strain. Mean value for foreleg grip strength was at the lower end of the historical control range. However, the lower foreleg grip strength was considered not to be toxicologically relevant as fore leg grip strength values in nulliparous 50 mg/kg/day/day females (Recovery animals) were within the normal range for female rats of this strain and age. Moreover, there were no corroborating changes in hind limb grip strength or other end points in the neuromuscular domain.
- At 15 and 50 mg/kg bw/day (males) and 50 mg/kg (Recovery females and non-lactating Main females combined), a statistically significantly lower motor activity (total movements) was recorded at the end of treatment (0.70x and 0.72x of control for males at 15 and 50 mg/kg bw/day, respectively, and 0.91x of control for females at 50 mg/kg bw/day). Mean counts for ambulations also appeared lower than controls for males at 15 and 50 mg/kg bw/day (0.67x of control for both dose levels), but were not statistically significant. No toxicological relevance was attached to these findings because of the following reasons: all groups showed a similar motor activity habituation profile (with high activity in the first interval that decreased over the duration of the test period); relatively high values were observed for the control group; no clear dose-related trend was recorded for males, and for females the difference to the control mean was slight; means for both treated males and females remained within the range considered normal for rats of this age and strain.
- Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related, non-adverse microscopic findings after treatment were noted in the lung, spleen, testes and uterus:
- In the lungs, an increased incidence and severity of alveolar macrophage aggregations (up to moderate degree) was present in both sexes at 15 and 50 mg/kg bw/day. The single incidence at minimal degree at 5 mg/kg bw/day was regarded to be within background range. In general, at 50 mg/kg bw/day the macrophages had a foamier (finely vacuolated) appearance, without black pigment. This finding was still present up to moderate degree in males and up to slight degree in females after a 15-day treatment free period.
- In the spleen, an increased incidence and severity of extramedullary hematopoiesis (up to slight) was present in males of the 50 mg/kg bw/day group. After a 15-day treatment free period, incidences and severities were within background range.
- In th testes, black-brown pigmented macrophages in the interstitium of the testes (up to slight) were present at 50 mg/kg bw/day. This finding was still present up to slight degree in males after a 15-day treatment free period
- In the Uterus, black-brown pigmented macrophages at the implantation sites (slight degree) were present in females at 50 mg/kg bw/day.
All the above findings were considered as non-adverse, based on their mild characters and absence of any other test-item related changes indicative of tissue/organ damage.
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:

effects observed, treatment-related

Description (incidence and severity):

THYROID HORMONE ANALYSIS
- At end of treatment, significantly higher serum levels of total T4 were noted in males at 15 and 50 mg/kg bw/day (1.3x and 1.2x of control, respectively), and in non-lactating females at 50 mg/kg bw/day (2.4x of control). Concurrently, there was a trend towards lower values of TSH in treated males as compared to concurrent controls (not statistically significant), but not in treated females. It should be noted that the variation in TSH level between animals within the same group was relatively high. Therefore, and since TSH levels were comparable in offspring at PND 13-15, an effect of the test compound on TSH levels in parental males is considered unlikely. At the end of the recovery period, serum levels of T4 and TSH in males and females at 50 mg/kg bw/day were comparable in all groups.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were not considered to have been affected by treatment. Most females had regular cycles of 4 to 5 days. An irregular cycle was noted for one female at 5 mg/kg bw/day at premating (with normal litter). Given that this occurred at low incidence, without a dose-related trend and absence of an apparent correlation to pregnancy status, this finding did not indicate a relation with treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

- Mating index was not affected by treatment. All females showed evidence of mating (see table 1)
- Precoital time was not considered to be affected by treatment. All females showed evidence of mating within 4 days (see table 2)
- Number of implantation sites was not considered to be affected by treatment (see table 3)
- Fertility index was not affected by treatment. Fertility index was 100 % for the control and 15 mg/kg bw/day group and 90% for the 5 and 15 mg/kg bw/day group. One female at 5 mg/kg bw/day and one female at 15 mg/kg bw/day were not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was not considered to be related to treatment (see table 1 and 4)

Details on results (P0)

DEVELOPMENTAL DATA (see table 5 and 6)
- Gestation index and duration of gestation at dose levels up to 15 mg/kg bw/day were not considered to be affected by treatment (see table 1).
- There was one couple treated at 5 mg/kg bw/day and one couple treated at 15 mg/kg bw/day. All couples at 50 mg/kg bw/day without pregnancy. Females at 50 mg/kg bw/day showed evidence of former pregnancy in the form of implantation sites in the uterus. Total litter loss was observed in one female of the 15 mg/kg bw/day group. No abnormalities were seen in the reproductive organs, which could account for the lack of offspring or total litter loss (see table 4). There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.
- Parturition/Maternal Care: For two females at 50 mg/kg bw/day, red fluid was noted in the cage on post-coitum Day 19 or Day 21. Additionally, one of these females showed red discharge (presumably vaginal) on post-coitum Day 20. None of these females delivered live offspring, and it was considered that this observation was indicative of fetal loss. No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of other pregnant females revealed no signs of fetal loss or premature birth. No deficiencies in maternal care were observed.
- Post-implantation survival index: At 50 mg/kg bw/day, none of the implantations developed into offspring. At 15 mg/kg bw/day, the total number of offspring born compared to the total number of uterine implantations was slightly lower than the control group (87% vs. 97% in the control group, and 92% at 5 mg/kg bw/day). The mean post-implantation survival index at this dose remained at the lower end of the range considered normal for rats of this age and strain. Post-implantation survival index at 5 mg/kg bw/day was not affected by treatment.
- Litter size: At 15 mg/kg bw/day, a statistically significantly lower mean number of living pups was recorded (9.6 vs. 12.5 in the control group and 12.3 at 5 mg/kg bw/day). Although the mean remained within the range considered normal for rats of this age and strain, this was considered to be a treatment–related effect.
- Live birth index: The number of live offspring on Day 1 after littering compared to the total number of offspring born was not affected by treatment. Live birth index was 99% in the control group and at 15 mg/kg bw/day, and 100% at 5 mg/kg bw/day. One pup of the control group and one pup at 15 mg/kg bw/day were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: whole body

Organ:

other: mortality

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):

At 5 and 15 mg/kg bw/day, blue discolouration of the whole body (attributed to discolouration by the test item) was recorded for most pups on one day up to all days of lactation. The number of days on which this observation was recorded was highest at 15 mg/kg bw/day. At 15 mg/kg bw/day, a total of three pups that were sacrificed in extremis on Day 5, appeared dehydrated and/or lean, and/or had no milk in the stomach on one or several days prior to sacrifice. The nature and incidence of other clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

- Viability index: At 15 mg/kg bw/day, the number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was lower than the control mean (90% vs. 98% in the control group). This mean was outside the range considered normal for pups of this age and strain. At this dose, a total of 8 pups were found dead or missing between PND 2 and 4 Pups missing were most likely cannibalised. One control pup and one pup at 5 mg/kg bw/day were found dead on Days 4 and 3, respectively. This mortality incidence remained well within the range considered normal for pups of this age and was therefore not considered to be related to treatment.
- Lactation index: At 15 mg/kg bw/day, the number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was lower than the control mean (94% vs.
100% in the control group). This mean exceeded the range considered normal for pups of this age and strain3. At this dose, a total of 4 pups died before scheduled sacrifice and one pup was sacrificed in extremis on Day 5, and one pup of litter was found missing on Day 7 and was most likely cannibalized. One pup at 5 mg/kg bw/day was found missing on Day 6 and was most likely cannibalized. This single mortality incidence remained well within the range considered normal for pups of this age and was therefore not considered to be related to treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Pup body weights were not affected by treatment. At 15 mg/kg bw/day, mean body weights of male and female pups combined appeared slightly lower than controls on Day 13 of lactation (approximately 5% lower than controls). However, this difference was slight and not statistically significant, and mean body weights of male and female pups remained within the range considered normal for rats of this age and strain. Therefore, this was not considered to represent a treatment-related difference.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

- At 15 mg/kg bw/day, sex ratio was statistically significantly lower (35/65 vs. 55/45 in the control group). Although this finding may have occurred by chance due to a lower mean litter size and subsequent higher relative contribution of a higher number of females in these litters, it could not be excluded that this was a treatment-related effect. Sex ratio at 5 mg/kg bw/day was not considered to be affected by treatment.
- Anogenital distance (absolute and normalized for body weight) in male and female pups was not considered to be affected by treatment.
- Treatment up to 15 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Blue discolouration of the whole body was recorded for a few pups at 5 mg/kg bw/day and for most pups at 15 mg/kg bw/day. At 15 mg/kg bw/day, a total of four) that were sacrificed in extremis on Day 5 appeared dehydrated. The nature and incidence of other macroscopic findings remained within the range considered normal for pups of this age, and were therefore not considered to be related to treatment.

Histopathological findings:

no effects observed

Description (incidence and severity):

Microscopic findings recorded for the thyroid gland of the F1-pups were within the range of background pathology encountered in rats of this age and strain.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

THYROID HORMONE ANALYSIS
At 5 and 15 mg/kg bw/day, higher serum levels of T4 in male and female PND 13-15 pups were recorded. For male pups, means were a factor 1.4 and 1.6 higher than the control mean at 5 and 15 mg/kg bw/day, respectively. For female pups, means were a factor 1.3 and 1.4 higher than the control mean at 5 and 15 mg/kg bw/day, respectively. At the microscopic level, no abnormalities were noted in the thyroid gland of the F1-pups that could be related to treatment. It should be noted that in this study the control mean value (both sexes) was at the lower end of the available historical control range , and mean values for pups at 15 and 50 mg/kg bw/day (both sexes) remained within the normal range of biological variation. Therefore, the observed changes in total T4 were considered to be non-adverse.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1 REPRODUCTION DATA SUMMARY

	GROUP 1 Control	GROUP 2 5 MG/KG	GROUP 3 15 MG/KG	GROUP 4 50 MG/KG
Females paired	10	10	10	10
Females mated	10	10	10	10
Non-pregnant females	0	1	1	0
Pregnant females	10	9	9	10
Females with implantations only	0	0	0	10
Females with living pups on Day 1	10	9	9	0
Mating index (%)	100	100	100	100
(Females mated / Females paired) * 100
Fertility index (%)	100	90	90	100
(Pregnant females / Females mated) * 100
Gestation index (%)	100	100	100	0
(Females with living pups on Day 1 / Pregnant females) * 100

Table 2 PRECOITAL TIME - F0-GENERATION - POST COITUM

DAY OF THE PAIRING PERIOD   NUMBER OF FEMALES MATED	GROUP 1 CONTROL	GROUP 2 5 MG/KG	GROUP 3 15 MG/KG	GROUP 4 50 MG/KG
1	2	3	-	3
2	3	2	6	3
3	1	1	-	2
4	4	4	4	2
MEDIAN PRECOITAL TIME	3	3	2	2
MEAN PRECOITAL TIME	2.7	2.6	2.8	2.3
N	10	10	10	10

+/++ Steel-test significant at 5% (+) or 1% (++) level

Table 3 IMPLANTATION SITES SUMMARY FEMALES

AT NECROPSY		GROUP 1 CONTROL	GROUP 2 5 MG/KG	GROUP 3 15 MG/KG	GROUP 4 50 MG/KG
Implantations	MEAN	13.0	13.4	11.6	11.2
	ST.DEV	1.3	1.9	1.4	3.2
	N	10	9	9	10

Table 4 Correlation of Histopathology Findings with In-Life Reason for Males that Failed to Sire and Females that Failed to Deliver Healthy Pups.

Group	Dose level mg/kg bw/day	Female/Male nos.	In-Life Reason	Histopathology
1	0	-/-	n.a.	n.a.
2	5	74/24	Not pregnant	-/-
3	15	77/27 83/33	Not pregnant Total litter loss	-/- -/-
4	50	86/36 87/37 88/38 89/39 90/40 91/41 92/42 93/43 94/44 95/45	Implantation sites only Implantation sites only Implantation sites only Implantation sites only Implantation sites only Implantation sites only Implantation sites only Implantation sites only Implantation sites only Found dead on Day 20 post-coitum (implantation sites present)	- -/- -/- -/- -/- -/- -/- -/- -/- -/-

Table 5 Developmental data - Laction F0 Generation 

	Group 1 Control	Group 2 5 mg/kg bw		Group 3 15 mg/kg bw
LITTERS TOTAL	10	9		9
DURATION OF GESTATION
MEAN (+)	21.3		21.2		21.7
ST.DEV.	0.5		0.4		0.5
N	10		9		9
DEAD PUPS AT FIRST LITTER CHECK
LITTERS AFFECTED (#)	1		0		1
TOTAL	1		0		1
MEAN (+)	0.1		0.0		0.1
ST.DEV.	0.3		0.0		0.3
N	10		9		9
LIVING PUPS AT FIRST LITTER CHECK
% OF MALES / FEMALES (#)	55 / 45		46 / 54		35 / 65 ##
TOTAL	125		111		86
MEAN (+)	12.5		12.3		9.6 ++
ST.DEV.	1.5		2.5		2.4
N	10		9		9
POSTNATAL LOSS
% OF LIVING PUPS	0.8		0.9		9.3
LITTERS AFFECTED (#)	1		1		4
TOTAL (#)	1		1		8 ##
MEAN (+)	0.1		0.1		0.9
ST.DEV.	0.3		0.3		1.2
N	10		9		9
CULLED PUPS
TOTAL	44		38		12
LIVING PUPS DAY 4 P.P.
TOTAL	80		72		66
MEAN (+)	8.0		8.0		7.3
ST.DEV.	0.0		0.0		2.0
N	10		9		9
BREEDING LOSS DAYS 5 - 13 P.P. % OF LIVING PUPS AT DAY 4 P.P.	0.0		1.4		6.1
LITTERS AFFECTED (#)	0		1		3
TOTAL (#)	0		1		4 #
MEAN (+)	0.0		0.1		0.4
ST.DEV.	0.0		0.3		0.7
N	10		9		9
LIVING PUPS DAY 13 P.P.
% OF MALES / FEMALES (#)	53 / 48		51 / 49		35 / 65
TOTAL	80		71		62
MEAN (+)	8.0		7.9		6.9
ST.DEV.	0.0		0.3		2.6
N	10		9		9

+/++ Steel-test significant at 5% (+) or 1% (++) level

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

Table 6 Developmental data

	GROUP 1 CONTROL	GROUP 2 5 MG/KG	GROUP 3 15 MG/KG	GROUP 4 50 MG/KG
Total number of offspring born	126	111	87	0
Total number of uterine implantation sites	130	121	104	112
Number of live offspring on Day 1 after littering	125	111	86	0
Number of live offspring on Day 4 (before culling)	124	110	78	0
Number of live offspring on Day 4 (after culling)	80	72	66	0
Number of live offspring on Day 13 after littering	80	71	62	0
Post-implantation survival index (%)	97	92	84	0
(Total number of offspring born/Total number of uterine implantation sites) * 100
Live birth index (%)	99	100	99	-
(Number of live offspring on Day 1 after littering/Total number of offspring born) * 100
Viability index (%)	99	99	91	-
(Number of live offspring on Day 4 (before culling)/Number of live offspring on Day 1 after littering)*100
Lactation index (%) (Number of live offspring on Day 13 after littering/Number of live offspring on Day 4 (after culling)) * 100	100	99	94	-