Registration Dossier

Administrative data

Description of key information

OECD 422: NOAEL= 15 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
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:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
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:
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. certificate)
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 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%).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
15 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

OECD 422

A Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening test was performed in Wistar Han rats in accordance with OECD 422 and GLP principles. Additional groups with a 15 day recovery period were included for the control and high dose levels to study the reversibility of possible effects. The recovery animals (used to study the potential reversibility of possible toxic effects) were not mated and consequently were not used for the assessment of reproduction/ developmentaltoxicity. The substance was administered orally (by gavage) at dose levels of 0, 5, 15 and 50 mg/kg bw/day (referred to as group 1, 2, 3 and 4, respectively) in propylene glycol. These dose levels were based on the results of a previously performed 14 day dose range finder. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. 10 animals/ dose/ sex were used for the main groups and 5 animals/ dose/ sex for the recovery groups. The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, functional observations (for 5 selected animals/sex/group and all Recovery group animals at the end of treatment), body weight and food consumption, oestrous cycle determination, clinical pathology (for 5 selected animals/sex/group and all Recovery group animals at the end of treatment and end of recovery), measurement of thyroxine (T4) and thyroid stimulating hormone (TSH), gross necropsy findings, organ weights and histopathologic examinations.

Formulation analyses confirmed that formulations of test item in propylene glycol were prepared accurately and homogenously. At 50 mg/kg bw/day, a total of 3 Main group females were found dead. No relevant signs were noted prior to the spontaneous death for these animals, and no clear 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. Significantly higher serum levels of total T4 were recorded in non-lactating females at 50 mg/kg bw/day (2.4 x 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 (2.4 x of control) the observed increase in total T4 was considered to represent a potentially adverse, compound-related effect. Also in males, a treatment-related increase inserum levels of total T4 was observed following treatment at 15 and 50 mg/kg bw/day. The observed changes in total T4 levels were not accompanied by either clear changes in TSH or findings for the thyroid gland (organ weight, morphology). Given the relatively slight magnitude of change (i.e. 1.3 x and 1.2 x of control, respectively) and the fact that group mean values for males at 15 and 50 mg/kg bw/day remained within the historical control range, it was regarded as non-adverse. 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.

At 50 mg/kg bw/day, lower body weight and body weight gain accompanied by lower food consumption (before and after correction for body weight) was recorded for females from Week 1 of the post-coitum period onwards. Over Days 17 to 20 of the post-coitum period, these females showed a slight weight loss (on average approximately 6%) 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 from the second week of the post-coitum period onwards, which was accompanied by a lower food consumption during the last week of lactation. These findings were not regarded to be adverse, given that they were reversible and of a generally mild degree, and were considered to be related to the lower litter size at this dose. Females at 15 and 50 mg/kg also displayed piloerection starting during the mating phase, which had resolved prior to or during the first week of the recovery period. Other parental changes that were considered to be treatment-related but non-adverse consisted of black or bluish discoloration of body parts/faeces, extramedullary haematopoiesis in the spleen with associated haematological changes, higher liver weight and clinical biochemistry changes, as described in more detail below. A non-adverse black or bluish discoloration of faeces, skin and/or eyes, and all examined organs was noted in all animals at 15 and 50 mg/kg bw/day, and remained present at the end of the recovery phase at 50 mg/kg bw/day. At 5 mg/kg bw/day, discoloration was confined to bluish discolouration of the liver in a few females. This discolouration most likely represented the test item/test item metabolite as the test item had a black appearance. These findings were considered as non-adverse based on the mild character and absence of any other test-item related change indicative of tissue/organ damage. A non-adverse slightly increased incidence and severity of extramedullary haematopoiesis was recorded in the spleen of males at 50 mg/kg bw/day. Severity was low (up to slight) and showed complete recovery. This finding was supported by changes in red blood cell parameters consisting of marginally lower red blood cell counts, and higher reticulocyte counts, red blood cell distribution width, mean corpuscular haemoglobin, and mean corpuscular haemoglobin concentration at the end of treatment. Recovery females at 50 mg/kg bw/day showed higher reticulocyte counts and red blood cell distribution width at the end of treatment, but red blood cell counts were unaffected and there were no morphological correlates. Except for higher reticulocyte counts, all these haematological. changes were of a minor degree. At the end of recovery, most of these parameters showed full recovery, except for mean corpuscular volume and mean corpuscular haemoglobin which were (still) higher in males at 50 mg/kg bw/day. Overall, given the generally slight severity of these findings these were not considered to represent an adverse effect on red blood cell turn over. Non-adverse changes in clinical biochemistry at the end of treatment at 50 mg/kg bw/day consisted of lower total protein in males and Recovery females, and higher sodium in Main and Recovery females. At the end of the recovery phase, total protein remained lower for Recovery group females. None of these changes had histopathological correlates, and given that these changes were generally slight they were not considered adverse.

A non-adverse higher liver weight (relative to body weight) was noted in 15 and 50 mg/kg bw/day. Main males at the end of treatment (approximately 8 or 14% higher than controls, respectively). In absence of correlating microscopic or clinical pathology findings, and based on reversibility of this finding after the 15-day recovery period, this higher liver weight was not considered to be adverse. In conclusion, NOAEL of 5 mg/kg bw/day is derived based on mortality (Charles River Laboratories 2018).

14 day dose-range finder

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 test of in Wistar Han rats by oral gavage. The oral route was selected as it is a possible route of human exposure during manufacture, handling or use of the test item. The substance was administered as a suspension in propylene glycol at dose levels of 1000, 200, 50 and 100 mg/kg bw/day (group 2, 3, 4 and 5) subsequently to 4 males and 4 females. A vehicle control group (group 1 was taken along). Chemical analyses of formulations for Groups 1, 2 and 3 were conducted once during the study to confirm accuracy and homogeneity. No chemical analyses was performed for the last added two Groups 4 and 5. The following parameters and end points were evaluated in this study: clinical signs, bodyweights, food consumption, clinical pathology (haematology, coagulation and clinical chemistry), gross necropsy findings and organ weights. Administration of the substance was not tolerated at dose levels of 100, 200 and 1000 mg/kg bw/day. Within 3 to 5 days (1000 and 200 mg/kg bw/day) and 9 to 11 days (100 mg/kg bw/day) all animals were either found dead or euthanizedin extremis(in the 200 mg/kg bw/day group only). This high toxicity was not expected based on the available data from an acute toxicity study indicating an LD50 > 10000 mg/kg bw/day. General observations prior to their deaths included piloerection (both sexes) and hunched posture (females only), body weight loss and reduced food consumption (absolute and relative to body weight). Severity increased with higher dose, and females seemed to be more sensitive to treatment than males. 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 bluish discoloration of their bodies, including internal tissues/organs, which most likely was related to the black colour of the test item. In these groups, organ weights of liver, kidneys, adrenals and spleen could only be determined for the animals at 200 mg/kg/day that were euthanizedinextremis. Incidental changes included increased weights of liver and kidneys (one male and one female), increased weight of adrenals (one male). No data on clinical laboratory parameters are available from all these animals that died preterm. Only limited toxicity was seen at 50 mg/kg bw/day. In-life findings included piloerection (both sexes) and hunched posture (females only). At end of treatment (Day 15), blood levels of cholesterol were markedly decreased (both sexes). In addition, higher levels of bile acids were recorded in one male and all females. There were no test item-related mortalities, bodyweight and food consumption remained within the normal range, and no clear changes in haematology parameters (incl. clotting) and organ weights were noted. In conclusion, based on the results of this dose range finder, doses of 5, 15 and 50 mg/kg bw/ day were selected for the combined 28-day repeated dose toxicity study with the reproduction/ developmental toxicity screening test (Charles River Laboratories 2018).

Justification for classification or non-classification

Based on the available data, the substance has to be classified as STOT-RE2 (H373: May cause damage to organs through prolonged or repeated exposure) in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.