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REACH

Bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate

EC number: 258-207-9 | CAS number: 52829-07-9

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Description of key information

In all repeated dose oral toxicity studies in rats and dogs, administration of test material was associated with decreased body weight gain. The 28-day NOAEL for oral toxicity (gavage) in rats was 50 mg/kg bw based on reduced body weight gain and distension of stomach and small intestine. In an OECD 422 study, additional histological changes in the heart in males were reported, which were, however, not confirmed in a follow-up OECD 443 study with 10 week premating treatment. In this OECD 443, effects on body weights were also reported with a NOAEL of 36 mg/kg body weight. The 90-day NOEL for oral toxicity (gavage) in rats was < 29 mg/kg bw based on reduced body weight gain in females. However, since the study also reported inflammation in all dose groups, the reliability of this study may have been compromised, as confirmed by ECHA in their final decision (Decision number: CCH-D-2114384240-56-01/F). Finally, the 90-day NOAEL for oral toxicity (feed) in dogs was 2600 ppm (69-78 mg/kg bw) based on decreased body weight and liver hypertrophy. The most reliable systemic NOAEL was is considered the NOAEL of 36 mg/kg body weight derived in the OECD 443 study (see chapter 7.8.1).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:: sub-chronic toxicity: oral
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
Qualifier:: according to guideline
Guideline:: other: OECD 443
GLP compliance:: yes (incl. QA statement)
Limit test:: no
Species:: rat
Strain:: Wistar
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source:
Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 6 weeks
- Weight at study initiation: (P) between 138 and 174 g (males) and between 106 and 141 g (females)
- Fasting period before study: no
- Housing: On arrival, prior to mating and during the post-weaning period, animals were group housed (up to 5 animals of the same sex and same dosing group and cohort together) in polycarbonate cages (Macrolon type IV; height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (type III; height 18 cm). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, type IV; height 18 cm) with a maximum of 5 males/cage). Females were individually housed in Macrolon plastic cages (type III, height 18 cm). During the lactation phase, females were housed in Macrolon plastic cages (type III, height 18 cm). Pups were housed with the dam until termination or weaning (on PND 21). During locomotor activity monitoring, F1-Cohort 2A 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 for a maximum of 2 hours. The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles.
- Diet:ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 20 to 22°C with an actual daily mean relative humidity of 45 to 74%. The values that were outside the targeted range occurred for 19 days with a maximum of 74% and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
Route of administration:: oral: feed
Details on oral exposure:: DIET PREPARATION
For preparation of the diets for Groups 2 to 4, the test item was mixed without the use of a vehicle, directly with the required amount of powder feed, i.e. standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Animals of Group 1 (control) received this standard powder rodent diet unprocessed and as received from the supplier. The diets prepared for animals of Groups 2 to 4 were stored in the freezer (≤-15°C) until use. On the day of use, it was provided to the animals in food hoppers, where it was kept at room temperature and under normal light conditions for a maximum of 5 days. Any remaining food left after filling the food hoppers might have been stored in the animal-room in a closed bag also for a maximum of 5 days, for supplementing food during the respective food consumption measurement interval. From study Week 8 onwards and based on the stability data obtained in this study, the diets provided in the food hoppers or stored in the animal room in closed bags were kept for a maximum of 10 days. The standard powder rodent diet for Group 1 animals was taken from the stock of the animal facility, which was stored as prescribed by the supplier.
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: The concentrations analyzed in the diets of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%). Small responses at the retention time of the test item were observed in the chromatograms of the Group 1 diet prepared for use in Week 1, Week 4, Week 13, Week 24 and Week 32. It was considered not to derive from the diet since a similar response was obtained in the analytical blanks.
Duration of treatment / exposure:: F0 Males: 11-13 weeks (including 10 weeks pre-mating)
F0 Females: 16-18 weeks (including 10 weeks pre-mating)
F0 Females which failed to deliver or had total litter loss: 13-14 weeks
F1 Cohort 1A : 10 weeks
F1 Cohort 1B Males: 11-13 weeks (including 11-12 weeks pre-mating)
F1 Cohort 1B Females: 16-18 weeks (including 11-12 weeks pre-mating)
F1 Cohort 2A: 7-8 weeks
F1 Cohort 2B: n/a
F1 Cohort 3: 5 weeks
F1 Females which failed to deliver: 14-16 weeks (including 11 weeks pre-mating)
Frequency of treatment:: daily
Dose / conc.:: 500 ppm
Dose / conc.:: 1 500 ppm
Dose / conc.:: 5 000 ppm
No. of animals per sex per dose:: see below
Control animals:: yes, plain diet
Details on study design:: - Dose selection rationale:
The dose levels in this study were selected based the results of a preliminary reproductive toxicity study with dietary exposure of the test item in rats and in an attempt to produce graded responses to the test item. In this study, disruption of the estrous cycle regularity and lower implantation sites were observed at 5000 ppm and higher (highest dose level used was 15000 ppm) and reduced body weight gain of pups of pups during lactation at 15000 ppm. In the preliminary study, parental toxicity was observed as dose-related increase in red blood cells in males (maximum increase of approximately 10% at 15000 ppm) and decreased white blood cell counts in treated females (approximately 30-40% decrease at all dose levels of 1500, 5000 and 15000 ppm, with no clear dose response). Furthermore, inflammatory changes in the heart were observed in a few males among all test item-treated groups. In a single male at 1500 and 15000 ppm this finding was accompanied by myofiber necrosis. As this finding is uncommon in Wistar (Han) rats at this age, further investigations was made in the current study to determine whether this finding was of toxicological significance for this compound. Reproduction and developmental toxicity observed in the preliminary study comprised lower the regularity of the estrous cycle was disrupted and slightly lower implantation sites in female rats at dose levels of 5000 ppm and higher. At 15000 ppm, lower litter sizes were observed, the pup growth was reduced during the lactation period and T4 levels in male and female pups (at age of approximately 2 weeks) were increased.
Observations and examinations performed and frequency:: IN-LIFE OBSERVATIONS

F0 Parental Animals:

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 the cage during observation, unless necessary for identification or confirmation of possible findings. Animals 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.

CLINICAL OBSERVATIONS
Clinical observations were performed at least once daily, beginning prior to first administration of the test item and lasting throughout the treatment periods up to the day prior to necropsy. 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. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables. Clinical observations were conducted in a standard arena once before the first administration of the test item and at weekly intervals during the treatment period.

BODY WEIGHT
Animals were weighed individually on the first day of treatment (prior to administration), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 postcoitum and during lactation on PND 1, 4, 7, 14 and 21. A terminal weight was recorded on the day of scheduled necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE
Food consumption was quantitatively measured twice weekly during the first 8 weeks of premating and weekly afterwards, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21. Food spillage was estimated in all cages over the first five weeks of the study and over Week 10 by means of sieving the bedding material, including the enrichments, with a metal sieve (mesh-size 1 mm) each time the cage was cleaned. The sieved amount, assumed to be mainly powder diet remains, was weighed and recorded. In addition, food spillage was estimated at discretion of the Study Director, e.g. in case of food hopper incidents.

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

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.

F1B Parental animals:

Body Weights – Cohort 1B
Mated females were weighed individually on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21. A terminal weight was recorded on the day of scheduled necropsy.

Food Consumption – Cohort 1B
Food consumption was not determined for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was quantitatively measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14, 17 and 21.

Estrous Cycle Determination – Cohort 1B
Estrous stages were determined by examining the cytology of vaginal lavage samples. Daily vaginal lavage was performed from start of the mating period until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.

Cohabitation/Mating Procedure – Cohort 1B
Animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating after at least 10 weeks of treatment. 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. Once mating had occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males. For two couples (Male No. 321 and Female No. 666 - Male No. 596 and Female No. 916), detection of mating was not confirmed in first instance. The actual mating date was determined based on a re-evaluation of the vaginal lavage for presence of sperm cells. Consequently, these couples were separated 2 and 3 days after the actual mating date, respectively. The actual mating date was designated Day 0 post-coitum.

General Reproduction Data – Cohort 1B
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.

CLINICAL PATHOLOGY

SAMPLE COLLECTION

F0-animals and Cohort 1A animals
Blood of 10 selected animals/sex/group5 of F0-animals and Cohort 1A animals 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 in the animal facility. Due to clotting of non-serum samples of individual animals, additional blood samples were obtained in the necropsy room. The selected F0-animals and Cohort 1A animals were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. Urine was collected into a specimen vial from the 10 selected animals/sex/group of F0-animals and Cohort 1A animals5 housed in individual metabolism cages overnight (approximately 15-20 hrs) with absence of food, but water was available.

HEMATOLOGY F0 and F1A
Blood samples at a target volume of 0.5 mL were collected into tubes containing K3-EDTA as anticoagulant. Samples were analyzed for the following parameters:
White blood cells (WBC), Neutrophils (absolute), Lymphocytes (absolute), Monocytes (absolute), Eosinophils (absolute), Basophils (absolute), Large unstained cells (LUC), (absolute) Red blood cells, Reticulocytes (absolute), Red Blood Cell Distribution Width (RDW), Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelets
A blood smear was prepared from each hematology sample. Blood smears were labelled, stained, and stored. Blood smears were evaluated when required to confirm analyser results.

COAGULATION F0 and F1A
Blood samples at a target volume of 0.45 mL were collected into tubes containing citrate as anticoagulant. Samples were processed for plasma, and plasma was analyzed for Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT)

CLINICAL CHEMISTRY F0 and F1A
Blood samples at a target volume of 0.5 mL were collected into tubes containing Li-heparin as anticoagulant. Samples at a target volume of 1.0 mL were collected in tubes without anticoagulant (same sample as for thyroid hormone measurement). Blood samples were processed for plasma or serum (bile acids), which was analyzed for the following parameters: Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Alkaline Phosphatase (ALP), Total protein, Albumin, Total Bilirubin, Bile Acids, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate (Inorg. Phos)

THYROIDE HORMONE F0 and F1A
Blood samples at a target volume of 1.0 mL (same sample as for bile acid measurement) were collected into tubes without anticoagulant. Blood samples were processed for serum. Serum was used for measurement of both T4 and TSH.

URINALYSIS F0 and F1A
Urine samples were analyzed for the following parameters: Volume Specific gravity, Clarity, Colour, pH, Blood, White blood cells (WBC), Bilirubin, Urobilinogen, Protein, Ketones, Glucose, Nitrite; Sediment: White blood cells (WBC-sed.), Red blood cells (RBC-sed.), Casts, Epithelial cells, Crystals, Bacteria, Other
Sacrifice and pathology:: TERMINAL PROCEDURES

Unscheduled Deaths – F0-Generation
If necessary for humane reasons, animals were euthanized as per Test Facility SOPs. These animals were deeply anaesthetized using isoflurane and subsequently exsanguinated. They underwent necropsy, and specified tissues were retained but not weighed.

Scheduled Euthanasia – F0-Generation
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 are summarized below:
- Males (which sired or failed to sire): After successful mating and a minimum of 10 weeks of treatment.
- Females which delivered: LD 23-25.
- Females which failed to deliver (Nos. 149, 151, 202, 218): With evidence of mating: Post-coitum Day 26-28.
- Female with total litter loss (No. 119): Within 24 hours after the last pup was found dead or missing.
Except for females with total litter loss, all animals surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy. Water was available.

Necropsy – F0-Generation
All animals 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 females. In case no macroscopically visible implantation sites were present, non-gravid 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. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

Organ Weights and Tissue Collection/Preservation – F0-Generation
The organs specified (see table below) were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for Male No. 1 euthanized in poor condition or in extremis. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated. Representative samples of the tissues identified (see table below) were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution), unless otherwise indicated.

Necropsy – F1B-Generation
Scheduled necropsy of the F1-Generation of Cohort 1B was conducted on the following days:
- F1-Males (which sired or failed to sire): Following completion of the mating period.
- F1-Females which delivered: LD 21-23
- F1-Females which failed to deliver with evidence of mating (No. 940): Post-coitum Day 27.
- F1-Females which failed to deliver without evidence of mating (No. 854): 24 days after the last day of the mating period.
Due to the preterm death of one Cohort 1A female in Groups 2 and 4 each, only 24 females (instead of 25) were available for mating in these two groups. Consequently, Male Nos. 409 (Group 2) and 585 (Group 4) were
not used for mating. The F1-animals of Cohort 1B were not deprived of food overnight before necropsy. The animals were weighed and deeply anaesthetized using isoflurane and subsequently exsanguinated.
The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites are present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea were recorded in addition.

HISTOLOGY
F0- and F1-Generation
Tissues mentioned in the table below from a selection of the F0- and F1-animals were embedded in paraffin, sectioned at a thickness of 2-4 micrometers, mounted on glass slides, and stained with hematoxylin and eosin (HE).

HISTOPATHOLOGY
F0- and F1-Generation
All tissues mentioned in the table below from a selection of the F0- and F1-animals were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. A peer review on the histopathology data was performed by a second pathologist.
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% or 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed according to sex and occasion. Descriptive statistics number, mean and standard deviation 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 3 observations.

The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Group 5 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). For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.

Non-Parametric: Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).

Incidence: An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Clinical signs:: effects observed, non-treatment-related
Description (incidence and severity):: No test item-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations up to 5000 ppm. Incidental findings that were noted included scabs, scales, wounds, alopecia, black discoloration and discharge of the eye, exophthalmos, and piloerection. These findings 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 considered not to be signs of toxicological relevance.
Mortality:: mortality observed, non-treatment-related
Description (incidence):: No test item-related mortality occurred during the study period up to 5000 ppm. One control male was (No. 01) sacrificed moribund for humane reasons (exophthalmos of the right eye).
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: Body weights and body weight gain of treated animals were affected by treatment with the test item at all tested diet concentrations.
Males:
In males at 500 ppm, body weights progressively decreased from 0.95x (Week 5) to 0.92x (Week 13) of control and body weight gain was decreased to 0.76-0.85x of control from Week 2 until 13, resulting in a terminal body weight of 0.92x of control. At 1500 ppm, body weights progressively decreased from 0.96x (Week 4) to 0.92x (Week 13) of control and body weight gain was decreased to 0.86-0.91x of control from Week 2 until 13, resulting in a terminal body weight of 0.92x of control. At 5000 ppm, body weights progressively decreased from 0.93x (Week 2) to 0.88x (Week 13) of control and body weight gain was decreased to 0.71-0.81x of control from Week 2 until 13 of treatment, resulting in a terminal body weight of 0.87x of control.

Females:
In females at 500 ppm, body weights and body weight gain were decreased compared with control to 0.95x and to 0.85-0.92x of control, respectively, during Week 3, 4 and 7 of the premating period. At the end of the post-coitum period, body weights were 0.95x of control. At 1500 ppm, body weights and body weight gain were lower than control from Week 2 of the pre-mating period onwards. Mean body weights were 0.93-0.96x of control during Week 2-9 and 11 and body weight gain 0.76-0.92x of control during Week 2-11. During the post-coitum and lactation period, body weights were decreased to 0.92-0.93x and 0.94-0.96x of control, respectively, resulting in a terminal body weight of 0.94x of control. No statistically significantly differences were observed in body weight gains. At 5000 ppm, body weights and body weight gain were lower than controls from Week 2 and 3 of the pre-mating period onwards, respectively. Mean body weights were decreased to 0.92-0.96x of control during Week 2-9 and 11 and body weight gain 0.83-0.90x of control during Week 3-9 and 11. The decreased body weights and body weight gains persisted throughout the post-coitum and the lactation period. Body weights were decreased up to 0.88x of control at the end of the post-coitum period and were decreased to 0.90x of control at the start of the lactation period up to 0.96x of control at the end of the lactation period. Body weight gains were lower than control (0.56-0.86x) during the post-coitum period and were increased during the lactation period reaching 1.70x of control at lactation Day 21, resulting in a terminal body weight of 0.93x of control.
Food consumption and compound intake (if feeding study):: effects observed, treatment-related
Description (incidence and severity):: Food consumption before or after correction for body weight were affected by treatment with the test item from 1500 ppm.

Males:
In males at 1500 ppm, absolute and relative food consumption were decreased during Week 1 of treatment (0.89x of control). Absolute food consumption recovered to normal levels in the following weeks, but relative food consumption was increased on several occasions (Week 2, 7-8 and 9-11, 1.07-1.09x of control). At 5000 ppm, absolute and relative food consumption were decreased during Week 1 of treatment (0.78x of control). While absolute food consumption levels were normal in the following weeks, relative food consumption was increased until Week 11 of treatment (1.05- 1.15x of control).

Females:
In females at 1500 ppm, absolute and relative food consumption were decreased during Week 1 of treatment (0.86x and 0.89x of control, respectively). While absolute food consumption recovered to normal levels in the following weeks, relative food consumption was increased on several occasions during the pre-mating period (Week 2, 3-4 and 8-10, 1.11-1.14x). During the post-coitum period, absolute food consumption was decreased during Days 0-11 to 0.84-0.88x of control while relative food consumption was only decreased during Days 7-11 to 0.88x of control. At 5000 ppm, absolute and relative food consumption were decreased during Week 1 of treatment (0.86x and 0.88x of control, respectively). During the following weeks, absolute food consumption recovered to normal levels until the end of the pre-mating period (0.93x of control), while relative food consumption was increased during most weeks of the pre-mating period (Week 2, 3-6 and 7-10, 1.09-1.15x of control) but was decreased at the end of the premating period (0.91x of control). During the post-coitum period, absolute (Days 0-11 and 17- 20) and relative (Days 0-11) food consumption levels were decreased to 0.79-0.86x and 0.87- 0.89x of control, respectively. Any other statistically significant changes in food consumption before or after correction for body weight were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment. In addition, changes observed in males during Week 4 and in females during lactation Days 1-4 were considered to be the result from slightly higher control.
Haematological findings:: effects observed, non-treatment-related
Description (incidence and severity):: No test item-related changes were observed in hematological parameters up to 5000 ppm. The statistically significant increase in mean corpuscular haemoglobin concentration (MCHC) in females at 500 ppm was considered unrelated to treatment as there was no dose related trend. Note: For the female control group, only 4 out of 10 samples were available due to clotting of the remaining samples. This somewhat lower number of samples did not affect the evaluation of the results since all data were within the historical control range.
Coagulation parameters of treated rats were unaffected by treatment with the test item up to 5000 ppm.
Clinical biochemistry findings:: effects observed, non-treatment-related
Description (incidence and severity):: Clinical biochemistry parameters of treated rats were not affected by treatment with the test item up to 1500 ppm.
In males at 5000 ppm, a statistically significant increase in total bilirubin (1.19x of control) and bile acid (2.14x) concentrations were observed. Mean values remained within the historical control range. In females at 5000 ppm, a statistically significant increase in alanine aminotransferase activity (ALAT; 1.75x of control) was observed. Mean values exceeded the historical control range. In females, potassium concentrations were increased at 500, 1500 and 5000 ppm. As all values were within the historical control range and in absence of a dose response, these changes were considered unrelated to treatment. The statistically significant decrease in albumin concentration was considered to be unrelated to treatment with the test item as it occurred in the absence of a dose-related trend.
Endocrine findings:: effects observed, non-treatment-related
Description (incidence and severity):: Thyroid hormone analyses:
Serum T4 levels were lower in males (0.82x of control) and females (0.66x) at 5000 ppm. Mean values at 5000 ppm remained within the historical control range for both males and
females. In males, this difference could be contributed to the relatively high serum T4 levels in concurrent controls compared to the historical control mean. Mean T4 levels at 5000 ppm were similar to historical control mean and therefore, the observed difference was considered unrelated to treatment. For females, similarly, a relatively high control mean compared to historical control mean was observed, partly contributing to the observed lower T4 values at 5000 ppm. However, as the mean T4 levels at 5000 ppm were also slightly decreased compared to historical control mean (0.84x), a treatment related effect could not be excluded. However, as values remained within the historical control range12, this potential effect was considered non-adverse. The statistically significantly decreased TSH serum levels in males at 1500 ppm, were considered unrelated to treatment with the test item as no dose response was observed.
Urinalysis findings:: no effects observed
Description (incidence and severity):: Urinalysis parameters of treated rats were unaffected by treatment with the test item up to 5000 ppm.
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: The organ weight changes as depicted in the table below were all considered to be the result of a lower terminal body weight. For some organs (brain, spleen, adrenal glands, testes, epididymides) an increased organ/relative to body weight was noted, with minor or no changes in absolute weights. In other organs (heart, liver, thyroid gland, kidneys, ovaries) the decrease in body weight resulted in lower absolute organ weights, with minor or no changes in the organ/relative to body weight. Any other organ weight changes (seminal vesicles, thymus) were observed in the lowest dose group only without a dose response. There were no macroscopic of microscopic findings in any of these organs, further indicating these organ weight changes were in line with the decrease in terminal body weight and not directly test item-related.
Gross pathological findings:: no effects observed
Description (incidence and severity):: There were no test item-related gross observations. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. Watery fluid in the uterus, found in four control and 500 ppm, in one 1500 ppm and in five 5000 ppm females, is related to a stage in the estrous cycle and is a normal finding.
Histopathological findings: non-neoplastic:: no effects observed
Description (incidence and severity):: There were no test item-related microscopic observations. All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Details on results:: For findings in F1 animals see chapter 7.8.1
Dose descriptor:: NOAEL
Effect level:: 500 ppm
Based on:: test mat.
Sex:: male/female
Basis for effect level:: body weight and weight gain; food consumption and compound intake
Remarks on result:: other: corresponding to 36 mg/kg bw in males and 41 mg/kg bw in females
Critical effects observed:: not specified

Reference 2

Endpoint:: sub-chronic toxicity: oral
Type of information:: experimental study
Adequacy of study:: key study
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: comparable to guideline study with acceptable restrictions
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:: yes
Remarks:: limited number of parameters investigated compared to current guidelines, stability and homogeneity of the test substance formulation was not determined, Inflammation observed in all animals, including control group
GLP compliance:: no
Limit test:: no
Species:: rat
Strain:: Sprague-Dawley
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: not indicated
- Weight at study initiation: males 156-210 g, females 148-187 g
- Housing: five to a cage (unless the number was reduced by mortality) In suspended cages with wire-mesh floors.
- Diet: powdered laboratory rat food, Spratt's Laboratory Diet 2, ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 50 ± 5
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:: oral: feed
Vehicle:: unchanged (no vehicle)
Details on oral exposure:: DIET PREPARATION
A pre-mix containing 20000 ppm was prepared each week and from this various dietary concentrations were obtained by direct dilution with further quantities of diet, homogeneity being achieved by mixing for 10 minutes in a rotary double-cone blender. The diets were then stored until use in heat-sealed, opaque polythene bags.
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: Samples of the diets fed to the rats containing test material were added to Methyl-ethylketon and boiled for 30 minutes at reflux. After cooling the mixture was made up with Methyl-ethylketon to the starting-weight and the content of test substance was determined by gaschromatography. The estimated relative reproducibility of this method is ± 10% or better. Within the limits of error of the sampling technique and the analytical method there was good correspondence between the concentrations found in the diets and the nominal values.
Duration of treatment / exposure:: 13 weeks
Frequency of treatment:: continuously
Dose / conc.:: 400 ppm
Remarks:: corresponding to 26 mg/kg bow in males and 29 mg/kg body weight in females
Dose / conc.:: 1 300 ppm
Remarks:: corresponding to 80 mg/kg bow in males and 90 mg/kg body weight in females
Dose / conc.:: 4 000 ppm
Remarks:: corresponding to 261 mg/kg bow in males and 277 mg/kg body weight in females
No. of animals per sex per dose:: - 20/sex/dose
- 5/sex additionally for control and high dose groups for recovery
Control animals:: yes, plain diet
Details on study design:: - Post-exposure recovery period in satellite groups: 4 weeks on 5 males and 5 females from control and high dosage level groups.
Observations and examinations performed and frequency:: CAGE SIDE OBSERVATIONS: Yes
All signs of ill-health or reaction to treatment together with any behavioural changes, were recorded. Any rat showing signs of severe debility or intoxication was isolated. Animals in extremis were sacrificed to prevent cannibalism. All mortalities were recorded as date and nature of death. All rats found dead, or killed for humane reasons, were subjected to detailed macroscopic examination in an attempt to define the cause of death. A full spectrum
of tissue samples was preserved and macroscopically abnormal tissues were subjected to histological examination.

BODY WEIGHT: Yes
The weight of each rat was recorded initially and at weekly intervals thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The quantity of food consumed by each cage of rats was recorded and the mean weekly intake calculated.

FOOD EFFICIENCY:
The efficiency with which food was utilized was assessed by calculation of mean food conversion ratios (FCR values), as weights of food consumed per unit gains in bodyweight.

WATER CONSUMPTION: Yes
Water consumption was measured during weeks 5 and 12 for each cage of Groups 1 (Control) and 4 (4000 ppm). and the mean daily intake calculated.

OPHTHALMOSCOPIC EXAMINATION: Yes
Before treatment commenced and during weeks 4, 8 and 12 the eyes of all male and female rats from Groups 1 (Control) and 4 (4000 ppm) were examined using a Keeler indirect ophthalmoscope.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During weeks 4, 8 and 12
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 10 males and 10 females from Groups 1 (Control) and 4 (4000 ppm)
- Parameters examined: Packed cell volume, Haemoglobin, Red cell count, Mean corpuscular haemoglobin concentration, mean cell volume, total white cell count, differential count, platelet count, thrombotest.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During weeks 4, 8 and 12
- Animals fasted: Yes
- How many animals: 10 males and 10 females from Groups 1 (Control) and 4 (4000 ppm)
- Parameters examined: Plasma Urea, Plasma Glucose, Total serum proteins, Serum protein electrophoresis and AG ratio, Serum alkaline phosphatase, Serum glutamic - pyruvic transaminase, Sodium, Potassium.

URINALYSIS: Yes
- Time schedule for collection of urine: During weeks 4, 8 and 12
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters examined: pH, Specific gravity, Protein, Reducing substances, Glucose, Ketones, Bile pigments, Urobilin, Blood Pigments. Microscopy of spun deposit - after centrifugation at 1000 rev/minute for 10 minutes, the deposit was examined for: epithelial cells, polymorphonuclear leucocytes, mononuclear leucocytes, erythrocytes, organisms, casts, abnormal constituents, sperms.
Sacrifice and pathology:: GROSS PATHOLOGY: Yes
After treatment for 13 weeks, 20 males and 20 females from each group were killed. The remaining 5 female and 5 male rats from the control and high dosage levels were killed after a further 4 week observation period.
- Organ weights: adrenals, brain, heart, kidneys, liver, ovaries, pituitary, spleen, testes, thymus, thyroid.
- Macroscopic: on all animals adrenals, aorta, brain, caecum, colon, duodenum, eye, femur, heart, ileum, jejunum, heart, kidneys, liver, lungs, lymph nodes, mammary gland, oesophagus, ovaries, pancreas, peripheral nerve (sciatic), pituitary, prostate (or uterus), salivary glands, seminal vesicles, skeletal muscle, skin, spleen, stomach, testes, thymus, thyroid, tongue, trachea and urinary bladder.

HISTOPATHOLOGY: Yes
Samples of the following tissues (together with any other macroscopically abnormal entity) were preserved in buffered 10% formalin (except eyes, which were preserved in Davidson's fixative): adrenals, aorta, bone marrow, brain, caecum, duodenum, eye, heart, ileum, heart, kidneys, liver, lungs, lymph nodes, oesophagus, ovaries, pancreas, pituitary, prostate (or uterus), spleen, stomach, testes, thymus, thyroid, tongue and urinary bladder. In the first instance, microscopic examination was confined to: (i) all rats that died, in an attempt to determine cause of death, (ii) all rats of Groups 1 (Control) and 4 (4000 ppm) killed after treatment for 13 weeks, (iii) all rats of Groups 1 (Control) and 4 (4000 ppm) killed at the end of the 4 week recovery period.
Statistics:: Student's t test was employed to assess the significance of intergroup differences where the data suggested evidence of a response to treatment.
Clinical signs:: no effects observed
Description (incidence and severity):: There were no visible signs of reaction to treatment.
Mortality:: mortality observed, non-treatment-related
Description (incidence):: One control male rat died during the removal of a blood sample during week 8. One female receiving 400 ppm was found dead in week 5 and one female receiving 1300 ppm was sacrificed during week 11 because of a suspected broken jaw. Autopsy of these rats revealed no macroscopic change that could be related to treatment.
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: Male rats receiving the test article at 4000 ppm and all three treated female sub-groups had a lower rate of bodyweight gain than that of the controls (statistically significant decreased (17%) in males at 4000 ppm and 13%, 23% and 24% in females at 400, 1300 and 4000 ppm respectively). Males receiving 400 or 1300 ppm gained weight at a rate comparable with that of the controls. During the four week withdrawal period, both males and females from the high dosage level group had a rate of bodyweight gain which was superior to that of the controls.
Food consumption and compound intake (if feeding study):: effects observed, treatment-related
Description (incidence and severity):: A marginally lower food intake was recorded during the treatment period for both sexes receiving the test material at 1300 or 4000 ppm, although the difference from the control intake only attained a level of statistical significance in females receiving 4000 ppm (reduction of 8%). During the withdrawal period, food consumption among the high dosage level males remained at a similar level, whereas females which had received 4000 ppm had a superior food intake to that of the controls.
Food efficiency:: effects observed, non-treatment-related
Description (incidence and severity):: During the treatment period, male rats receiving 4000 ppm and all females receiving the test item had an inferior efficiency of food utilization when compared with that of the controls. During the withdrawal period males and females which had received 4000 ppm had a superior efficiency of food utilization when compared with that of the controls.
Water consumption and compound intake (if drinking water study):: no effects observed
Description (incidence and severity):: Water intake recorded during weeks 5 and 12 was comparable in controls and rats receiving test material at 4000 ppm.
Ophthalmological findings:: no effects observed
Description (incidence and severity):: No abnormalities of the eyes were detected that were considered to be related to treatment.
Haematological findings:: effects observed, non-treatment-related
Description (incidence and severity):: When compared with control values, minimal increases in RBC count, PCV and Hb levels were observed at weeks 4, 8 and 12 in females receiving 4000 ppm. Although attaining a level of statistical significance, all values were within 'normal' limits for the strain and age of rat employed. The differences were, therefore, considered to be fortuitous and without biological significance. All other parameters measured were within 'normal' limits.
Clinical biochemistry findings:: effects observed, non-treatment-related
Description (incidence and severity):: Some small differences between values for controls and rats receiving the test compound at 4000 ppm attained a level of statistical significance. As these differences were not seen consistently at the 4, 8 and 12 weeks investigations and all values were within 'normal' limits, these intergroup differences were not considered to be of toxicological significance.
Urinalysis findings:: no effects observed
Description (incidence and severity):: All parameters studied were comparable in controls and rats receiving test material at 4000 ppm.
Behaviour (functional findings):: no effects observed
Immunological findings:: not examined
Organ weight findings including organ / body weight ratios:: effects observed, non-treatment-related
Description (incidence and severity):: There were no organ weight changes recorded in rats sacrificed at 13 weeks which could clearly be attributed to treatment. The differences between control and treated rats in both absolute and relative organ weight values, which attained a level of statistical significance were considered to reflect the lower bodyweight of the treated rats rather than a direct effect of treatment (liver decreased in males (13%) at 4000 ppm and in females (10%) at 4000 ppm; kidney statistically significant decreased in males (15%) at 4000 ppm and in females (9-12%) at 1300 and 4000 ppm; adrenals statistically significant decreased in females (15-16%) at 1300 and 4000 ppm). When expressed relative to body weight, the brain was statistically significant increased in males (7%) at 4000 ppm and in females (8-13%) at 1300 and 4000 ppm. The gonads were statistically significant increased in males (9%) at 4000 ppm and in females (14%) at 1300 ppm. When expressed relative to brainweight, the only values which differed significantly from control values were organs in which a decreased weight was recorded from control values in the absolute data. Thus indicating that organ weight differences were primarily the result of disparities in the group bodyweights. At the end of the recovery period, there were no treatment-related differences in organ weights between control rats and rats that had received the test substance at 4000 ppm. The increased thyroid weight recorded in the treated male group was considered to be fortuitous, since at the 13 week kill, the weight of this organ in treated rats was comparable with that of the controls.
Gross pathological findings:: no effects observed
Description (incidence and severity):: There were no treatment-related macroscopic findings at autopsy after 13 weeks of treatment and after the recovery period.
Neuropathological findings:: not examined
Histopathological findings: non-neoplastic:: effects observed, non-treatment-related
Description (incidence and severity):: After both 13 weeks and 17 weeks, no histopathological change or variation from normal was seen in the tissues examined that was considered to be related to the administration of test material. In both treated and control animals foci of inflammatory cell infiltration, peribronchiolar lymphoid hyperplasia and perivascular accumulation of lymphocytes in the lungs, vacuolated and distended hepatocytes (males) and incidentally minimal chronic inflammatory cell infiltration in the interstitial tissue of the kidneys were observed. These changes were considered to be those that occur commonly in the rat and were of no significance.

Details on histopathology, week 13
The following comments are made in summary:
Lungs: Foci of chronic inflammatory cell infiltration, peribronchiolar lymphoid hyperplasia and perivascular accumulations of lymphocytes were seen in the lungs of the majority of animals and were associated in some instances with patchy consolidation, groups of distended macrophages, bronchiectasis, collapse and bronchiectatic abscess formation. In one male control rat pleurisy was seen; there was evidence of haemorrhage in a male high dose rat and sparse cellular exudate in the bronchioles in a female high dose rat. Foci of dystrophic mineralisation were seen in the media of the pulmonary artery of several animals from all groups. These changes were recognised as those that occur commonly in the lungs of the rat. They were related to chronic respiratory infection and were of no toxicological significance.
Liver: Vacuolated and distended hepatocytes were seen in males from both control and treated groups and foci of chronic inflammatory cell Infiltration were also noted in occasional animals. An Infarct was seen in a female control rat and a trace of fat was seen in another female control rat. These changes were considered to be those that occur commonly in the liver of the rat and were of no significance.
Kidneys: Minimal chronic Inflammatory cell infiltration was seen in the Interstitial tissue of the kidneys of occasional animals from both control and treated groups and was associated with occasional distended tubules containing eosinophilic material and tubules characterised by the basophilic staining of the cells of the epithelium. Foci of dystrophic mineralisation were seen at the corticomedullary junction in several females from all groups. Hydronephrosis was noted in female control rat. These changes were considered to be those that occur commonly in the kidneys of the rat and were of no significance.
Other morphological entities observed but not considered to be significant included:
chronic inflammatory cell Infiltration in the pancreas in one male control and one make high dose rat; macrophages containing brown pigment and foci of extra-medullary haemopoiesis in the spleens of occasional animals from both control and treated groups; foci of chronic inflammatory cell infiltration In the myocardium and the pericardium In occasional rats from both control and treated groups; hyperplastic lymph nodes from occasional animals in both control and
treated groups; congestion in thymus of a frmale control and a male high dose rat; epithelial whorl In the thyroid of another male control rat.

Details on histopathology, week 17
The following comments are made in summary:
No histopathological change or variation from normal was seen in the tissues examined that was considered to be related to the administration of the compound under test.
Lungs: Peribronchiolar lymphoid hyperplasia and perivascular and subpleural accumulations of lymphocytes were seen in the lungs of the majority of animals examined. Bronchopneumonic consolidation was seen in two high dose males. Foci of dystrophic mineralisation were seen In the media of the pulmonary artery in a female control. These changes were recognised as those that occur commonly in the lungs of laboratory rats, they showed no evidence of relationship to treatment and hence were disregarded from a point of view of the experiment.
Dose descriptor:: NOEL
Effect level:: < 29 mg/kg bw/day (nominal)
Sex:: male/female
Basis for effect level:: body weight and weight gain
Dose descriptor:: LOEL
Effect level:: 29 mg/kg bw/day (nominal)
Sex:: male/female
Basis for effect level:: body weight and weight gain
Critical effects observed:: not specified
Conclusions:: A LOEL of 29 mg/kg was derived based on body weight changes.
Executive summary:: In a 90-day oral similar to OECD 408, study groups of 20 male and 20 female Sprague-Dawley rats were exposed daily to the test article at 0, 400, 1300 and 4000 ppm (males 0, 26, 80 and 261 mg/kg body weight; females 0, 29, 90 and 277 mg/kg body weight) via their diet during 90 days. Additional 5 male and female animals in the control and high-dose group (4000 ppm) were allowed a 4-week recovery period. No treatment-related effect was seen on mortality or clinical observations. A statistically significant decrease in body weight gain compared to the control group was observed in males exposed to 4000 ppm (17%) and all treated females (13%, 23% and 24% at 400, 1300 and 4000 ppm respectively). Food consumption was statistically significantly reduced in females (8%) during week 1-13 at 4000 ppm. No treatment-related effect was observed for hematological parameters, urinalysis and clinical chemistry (conducted only in control and highest dose group). Liver, kidney, adrenals, showed decreased absolute weights compared to the control group at 4000 ppm in males and 1300 and 4000 ppm in females, however this is expected to be due to the body weight gain decrease rather than to be due to the test substance and is also reflected in an relative increase compared to control of brain and gonads. No treatment-related effects were noted after the recovery period. At macroscopic or histopathological examination no treatment-related abnormalities were noted. The LOEL was determined at 29 mg/kg bw based on the decreased body weight gain in females. The study had some limitations due to a background inflammation in all animals including control (inflammatory cell infiltration in lungs and kidney). However these findings did not seem to impair the results of the study and were considered of no significance.

Reference 3

Endpoint:: sub-chronic toxicity: oral
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 1974
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: comparable to guideline study with acceptable restrictions
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
Deviations:: yes
Remarks:: limited parameters, stability, homogeneity and accuracy of the test substance formulation was not determined
GLP compliance:: no
Limit test:: no
Species:: dog
Strain:: Beagle
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: internal breeding facility
- Age at study initiation: 7-13.5 months
- Weight at study initiation: males 11-15.1 kg, females 8.7-13 kg
- Housing: individually housed in metabolism cages
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
not specified
Route of administration:: oral: feed
Vehicle:: unchanged (no vehicle)
Details on oral exposure:: DIET PREPARATION
The diet was prepared from powdered Purina Dog meal mixed with 15% dry malt (Diamalt OCD) and 10% water.
A 2 kg pre-mix of the dog meal with compound at the appropriate levels was prepared and mixed for 1 hour. This was then added to the bulk diet and the whole mixed for a further 0.5 hour (water being added during this time). The mixture was then pelleted and dried for 12 hours at a temperature not exceeding 45°C. Control diet was prepared in the same manner without addition of test substance.
Analytical verification of doses or concentrations:: no
Duration of treatment / exposure:: 13 weeks
Frequency of treatment:: continuously
Dose / conc.:: 800 ppm
Remarks:: corresponding to actual intake males 27 mg/kg bw and females 27 mg/kg bw
Dose / conc.:: 2 600 ppm
Remarks:: corresponding to actual intake males 69 mg/kg bw and females 78 mg/kg bw
Dose / conc.:: 5 000 ppm
Remarks:: corresponding to actual intake males 150 mg/kg bw and females 155 mg/kg bw
The initial high dose of 8000 ppm was unpalatable and was therefore reduced to 2600 ppm on day 43 and increased to 5000 ppm on day 50 until the end of th estudy period.
No. of animals per sex per dose:: 4/sex/dose for low and intermediate dose
5/sex/dose for control and high dose (extra animals for recovery period)
Control animals:: yes
Details on study design:: Post-exposure period: 4 weeks
Observations and examinations performed and frequency:: CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE
- Time schedule for examinations: daily

FOOD EFFICIENCY:
Calculated for 2 weeks pre-test and then for each month of the trial using the formula (Body weiqht gain x 100)/ (Total food consumed)

OPHTHALMOSCOPIC EXAMINATION: Yes
An ophthalmic and hearing examination was carried out on all animals pre-test and during weeks 6, 9 and 13. The animals on the recouery experiment were also examined during week 17.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: pre-test and during weeks 5, 9 and 13. Samples were also taken from the dogs on the recovery experiment (two animals from each of groups 1 and 4) during week 17.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all animals
- Parameters checked: Haemoglobin, Erythrocytes, Haematocrit, Reticulocytes, Inclusion Bodies, Thrombocytes, Leucocytes (total count and differential count), Prothrombin time, ESR

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: pre-test and during weeks 5, 9 and 13. Samples were also taken from the dogs on the recovery experiment (two animals from each of groups 1 and 4) during week 17.
- Animals fasted: No data
- How many animals: all animals
- Parameters checked: Sodium, Potassium, Glucose, Urea, SGOT, SGPT, SAP, Serum proteins (total and electrophoresis), Cholesterol

URINALYSIS: Yes
- Time schedule for collection of urine: pre-test and during weeks 5, 9 and 13. Samples were also taken from the dogs on the recovery experiment (two animals from each of groups 1 and 4) during week 17.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked: pH, specific gravity, protein, glucose, bilirubin, ketones, blood, urine sediment

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:: - Organ weights: adrenals, brain, heart, kidneys, liver, ovaries, pituitary, spleen, testes, thymus, thyroid, prostate and uterus

- Macroscopic: on all animals: adrenals, aorta, brain, colon, eye, heart, kidneys, liver, lungs, lymph nodes, mammary gland, ovaries, pancreas,
peripheral nerve (sciatic), pituitary, prostate (or uterus), small intestine, skeletal muscle, spleen, spinal cord, stomach, testes, thymus, thyroid and
urinary bladder

- Microscopic: on all animals: adrenals, aorta, bone marrow, brain, colon, eye, heart, kidneys, liver, lungs, lymph nodes, mammary gland, ovaries,
pancreas, peripheral nerve (sciatic), pituitary, prostate (or uterus), small intestine, skeletal muscle, spleen, spinal cord, stomach, testes, thymus,
thyroid and urinary bladder
Statistics:: Laboratory parameters and organ weight data were analysed by the Mann Whitney U test, in those cases where it was considered that meaningful results might be obtained.
Clinical signs:: no effects observed
Description (incidence and severity):: No mortalities and no treatment related abnormalities occurred.
Mortality:: no mortality observed
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: At 8000 ppm the diet proved so unpalatable that the dogs refused to eat it, consequently food intake and body weight fell and became so extreme as to threaten survival. On day 43 the dietary concentration of the test compound was reduced to 2600 ppm. At this level food intake and body weights improved dramatically. On day 50 the dietary concentration was increased to 5000 ppm. At this level food intake fell slightly but improved as the trial progressed. From this time body weight gains were satisfactory. At 2600 ppm and 800 ppm there was no significant difference in food consumption and body weight gains compared to controls.
Food consumption and compound intake (if feeding study):: effects observed, treatment-related
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: effects observed, non-treatment-related
Description (incidence and severity):: Mild conjunctivitis in all groups (including controls), not related to treatment.
Haematological findings:: effects observed, treatment-related
Description (incidence and severity):: In the high dose group at week 5 a slight decline in red cell parameters (haemoglobin, erythrocyte and haematocrit) was recorded in females only (P < 0.05). The ESR was increased in a few individuals. By week 13 these differences were no longer significant and were considered to be due to the poor food intake during the first few weeks of the trial. At week 5 there was a marked increase in ESR in one male animal of the 2600 ppm group only.
Clinical biochemistry findings:: effects observed, treatment-related
Description (incidence and severity):: In female animals of the high dose the serum protein levels were slightly below controls throughout the test. This may also be attributed to poor food intake. At 2600 ppm, serum proteins in females were marginally lower than controls, all values were within normal limits.
Urinalysis findings:: no effects observed
Description (incidence and severity):: No treatment related effects recorded.
Behaviour (functional findings):: no effects observed
Immunological findings:: not examined
Organ weight findings including organ / body weight ratios:: effects observed, non-treatment-related
Description (incidence and severity):: Differences observed in some organ weights (adrenals, ovaries, uterus, heart and spleen) could not be attributed to treatment due to high inter-individually variations and small group size with large standard deviations.
Gross pathological findings:: effects observed, non-treatment-related
Description (incidence and severity):: At autopsy no changes caused by the administration of the test compound were seen. Lungs granuloma in 1 female at 8000 ppm, 1 female at 2600 ppm and 2 males and 1 female at 8000 ppm.
Neuropathological findings:: not examined
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: On histopathological examination the only change due to the administration of the test compound was a minimal hypertrophy of periportal hepatocytes in high dose animals which is considered to be an adaptive phenomenon.
Dose descriptor:: NOAEL
Effect level:: 69 mg/kg bw/day (nominal)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: body weight and weight gain; histopathology: non-neoplastic
Dose descriptor:: LOAEL
Effect level:: 150 mg/kg bw/day (nominal)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: body weight and weight gain; histopathology: non-neoplastic
Critical effects observed:: not specified
Conclusions:: NOAEL 2600 ppm (69-78 mg/kg bw) based on effects on body weight gain decrease and liver hypertrophy.
Executive summary:: In a repeated dose toxicity study similar to OECD TG 409, male and female dogs (4/sex/dose) were exposed to the test substance at 0, 800, 2600 and 8000 (the latter reduced to 2600 ppm in week 7 and then increased up to 5000 ppm from week 8 onwards) ppm via their diet during 90 days. This equals daily dosages of 0, 27, 69 and 150 mg/kg bw for males and 0, 27, 78 and 155 mg/kg bw for females. In addition, two animals (one male and one female) in the control and high-dose groups were included for a 4-week recovery period. No mortality occurred. No treatment-related clinical signs were observed. At 8000 ppm the diet proved so unpalatable that the dogs refused to eat it, consequently food intake and body weight decreased and threatened survival. In consequence, the dietary concentration of the test compound was adjusted as described above. However, the average daily test substance intake in the high dose group remained relatively constant. After 13 weeks, body weight gain was decreased in males and females in the high dose group (11 and 16%, respectively). A slight and transient (5th and 9th week) decrease in red cell parameters (haemoglobin, packed cell volume and red blood cells) was noted in females in the high dose group (week 5 and 9). Differences observed in some organ weights could not be attributed to treatment due to high inter-individual variations and small group size with large standard deviations. Histopathological examination revealed lung granulomas (males 2/4 in the high-dose group; females 1/4 at each treatment level) which is considered as a background finding in that species. In addition, minimal hepatic periportal hypertrophy in the high dose group was noted. No treatment related changes were seen in the recovery group after 4 weeks without exposure. The NOAEL was established to be 2600 ppm (69-78 mg/kg bw/day) based on decreased body weight and liver hypertrophy.

Endpoint conclusion

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 36 mg/kg bw/day
Study duration:: subchronic
Species:: rat
System:: other: general toxicity: body weight gain

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

The target chemical was assessed in a 90 days feeding study in dogs (1974). At a concentration of 8000 ppm the test article was so unpalatable that dogs refused to eat. On day 43 the dietary concentration of the test compound was reduced to 2600 ppm and on day 50 it was increased again to 5000 ppm, whereupon a reasonable food intake was maintained. A slight reduction in red cell parameters until the 12th week and a reduction of serum protein throughout the test were recorded in females only. These changes in the laboratory parameters were probably caused by the poor food intake during the first part of the test. A minimal hepatocyte hypertrophy was observed. This is considered to be a phenomenon of adaptive nature. At 2600 ppm and at 800 ppm no changes were seen which could be considered as related to the administration of test material. In a recovery experiment of four weeks (8000 -> 5000 ppm) neither changes of laboratory parameters nor histopathological changes were seen.

In another 90d study the target was administered in feed to groups of male and female Sprague-Dawley rats (20 per sex) for 13 weeks at dose levels of 0, 400, 1300 and 4000 ppm (1974). Five additional male and female animals were included in the control and high dose groups and were kept without treatment for an additional 4 weeks after the treatment schedule (recovery animals). The following observations were performed: clinical signs, viability, food and water consumption, body weights and food utilization and ophthalmoscopic examination. Laboratory analyses were performed during weeks 4, 8 and 12 on animals of the control and high dose groups. At necropsy, organ weights were recorded and microscopic analyses were performed. Marginally lower food intake was observed among rats receiving 1300 or 4000 ppm. Food intake was improved on removal from treatment among females that had received 4000 ppm. A decreased rate of bodyweight gain and reduced efficiency of food utilization was recorded in all treated female groups and in males receiving 4000 ppm. During the withdrawal period, superior bodyweight gain and efficiency of food utilization in rats of the high dosage group was observed. The performance of rats treated at 400 ppm was comparable with that of the controls. Liver, kidney, adrenals, showed decreased absolute weights compared to the control group at 4000 ppm in males and 1300 and 4000 ppm in females, however this is expected to be due to the body weight gain decrease rather than to be due to the test substance and is also reflected in an relative increase compared to control of brain and gonads. No treatment-related effects were noted after the recovery period. At macroscopic or histopathological examination no treatment-related abnormalities were noted. The LOEL was determined at 29 mg/kg bw based on the decreased body weight gain in females. The study had some limitations due to a background inflammation in all animals including control (inflammatory cell infiltration in lungs and kidney).

In a guideline compliant OECD 443 study groups of male and female Wistar rats were exposed to the target chemical by feed at dose levels of 0, 500, 1500, 5000 ppm. Dietary concentrations were lowered by 50% during the lactation period of the F0-females. Parental effects were observed at all dose levels tested. In males, body weight and body weight gain were lower in all treatment groups compared to concurrent controls on most days of treatment. Notably, there was no dose response between 500 and 1500 ppm. The lowered body weights and body weight gains occurred without a concurrent decrease in food consumption. In fact, increased relative food consumption was noted compared to concurrent controls at 1500 ppm on several occasions and at 5000 ppm throughout the treatment period. The delayed growth of 1500 and 5000 ppm males in the presence of higher relative food intake is indicative for a lower food efficiency, i.e. more food has to be ingested to reach the same growth in the animal’s mass. Given the magnitude of the effect at 1500 and 5000 ppm (approximately 8-13% lower terminal weight compared to controls) in combination with the decreased food efficiency, it was considered related to treatment with the test item and adverse from 1500 ppm. As food consumption was unaffected at 500 ppm, the effect on body weight was considered minimal and non-adverse at this dose level. For females at 5000 ppm, body weights and body weight gains were decreased compared to controls throughout the premating, mating and post-coitum period. Although body weight gains were increased during the last part of the lactation period, terminal body weights were lower compared to concurrent controls. Furthermore, increased relative food consumption was noted compared to concurrent controls at 5000 ppm throughout the treatment period, indicative for a lower food efficiency. Based on the magnitude of the effect that lasted throughout the largest part of the treatment period, together with the decreased food efficiency this was considered adverse. For females at 500 and 1500 ppm, a similar trend with lower body weights and body weight gains was observed during the premating and mating period. Body weight gains were not affected during the post-coitum and lactation period. No concurrent decrease in food consumption was observed. Food consumption was actually increased on several occasions during the treatment period at 1500 ppm. Based on the combination of lower body weights and a lower food efficiency, this was considered adverse at 1500 ppm. Whereas food consumption was unaffected at 500 ppm. In combination with the relatively small effect on body weight this dose level, this was considered non-adverse. The observed reduction in food consumption during Week 1 of the study in both males and females at 1500 and 5000 ppm, was most likely contributed a palatability issue with the diet containing test item, as it recovered to normal levels thereafter. Differences in clinical biochemistry parameters were observed at 5000 ppm. These consisted of higher total bilirubin and bile acid levels in males and higher alanine aminotransferase activity in females. These differences were considered non-adverse as they occurred in one sex only and in absence of a microscopic correlate at the organ level. Several changes were observed in organ weights (brain, spleen, adrenal glands, heart, liver, thyroid gland, kidneys, thymus, seminal vesicles, testes, epididymides and ovaries) at all dose levels. As changes in organ/relative to body weight occurred with minor or no changes in absolute weight or vice versa, these changes were regarded to be the result of a lower terminal body weight and not directly affected by treatment with the test item. There were no macroscopic or microscopic findings correlates for these organ weight changes. No test item-related changes were noted in any of the remaining parameters investigated in this study (i.e. mortality/viability, clinical appearance, hematology and coagulation parameters, thyroid hormone analysis, urinalysis, macroscopic examination and microscopic examination). In conclusion, based on the results of this extended one generation reproductive toxicity study the following no-observed-adverse-effect level (NOAEL) of the test item were established: F0 -generation: 500 ppm (on average corresponding to 36 mg/kg/day in males and 41 mg/kg/day in females of the F0-generation; based on a decreased body weights and food consumption at 1500 ppm).

In a 28 day repeated dose toxicity study performed in rats the target compound was administered orally by gavage at daily doses of 600, 1000 and 2000 mg/kg (5 animals per sex and group) (1976). Mortalities and symptoms were recorded daily, bodyweight gain 3 times and food consumption once weekly. Laboratory investigations were performed at the end of the experiment. All animals of the 2000 mg/kg group died between day 2 and 17. Two male rats of the 1000 mg/kg group died on day 5 or 28. No mortalities occurred at the 600 mg/kg dose level. The main symptoms observed in all dosed groups were ptosis of eye lids, muscular hypotonia and rough coat. Sedation, brownish eye discharge and kyphotic carriage were seen in the 1000 and 2000 mg/kg/day group. The latter showed also soiled snout and dyspnoea. Some individual rats of all dosed groups showed salivation, stiff movements, slow or irregular respiration, ventrior laterocumbency, slight cyanosis, tremor, meteorism, dyspnoea, sedation, diarrhea and soiled snout. Loss of bodyweight was observed in the male rats of the 2000 mg/kg group. The females of this group showed stagnation of growth during the first days of the study followed by reduced weight gain up to day 12 and practically normal weight until day 16 after which all animals of this group had died. Male and female rats of the 1000 and 600 mg/kg/day group showed a physiological pattern of growth. Food consumption was low in the males of the 1000 mg/kg/day group during the first two weeks of the experiment. In all other dosed groups this parameter was found to be within physiogical limits. Laboratory investigations revealed an increase of neutrophilic granulocytes and a relative decrease of lymphocytes in the differential blood count. All other parameters investigated were found to be within physiological limits valid for the strain of rats used. At termination of the study the animals were referred to pathology and autopsied. Special attention was paid to the sympathetic nervous system and ganglia. In the latter, transmitter function was checked in some rats by histochemical techniques. Histologically increased amount of eosinophilic and neutrophilicleucocytes in the spleen and in the blood vessels and perivascular tissue in the lungs was observed in all treated animals. The quantitative neurohistochemical examination showed that the average noradrenaline content of the principal perikarya of the superior cervical ganglion of treated rats was distinctly lower than in the controls. The measured values recorded in the striatum and the vas deferens did not deviate from the control values. No NOAEL was established.

In a repeated dose toxicity study similar to OECD TG 407, male and female CFY rats (10/sex/group) were orally exposed to target material (suspended in 0.5% CMC) via gavage at 0, 50, 200 and 600 mg/kg bw/day for 28 days (1972). One male in the control group (day 17) and two females in the 600 mg/kg bw/day group (day 8) were found dead. Reduced grooming, urine-stained fur, salivation after dosing and respiratory distress during week 3 and 4 were observed at 600 mg/kg bw/day. Statistically significant reduction of body weight gain was observed in males and females at 600 mg/kg bw/day (25 and 45%, respectively) and in females exposed at 200 mg/kg bw/day (21%). Body weight was decreased in males at 200 mg/kg/day, but did not reach statistical significance. No treatment-related food consumption effects were observed. No treatment-related effects were observed for haematological parameters, clinical chemistry or urinalysis. Absolute and relative adrenal weights were statistically significantly increased at 600 mg/kg bw/day (16 and 29%, respectively) in males only without histopathological changes. Necropsy examinations showed distension of small intestine in one and two males at 200 and 600 mg/kg bw, respectively, and in one female at 200 mg/kg bw. Histopathology showed no relevant effects. The NOAEL was established to be 50 mg/kg bw/day based on the reduced body weight gain and gross pathology.

In an OECD 422 study, Wistar Han rats were treated with the target chemical by daily dietary administration at dose levels of 1500, 5000 and 15000 ppm. The rats of the control group received standard powder rodent diet. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 14 days of lactation (51-63 days). The females that failed to deliver pups were exposed for 42 to 54 days. The amount of test item incorporated in the diet was kept at a constant level in terms of ppm, throughout the study period. Diets were considered accurately and homogeneously prepared and stable for at least 10 days at room temperature under normal laboratory light conditions in open containers.

After start of exposure, a transient body weight loss was observed in high dose animals (exposed to 15000 ppm), of maximally 7-8% lower body weights on Day 4 of treatment. During the further progress of the study, reduced body weight gain was observed in both sexes, resulting in 11% lower body weights in males and 18% lower body weights in females at termination. High dose animals also showed transient signs of ill health, comprising piloerection, hunched posture, tremor and/or narrowed palpebral fissures. The effects on body weight in high dose animals were considered adverse, considering the magnitude of change. Similar effects on body weight gain and body weights were observed in mid dose males and females (exposed to 5000 ppm) and low dose females (1500 ppm), but to a lesser extent and therefore considered not adverse. Food spillage was observed by the animals of all groups receiving test item containing diet. The spillage was increased at higher test item concentrations in the diet and was considered to be the result of a refusal to eat the diet because of a bad taste caused by test item. It was concluded that the food spillage was responsible for the increased food consumption data in comparison with the control and low dose food consumption. The actual difference in food consumption by each animal receiving test diet was considered not toxicologically relevant. A lower motor activity (total movements and ambulations) was observed in males, most prominent during the first stage of the 1-hour recording period, whereas their habituation profile was considered similar to that of the other dose groups. As mean values remained within the normal range of biological variation, this was considered not adverse.

Test item-related changes in the haematology parameters comprised decreased total white blood cell counts, and corresponding lymphocytes and neutrophils in females of all three test groups. The changes in clinical biochemistry parameters comprised increased chloride (in high dose males) and decreased levels of inorganic phosphate (mid and high dose males), potassium (high dose males) and total bilirubin (high dose females). Considering the magnitude of change and/or as values were outside the normal range of biological variation, these changes were considered adverse. In the heart of males, inflammatory changes, together with myofiber necrosis in a single male treated at 1500 and 15000 ppm each, were observed in all test item-treated groups. This finding is uncommon in Wistar (Han) rats at this age. These heart findings were considered to be adverse based on the inflammatory and degenerative nature. However, in the follow-up OECD 443 study with longer exposure, these heart effects could not be reproduced, therefore they are considered a chance finding in this study. Low degrees of adipocytes in the bone marrow could be seen as a spontaneous finding and the small increase in the absence of any additional inflammatory, degenerative or proliferative changes was considered non-adverse. No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. mortality, functional observations (grip strength, hearing ability, pupillary reflex and static righting reflex), clotting parameters, male T4 thyroid hormone levels, macroscopic examination and organ weights).

Based on the results of this study, the following no-observed-adverse-effect level (NOAEL) was established:

Parental NOAEL: < 1500 ppm (corresponding to an actual test article intake of 221 mg/kg/day based on adverse histopathology findings).

In addition, two repeated dose studies are available with a structurally related compound (source chemical). The use of the target and the source chemical in a read-across approach to address repeated dose toxicity was agreed upon in ECHA decision CCH-D-2114384240-56-01/F from January 8th, 2018.

The source chemical was assessed in a OECD 422 study in parallel. Wistar Han rats were treated with the test item by dietary administration at dose levels of 1500, 5000 and 15000 ppm. The rats of the control group received standard powder rodent diet.

Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (51-63 days). The females that failed to deliver pups was exposed for 42 days. The amount of test item incorporated in the diet was kept at a constant level in terms of ppm, throughout the study period. After start of exposure, a transient body weight loss was observed in high dose animals (exposed to 15000 ppm), being more prominent in females. This effect was likely to be related to a refusal, or at least a restrain, of the animals to eat the test item containing diet in first instance. During the further progress of the study, reduced body weight gain was observed in both sexes, resulting in 10% lower body weights in males and 20% lower body weights in females at termination, when compared to control. High dose animals also showed narrowed palpebral fissures for varying periods of a few to several days. Some of these animals also showed transitory piloerection and/or hunched posture. The effects on body weight in high dose animals were considered adverse, considering the magnitude of change. Similar effects on body weight gain and body weights were also observed in mid dose males and females (exposed to 5000 ppm), but to a lesser extent and therefore considered not adverse. Food spillage was observed by the animals receiving test item containing diet. The spillage was increased at higher test item concentrations in the diet and was considered to be the result of a refusal to eat the diet because of a bad taste caused by test item. In mid and high dose animals, exposed to 5000 and 15000 ppm in the diet respectively, it was concluded that the food spillage was responsible for the increased food consumption data in comparison with the control and low dose food consumption. The actual food consumption by each animal receiving test diet was assumed comparable to that expected for Wistar Han rats of corresponding weight.

Treatment-related changes in the clinical pathology parameters comprised decreased total protein and albumin levels in both sexes at 15000 ppm. Moreover, decreased reticulocyte counts, haemoglobin concentrations and total bilirubin levels were noted in females and increased cholesterol levels in males. Except for decreased reticulocyte counts (starting at 1500 ppm) and decreased haemoglobin levels (starting at 5000 ppm) in females changes were limited to the high dose animals exposed at 15000 ppm. In the absence of corroborating findings, these were considered not adverse. Decreased thymus weights after treatment with 15000 ppm correlated with the macroscopic finding of reduced thymus size and was the consequence of lymphoid depletion. The depletion was not reflected in the haematology data (no decreased lymphocytes) and therefore not considered adverse. Based on low severity and the absence of any degenerative, proliferative or inflammatory changes, the acinar hypertrophy of the parotid gland was not considered adverse. No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. mortality, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), clotting parameters, male T4 thyroid hormone levels). The NOAEL was 5000 ppm (corresponding to an actual test article intake of 469 and 804 mg/kg/day for males and females, respectively, based on reduced body weights at 15000 ppm).

The source chemical was further assessed in two repeated dose studies. In a repeated-dose 28-day oral toxicity study with a 14-day recovery the test substance was administered daily by gavage to male and female Wistar rats at dose levels of 0, 100, 300 and 750/1000 mg/kg bw/d. Regarding clinical examinations, no signs of general systemic toxicity were observed at dose levels up to 750/1000 mg/kg bw/d. Salivation was seen after dosing all rats of test groups 2 and 3 (300 and 750/1000 mg/kg bw/d) and sporadically in 5 male animals and three females animals of test group 1 (100 mg/kg bw/d). From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by the taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect. Dilation of pupils or delayed closure observed in several animals of test group 3 (750/1000 mg/kg bw/d). In addition, ptosis of eyelids was observed and was considered as secondary to the mydriatic effect. It was suggested that the test substance and/or its metabolite(s) interact with the neurotransmitter system provided sufficiently high plasma concentrations could be obtained. For chemically-related substances, binding to acetylcholine receptors was shown (PAPKE et al., 1994; GRAHAM et al., 2005). This receptor activity might lead to inhibitory effects (e.g. mydriasis) modulated by the parasympathic nervous system. How the neurological changes induced ultimately causes pupil dilation is unknown. This effect was obviously concentration-dependent and was not observed during the recovery period. Post mortem examinations did not indicate a damage of the involved organ(s). Therefore, this effect represents a transient functional alteration only induced at peak concentrations not leading to tissue damage. Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 750 mg/kg bw/d in male rats and 1000 mg/kg bw/d in female rats. With regard to pathology, no clear histopathological correlate was found in the liver of female rats of test group 3 (1000 mg/kg bw/d) that could explain the slight increase of the absolute and relative organ weight. Nevertheless, the weight increase was regarded as a treatment related adaptive effect due to an enzyme induction as a detoxifying mechanism in the hepatocytes. No histopathological findings were observed in the spleen of males of test group 3 (750 mg/kg bw/d). Therefore, the increase in the relative weight of the spleen was not considered to be treatment-related. In conclusion, a 4 week oral administration of test substance up to 750 mg/kg/day in males and 1000 mg/kg/day in females led to no pathological findings in the rat. Thus, under the conditions of the present study, the no observed adverse effect level (NOAEL) was 300 mg/kg bw/d for male and female Wistar rats taking into account the eye findings (mydriasis) at dose levels of 750 mg/kg bw/d (males) and 1000 mg/kg bw/d (females). Increased liver weights in females were assessed as adaptive and non-adverse as no histopathological findings were observed. Salivation was considered to be related to either the bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect.

REFERENCES

RL Papke, AG Craig and SF Heinemann: Inhibition of nicotinic acetylcholine receptors by bis (2,2,6,6- tetramethyl- 4-piperidinyl) sebacate (Tinuvin 770), an additive to medical plastics, Volume 268, Issue 2, pp. 718-726,1994

John H. Graham, Roger L. Papke and Jerry J. Buccafusco: Functional Central Nicotinic Acetylcholine Receptor Antagonism by Systemic Administration of Tinuvin 770 (BTMPS) Current Alzheimer Research Volume 2, Number 2, 2005

Justification for classification or non-classification

There are conclusive but not sufficient data for classification of the test item with regard to target organ toxicity. The test item is not classified for this endpoint in accordance to Directive 67/548/EEC or the CLP Regulation (EC) No 1272/2008 as well as GHS regulations.

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

Group No.		Mean over means intake [mg test item/kg body weight] (mean range indicated within brackets)
		2		3		4
Nominal dietary inclusion level (ppm) during (pre-/post-) mating and post-coitum		500		1500		5000
Nominal dietary inclusion level (ppm) during lactation		250		1250		2500
Sex	Study period
Males	Pre-mating	38	(28-53)	115	(88-155)	400	(302-543)
	Post-mating	26	(25-27)	81	(80-82)	266	(264-268)
	Mean of means^a	36		109		378
Females	Pre-mating	42	(33-54)	130	(100-158)	440	(317-539)
	Post-coitum	33	(31-36)	100	(96-107)	342	(310-447)
	Lactation	45	(29-59)	136	(86-175)	467	(291-609)
	Mean of means^a	41		126		427

MALES		GROUP 1	GROUP 2	GROUP 3	GROUP 4
MALES		CONTROL	500 PPM	1500 PPM	5000 PPM
PRE MATING
DAY 1	MEAN	153	158	153	154
WEEK 1	ST.DEV	8.3	7.5	9.5	7.4
	N	28	28	28	28
DAY 9	MEAN	205	199	200	190 **
WEEK 2	ST.DEV	11.4	12.3	13.6	10.7
	N	28	28	28	28
DAY 15	MEAN	238	230	230	219 **
WEEK 3	ST.DEV	14.6	13.9	15	14.5
	N	28	28	28	28
DAY 22	MEAN	255	248	244 *	229 **
WEEK 4	ST.DEV	15.9	13	15.3	16.5
	N	28	28	28	28
DAY 29	MEAN	298	282 **	284 *	271 **
WEEK 5	ST.DEV	21.3	17.8	20.2	21
	N	28	28	28	28
DAY 36	MEAN	315	297 **	297 **	283 **
WEEK 6	ST.DEV	22.5	19.1	22.9	24
	N	28	28	28	28
DAY 43	MEAN	334	312 **	312 **	298 **
WEEK 7	ST.DEV	26.1	21	25.8	26.6
	N	27	28	28	28
DAY 50	MEAN	346	323 **	324 **	307 **
WEEK 8	ST.DEV	29.1	22	28.5	28.8
	N	27	28	28	28
DAY 57	MEAN	361	334 **	335 **	319 **
WEEK 9	ST.DEV	30.8	23.8	30.3	30.4
	N	27	28	28	28
DAY 64	MEAN	370	341 **	342 **	326 **
WEEK 10	ST.DEV	31.3	25.1	32	33.4
	N	27	28	28	28

MATING PERIOD	MEAN	379	351 **	351 **	335 **
DAY 1
WEEK 1	ST.DEV	33.2	25.8	33.8	34.2
	N	27	28	28	28
DAY 8	MEAN	383	355 **	354 **	339 **
WEEK 2	ST.DEV	33.8	25.5	32.9	34.2
	N	27	28	28	28
DAY 15	MEAN	391	360 **	361 *	346 **
WEEK 3	ST.DEV	34.3	28.6	32.1	33.5
	N	19	20	20	20
DAY 22	MEAN	401	349	397	352
WEEK 4	ST.DEV	25.5	10.9	44.4	16.6
	N	3	4	4	4

FEMALES		GROUP 1	GROUP 2	GROUP 3	GROUP 4
FEMALES		CONTROL	500 PPM	1500 PPM	5000 PPM
PRE MATING
DAY 1	MEAN	122	121	122	120
WEEK 1	ST.DEV	6.1	6.6	8.1	5.6
	N	28	28	28	28
DAY 9	MEAN	148	143	142 *	142 *
WEEK 2	ST.DEV	8.2	7.2	10.5	9.2
	N	28	28	28	28
DAY 15	MEAN	165	157 *	156 **	155 **
WEEK 3	ST.DEV	9.2	10.2	11.9	10.5
	N	28	28	28	28
DAY 22	MEAN	180	171 *	168 **	167 **
WEEK 4	ST.DEV	9.9	12.4	13.8	10
	N	28	28	28	28
DAY 29	MEAN	192	187	179 **	180 **
WEEK 5	ST.DEV	10.5	12.9	14.8	12.6
	N	28	28	28	28
DAY 36	MEAN	198	193	187 **	187 **
WEEK 6	ST.DEV	11.4	14.1	15.5	12.2
	N	28	28	28	28
DAY 43	MEAN	210	200 *	196 **	194 **
WEEK 7	ST.DEV	11.2	15.1	14.9	12.8
	N	28	28	28	28
DAY 50	MEAN	217	210	205 **	201 **
WEEK 8	ST.DEV	11.7	15.3	14.3	12
	N	28	28	28	28
DAY 57	MEAN	221	213	205 **	204 **
WEEK 9	ST.DEV	12.2	15.7	15.3	13.7
	N	28	28	28	28
DAY 64	MEAN	224	221	215	216
WEEK 10	ST.DEV	12	15.9	15.8	13.8
	N	28	28	28	28

MATING PERIOD
DAY 1	MEAN	226	219	214 **	212 **
WEEK 1	ST.DEV	11.8	16.7	16.2	14.4
	N	28	28	28	28
DAY 8	MEAN	264	233	232	224
WEEK 2	ST.DEV	---	---	5.7	---
	N	1	1	2	1
DAY 15	MEAN	260
WEEK 3	ST.DEV	---
	N	1

POST COITUM
DAY 0	MEAN	228	222	212 **	211 **
	ST.DEV.	13.4	16.1	16	14.2
	N	28	27	28	26
DAY 4	MEAN	242	236	226 **	220 **
	ST.DEV.	15	15.8	15.1	16
	N	28	27	28	26
DAY 7	MEAN	248	242	228 **	222 **
	ST.DEV.	14.1	15.4	16.4	16
	N	28	27	28	26
DAY 11	MEAN	262	255	244 **	237 **
	ST.DEV.	14.8	13.8	15.2	17.4
	N	28	27	28	26
DAY 14	MEAN	272	263	250 **	242 **
	ST.DEV.	14.8	16.2	15.7	18.6
	N	28	27	28	26
DAY 17	MEAN	294	283	274 **	264 **
	ST.DEV.	17.5	18.3	17	19.1
	N	28	27	28	26
DAY 20	MEAN	330	314 *	304 **	289 **
	ST.DEV.	18.2	20	21.9	22.4
	N	28	27	28	26

LACTATION
DAY 1	MEAN	251	244	237 **	227 **
	ST.DEV.	13.9	15.3	17	17.2
	N	28	26	28	26
DAY 4	MEAN	264	260	250 **	243 **
	ST.DEV.	15.1	16.1	19.2	17.2
	N	27	26	28	26
DAY 7	MEAN	273	271	259 *	252 **
	ST.DEV.	15.9	16.4	18.8	18.5
	N	27	26	28	26
DAY 14	MEAN	274	275	264	256 **
	ST.DEV.	22.6	15.3	18.2	19.5
	N	27	26	28	26
DAY 21	MEAN	276	276	266 *	264 *
	ST.DEV.	15.6	14.6	15.5	17.9
	N	27	26	28	26

MALES		GROUP 1	GROUP 2	GROUP 3	GROUP 4
MALES		CONTROL	500 PPM	1500 PPM	5000 PPM
PRE MATING
DAY 1	MEAN	0	0	0	0
WEEK 1	ST.DEV	0	0	0	0
	N	28	28	28	28
DAY 9	MEAN	34	26 **	30 **	24 **
WEEK 2	ST.DEV	4.3	3.9	4.8	3.6
	N	28	28	28	28
DAY 15	MEAN	55	46 **	50 **	43 **
WEEK 3	ST.DEV	6.8	5.4	5.8	6.6
	N	28	28	28	28
DAY 22	MEAN	67	57 **	59 **	49 **
WEEK 4	ST.DEV	8	6	7.8	8.3
	N	28	28	28	28
DAY 29	MEAN	95	79 **	85 **	77 **
WEEK 5	ST.DEV	12.4	8.9	10.2	11
	N	28	28	28	28
DAY 36	MEAN	106	88 **	93 **	84 **
WEEK 6	ST.DEV	13.6	9.4	11.9	12.7
	N	28	28	28	28
DAY 43	MEAN	119	98 **	103 **	94 **
WEEK 7	ST.DEV	14.7	11.7	12.4	14.2
	N	27	28	28	28
DAY 50	MEAN	127	105 **	111 **	100 **
WEEK 8	ST.DEV	16.8	12.7	14.6	15.6
	N	27	28	28	28
DAY 57	MEAN	136	112 **	118 **	108 **
WEEK 9	ST.DEV	17.9	14	15.5	16.6
	N	27	28	28	28
DAY 64	MEAN	143	116 **	123 **	112 **
WEEK 10	ST.DEV	19	15.1	16.3	18.6
	N	27	28	28	28

MATING PERIOD
DAY 1	MEAN	148	123 **	129 **	118 **
WEEK 1	ST.DEV	20.5	16	17.5	18.9
	N	27	28	28	28
DAY 8	MEAN	151	125 **	131 **	121 **
WEEK 2	ST.DEV	20.4	15.6	16.4	19.2
	N	27	28	28	28
DAY 15	MEAN	155	129 **	135 **	126 **
WEEK 3	ST.DEV	22.5	17.1	14.1	18.8
	N	19	20	20	20
DAY 22	MEAN	161	123 **	147	131 *
WEEK 4	ST.DEV	16.7	15.3	11.1	7.3
	N	3	4	4	4

FEMALES		GROUP 1	GROUP 2	GROUP 3	GROUP 4
FEMALES		CONTROL	500 PPM	1500 PPM	5000 PPM
PRE MATING
DAY 1	MEAN	0	0	0	0
WEEK 1	ST.DEV	0	0	0	0
	N	28	28	28	28
DAY 9	MEAN	21	19	16 **	19
WEEK 2	ST.DEV	4.1	5.1	4.6	5.4
	N	28	28	28	28
DAY 15	MEAN	35	30 **	28 **	29 **
WEEK 3	ST.DEV	4.7	6.8	5.8	7.1
	N	28	28	28	28
DAY 22	MEAN	48	41 **	37 **	40 **
WEEK 4	ST.DEV	6.1	6.9	8	7.2
	N	28	28	28	28
DAY 29	MEAN	58	55	46 **	51 **
WEEK 5	ST.DEV	7.6	7.9	8.6	8.6
	N	28	28	28	28
DAY 36	MEAN	63	60	53 **	56 *
WEEK 6	ST.DEV	8.5	8.8	8.9	8.1
	N	28	28	28	28
DAY 43	MEAN	72	66 *	61 **	62 **
WEEK 7	ST.DEV	8.5	10.8	8.7	9.8
	N	28	28	28	28
DAY 50	MEAN	79	74	68 **	68 **
WEEK 8	ST.DEV	8.3	9.8	8.7	9.3
	N	28	28	28	28
DAY 57	MEAN	82	77	68 **	71 **
WEEK 9	ST.DEV	9	10.1	9.5	10.5
	N	28	28	28	28
DAY 64	MEAN	84	83	77 *	81
WEEK 10	ST.DEV	9.1	10.8	9.4	9
	N	28	28	28	28

MATING PERIOD
DAY 1	MEAN	86	82	75 **	77 **
WEEK 1	ST.DEV	9.5	12.2	9.8	10.4
	N	28	28	28	28
DAY 8	MEAN	115	99	84	96
WEEK 2	ST.DEV	---	---	15.1	---
	N	1	1	2	1
DAY 15	MEAN	111
WEEK 3	ST.DEV	---
	N	1

POST COITUM
DAY 0	MEAN	0	0	0	0
	ST.DEV.	0	0	0	0
	N	28	27	28	26
DAY 4	MEAN	6	6	6	4 *
	ST.DEV.	2.2	2.4	3	2.9
	N	28	27	28	26
DAY 7	MEAN	9	9	8	5 **
	ST.DEV.	2.4	2.7	3.2	3.4
	N	28	27	28	26
DAY 11	MEAN	15	15	15	12 **
	ST.DEV.	3.1	4.2	3.7	3.9
	N	28	27	28	26
DAY 14	MEAN	19	19	18	15 **
	ST.DEV.	3.5	3.8	3.9	4.1
	N	28	27	28	26
DAY 17	MEAN	29	28	29	25 *
	ST.DEV.	3.8	5.3	5.2	4.4
	N	28	27	28	26
DAY 20	MEAN	45	42	43	37 **
	ST.DEV.	4.6	8.1	6.2	6
	N	28	27	28	26

LACTATION
DAY 1	MEAN	0	0	0	0
	ST.DEV.	0	0	0	0
	N	28	26	28	26
DAY 4	MEAN	5	6	6	7
	ST.DEV.	4.4	3.5	4	3.5
	N	27	26	28	26
DAY 7	MEAN	9	11	9	11
	ST.DEV.	4.1	4.3	4.3	4.4
	N	27	26	28	26
DAY 14	MEAN	9	13	12	13
	ST.DEV.	7.1	4.1	7.9	5.8
	N	27	26	28	26
DAY 21	MEAN	10	13	12	17 **
	ST.DEV.	4.8	5	6.3	5
	N	27	26	28	26

	Males			Females
Dose level (ppm):	500	1500	5000	500	1500	5000
Body Weight	-8**	-8**	-13**	-3	-6**	-7**
Brain	-8**	-8**	-13**	-3	-6**	-7**
Absolute	-1	0	-2	1	-1	-1
Relative to body weight	5*	9**	12**	4	5*	6**
Pituitary gland
Absolute	-10	-10*	-10**	-8	-8	-8*
Relative to body weight	0	0	0	0	0	0
Heart
Absolute	-5	-6*	-8**	-3	-6*	-6*
Relative to body weight	2	2	5*	1	0	2
Liver
Absolute	-8*	-9*	-11*	-11**	-11**	-15**
Relative to body weight	0	0	2	-7*	-6	-9**
Thyroid gland
Absolute	-5	-16*	-11	-6	-18**	-12**
Relative to body weight	0	0	0	-13	-13*	-13
Kidneys
Absolute	-4	-7*	-8**	-3	-6**	-6**
Relative to body weight	3	2	5**	-1	-1	0
Adrenal glands
Absolute	4	6	-2	-7	-6	-8**
Relative to body weight	7*	14**	7**	-3	0	0
Spleen
Absolute	0	0	1	n.s.	n.s.	n.s.
Relative to body weight	7	9*	15**	n.s.	n.s.	n.s.
Testes
Absolute	0	0	-3	n.a.	n.a.	n.a.
Relative to body weight	7*	9**	11**	n.a.	n.a.	n.a.
Epididymides
Absolute	1	-1	-3	n.a.	n.a.	n.a.
Relative to body weight	9**	8**	12**	n.a.	n.a.	n.a.
Ovaries
Absolute	n.a.	n.a.	n.a.	-3	-14**	-15**
Relative to body weight	n.a.	n.a.	n.a.	-2	-9	-8
Seminal vesicles
Absolute	4	-6	-7	n.a.	n.a.	n.a.
Relative to body weight	13*	3	7	n.a.	n.a.	n.a.
Thymus
Absolute	-12*	-2	-11	n.s.	n.s.	n.s.
Relative to body weight	-5	6	2	n.s.	n.s.	n.s.

Males	Body Weight	Brain	Pituitary (mg)	Heart	Liver	Spleen	Thymus	Kidney	Thyroid (mg)	Adrenals (mg)	Gonads
Group 1	527	2	16	1.5	21.6	0.9	0.5	3.9	26	63	5
Group 2	506	2	17	1.5	20.1	0.8	0.5	3.7	26	62	5
Group 3	521	2.1	15	1.5	21.1	0.9	0.5	3.6	24	58	5.2
Group 4	469**	2	15	1.4	18.7**	0.8	0.4	3.3**	24	61	4.9

Females	Body Weight	Brain	Pituitary (mg)	Heart	Liver	Spleen	Thymus	Kidney	Thyroid (mg)	Adrenals (mg)	Gonads
Group 1	306	1.9	17	1	11	0.6	0.3	2.4	20	82	87
Group 2	287*	1.9	17	0.9	10.4	0.6	0.3	2.3	22	75	88
Group 3	274***	1.8	16	0.9	10.3	0.5	0.3	2.2*	22	69***	89
Group 4	266***	1.9	16	0.9	9.9**	0.6	0.3	2.1**	19	70**	84

Males	Body Weight	Pituitary	Heart	Liver	Spleen	Thymus	Kidney	Thyroid	Adrenals	Gonads
Group 1	527	0.8	73	1068	45	23	193	1.3	3.1	248
Group 2	506	0.8	73	983	41*	24	180	1.3	3	245
Group 3	521	0.7	71	1014	43	23	176	1.2	2.8	249
Group 4	469**	0.8	70	955*	43	22	171	1.2	3.1	250

Females	Body Weight	Pituitary	Heart	Liver	Spleen	Thymus	Kidney	Thyroid	Adrenals	Gonads
Group 1	306	0.9	53	589	32	18	128	1.1	4.3	4.7
Group 2	287*	0.9	51	562	30	18	123	1.2	4.1	4.8
Group 3	274***	0.9	51	564	30	14	120	1.2	3.8**	4.9
Group 4	266***	0.8	51	535*	31	16	115*	1	2.8**	4.5

Registration Dossier

Registration Dossier

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Diss Factsheets

Bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate

Endpoint summary

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

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Reference 1

Reference 2

Reference 3

Endpoint conclusion

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Additional information

Justification for classification or non-classification

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