4-(1,1-dimethylethyl)cyclohexyl acrylate

Endpoint summary

Description of key information

NOAEL (systemic): 150 mg/kg bw/day (OEDC 422)
NOAEL (systemic): 500 mg/kg bw/day (OECD 408)

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

CRL: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 141-187g (males), 108-142g (females)
- Housing: groups of up to 5 animals
- Diet (e.g. ad libitum): ad lib.
- Water (e.g. ad libitum): ad lib.
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21 °C
- Humidity (%): 44-74% (The values that were outside the targeted mean humidity range of 40-70% occurred occasionally and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study.)
- Air changes (per hr): >= 10
- Photoperiod (hrs dark / hrs light): 12h/12h

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

VEHICLE
- Justification for use and choice of vehicle: solubility
- Concentration in vehicle: 0 -100 mg/mL
- Amount of vehicle: 5mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

All analyzed formulation of all dose groups (day 1 and in weeks 6 and 12) were within 90 to 110% of the target concentration. No test material was detected in the control formulations. All formulations were homogeneous, i.e., coefficient variation <=10%)

Duration of treatment / exposure:

at least 90 days

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on previous OECD 422

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily, 0-1h post dose

CHECK FOR MORTALITY: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

ARENA OBSERVATION
- before first administration and weekly during treatment

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: regular basis via visual inspection of water bottles

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once prior to dosing (all groups) and during week 13 (group 1 and 4, remaining animals only, if differences noted)


HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: WBC, neutrophils, lymphocytes, monocytes, eosinophils, basophils, large unstained cells, RBC, reticulocytes, hemoglobin, hematocrit, MCV, MCH, MCHC, platelets, red blood cell distribution width

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of treatment
- Animals fasted: Yes
- How many animals: all
- Parameters examined: prothrombin time, APTT, ALT, AST, ALP, total protein, albumin, bilirubin, urea, creatinine, glucose, cholesterol, triglycerides, HDL and LDL cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate, T3, T4, TSH

URINALYSIS: Yes
- Time schedule for collection of urine: end of treatment
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: volume, specific gravity, clarity, color, pH, blood, leukocyte esterase, bilirubin, urobilinogen protein, ketones, glucose, nitrite

NEUROBEHAVIOURAL EXAMINATION (FOB): Yes
- Time schedule for examinations: once during weeks 12-13
- Dose groups that were examined: 5 animals per group
- Battery of functions tested: sensory activity, grip strength, motor activity, general appearance/reactions to handling

ESTROUS STAGE
- Time schedule: day of necropsy

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes (for low and mid dose only gross lesions and target tissues unless differences observed between high dose and control)

Optional endpoint(s):

staining for alpha-2µ in high dose and control males

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg: Slight, transient tremors of the forelimbs, head and shoulder muscles, and closed eyes were observed on Days 86-88 in all males (prior to functional observations on Day 86) and females.

Salivation was observed in all dose groups, which is considered a consequence of local irritation and/or bad taste of the test substance due to the temporary and short appearance right after dosing.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

1 control animal was euthanized on Day 46 due to complication with the gavage procedure

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 500mg/kg, females body weights were higher from day 57 onward, resulting in a 12% increase by day 91. In absence of corroborating findings, this change was considered not adverse.

In addition, male body weights were 5% lower on day 91 as a consequence of lower body weight gain after day 50. The difference was not statistically significant and because of the low magnitude of the change likely represents normal variation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

at 500mg/kg: higher food consumption in females starting on day 50 (+10% over entire treatment period). This finding was not considered adverse.

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg bw/day, females showed a higher white blood cell count (1.43x of control), consisting of higher neutrophil (1.51x, not statistically significant), lymphocyte (1.41x), monocyte (1.70x) and eosinophil (1.42x) count. In addition, higher large unstained cell (2.22x) and reticulocyte count (1.20x) were noted in females. Also, a non-significant shorter activated partial thromboplastin time was observed (0.89x of control). In the absence of corroborating findings, these changes were considered not adverse.

Females at 500 mg/kg bw/day also showed a higher reticulocyte count, which correlated with an increased incidence and/or severity of extramedullary hematopoiesis and pigment in the spleen. The minor increase in extramedullary hematopoiesis and pigment were considered non-adverse due to their adaptive nature.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males, lower albumin concentration at 50, 150 and 500 mg/kg bw/day (0.97, 0.95 and 0.95x of control, respectively), and lower total protein concentrations at 150 and 500 mg/kg bw/day (both 0.95x) were noted. In addition, higher urea (1.16x) and inorganic phosphate (1.13x) concentrations, and lower glucose concentrations (0.80x) were observed at 500 mg/kg bw/day.
In females, higher cholesterol (1.28x; not statistically significant), HDL cholesterol (1.26x; not statistically significant), and potassium (1.11x) concentrations were observed at 500 mg/kg bw/day.
The changes were generally minor, there were no corroborating findings, and might largely be related to enhanced liver metabolism. They are not considered adverse.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

In females at 500 mg/kg bw/day, higher T4 concentrations were observed (1.80x of control). The T4 value for 7/10 females at 500mg/kg remained within the normal range historically observed in rats under similar study conditions. In parallel studies, higher control values matching those of the high dose group were observed.
T3 and TSH concentrations were not affected, there were no histopathological findings.
There was no effect in males.
Overall, the change is not considered adverse and relation to treatment is unlikely.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Higher kidney weights in males (relative at 150mg/kg, absolute and relative at 500mg/kg)
Higher liver weights (relative in males at 500mg/kg; absolute and relative in females at 150 and 500mg/kg). This change was considered adaptive and non-adverse.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Pale discoloration of kidneys in 2 high dose males (500mg/kg), as a consequence of α2µ-globulin nephropathy

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the kidney of males, microscopic findings were noted starting at 50 mg/kg/day. These consisted of an increased incidence and severity of hyaline droplet accumulation (minimal to marked), granular casts (minimal to marked), tubular degeneration (minimal to mild) and an increased incidence and severity of tubular basophilia (minimal to marked). All these changes were observed to be in a dose-related manner. Immunohistochemistry for alpha 2u-globulin was performed on the kidney of all control and treated males. Hyaline droplets as well as the granular casts stained positive for alpha 2u-globulin in all control (hyaline droplets only) and treated males.

In the liver, hepatocellular hypertrophy (minimal) was noted in 2/10 males and females at 500 mg/kg bw/day. In the thyroid gland, an increased incidence of follicular cell hypertrophy (minimal) was noted in males at 500 mg/kg bw/day. Because of the low grade of these findings they were not considered adverse.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

> 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: highest dose tested

Dose descriptor:

LOAEL

Effect level:

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

Sex:

male

Basis for effect level:

other: a2µ nephropathy

Remarks on result:

other: male rat specific finding, not relevant for human risk assessment

Key result

Critical effects observed:

no

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines; OPPTS 870.3650: Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test (Jul 2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10 - 11 weeks (males/females)
- Housing: individually (polycarbonate cages type III supplied by TECNIPLAST)
- Exceptions:
- During overnight matings, male and female mating partners were housed together in polycarbonate cages type III.
- Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- Diet (e.g. ad libitum): ad libitum (ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): ad libitum (dringing water)
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Laromer TBCH was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, corn oil was filled up to the desired volume, subsequently released with a magnetic stirrer. The test substance preparations were produced weekly and stored at room temperature.

VEHICLE
- Justification for use and choice of vehicle: In the context of toxicological studies the stability of the test substance Laromer TBCH in the vehicle corn oil has to be verified.
- Concentration in vehicle: 1.25 g/100 mL (50 mg/kg bw/d); 3.75 g/100 mL (150 mg/kg bw/d); 12.50 g/100 mL (500 mg/kg bw/d)
- Amount of vehicle: 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In the context of toxicological studies the stability of the test substance Laromer TBCH in the vehicle corn oil has to be verified.
Concentration control analyses of the test substance preparations were performed in samples of all concentrations at the start of the administration period. Given that the test substance is completely miscible with corn oil, solutions were considered to be homogenous and no homogeneity analyses were carried out.

Duration of treatment / exposure:

males: 28 days
female: 57 days

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The doses were selected based on a 14-day range finder (study number 10C0580/09S051). Four male and female rats were treated with 300 and 1000mg/kg of the registered substance. At 1000mg/kg, food consumption was severely reduced (app- -50%), leading to a reduced terminal body weight inmales and body weight loss during the first 3 days of the study in males and females. Liver weights were increased in both sexes by about 20%. At 300mg/kg, the liver weight increase was still significant, but no effects on body weight were observed.
- Route of administration: The oral route was selected since administration by gavage has been proven to be appropriate for the detection of a toxicological hazard.

Observations and examinations performed and frequency:

MORTALITY / CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity.Abnormalities and changes were documented daily for each affected animal. The littering and lactation behavior of the dams was generally evaluated in the
mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis. On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of littering was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were examined: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level , tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/consistency), assessment of the urine discharged during the examination, pupil size

BODY WEIGHT: Yes
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).
The body weight change of the animals was calculated from these results. The following exceptions are notable for the female animals:
- During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
- Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
- Females without a litter and without positive evidence of sperm in the vaginal smear or waiting for necropsy were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION:
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0-7, 7-14 and 14-20.
- Food consumption of F0 females, which gave birth to a litter was determined for PND 1-4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

WATER CONSUMPTION:
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: In the morning towards the end of tha administrative period.
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes
- How many animals: first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: In the morning towards the end of tha administrative period.
- Animals fasted: Yes
- How many animals: first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group

URINALYSIS: Yes
- Time schedule for collection of urine: overnight towards teh end of the administrative period.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
A functional observational battery (FOB) was performed in the first five surviving male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period
- Dose groups that were examined: all amimals
- Battery of functions tested:
Home cage observations:
The animals were observed in their closed home cages; during this period any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals.
Attention was paid to: Posture, tremors, convulsions, abnormal movements, impairment of gait
Open field observations:
The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined: Behavior on removal from the cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors,
convulsions, abnormal movements/stereotypes, impairment of gait, activity/arousal level, feces excreted within 2 minutes (appearance/consistency), urine excreted within 2 minutes (amount/color, rearing within 2 minutes.s
Sensory motor tests/ reflexes:
The animals were then removed from the open field and subjected to following sensory motor or reflex tests: 1. Reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory startle response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), grip strength of forelimbs and hindlimbs, landing foot-splay test.

Motor activity (MA) was also measured on the same day as the FOB was performed in the first five parental males and the first five surviving females with litter (in order of delivery) per group.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.
Organ weights
- The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, epididymides, tstes.
- The following weights were determined in 5 animals/sex and test group (females with litters, same animals as used for clinical pathology examinations): Adrenal glands, brain, heart, kidneys, liver,spleen, thymus.

The following organs or tissues of all parental animals were fixed in in 4% neutralbuffered formaldehyde or in modified Davidson’s solution: All gross lesions, adrenal glands, aorta, bone marrow (femur), brain, cecum, cervix, coagulating glands, colon, duodenum, eyes with optic nerve, esophagus, extraorbital lacrimal glands, epididymides (modified Davidson’s solution), femur with knee joint, heart, ileum, jejunum (with Peyer’s patches), kidneys, larynx, liver, lungs, lymph nodes (axillary and mesenteric), mammary gland (male and female), nose (nasal cavity), ovaries (modified Davidson’s solution), oviducts, pancreas, parathyroid glands, pharynx, pituitary gland, prostate gland, rectum, salivary glands (mandibular and sublingual), sciatic nerve, seminal vesicles, skeletal muscle, spinal cord (cervical, thoracic and lumbar cord), spleen, sternum with marrow, stomach (forestomach and glandular stomach), testes (modified Davidson’s solution), thymus, thyroid glands, trachea, urinary bladder, uterus (uteri of all cohabited female F0 parental animals were stained according to Salewski’s method), vagina.

Statistics:

Statistics of clinical pathology
Means, medians and standard deviations of each test group were calculated for several parameters.
Blood parameters: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (twosided) for the hypothesis of equal medians For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians
Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity): Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
Urine pH, volume, specific gravity, color and turbidity: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (twosided) for the hypothesis of equal
medians. Urine color and turbidity are not evaluated statistically.

Statistics of pathology
Weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting pvalue was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Tonic-clonic convulsions were observed in 5 high dose female animals on different days during lactation period. Twitching (mostly slight, sometimes moderate) was observed during the entire administration period (except gestation and lactation) in 9 male and all female animals of test group 3 (500 mg/kg bw/d) from premating day 8 onwards on different study days (mostly 0-2 hours, partly 2-5 hours after treatment). The findings were assessed as treatement related and adverse.
Piloerection was observed in 2 male animals of test group 3 (500 mg/kg bw/d) from mating day 2 (study day 15) onwards on different study days. The finding was assessed as being related to treatment and adverse. A single male animal of test group 2 (150 mg/kg bw/d) showed piloerection just on mating day 2. In this case, a relation to treatment was not assumed because of the transient and short occurrence of the finding. Plough nose-first into bedding was observed shortly after treatment during the entire administration period (except gestation and lactation) on different study days in all males and 4 females of test group 3 (500 mg/kg bw/d) as well as in 6 males and 1 female of test group 2 (150 mg/kg bw/d). Salivation shortly after treatment, mostly slight, sometimes moderate, was observed in all phases of the study for all male and female animals of test group 3 (500 mg/kg bw/d) as well as for 7 male and 2 female animals of test group 2 (150 mg/kg bw/d). From the temporary, short appearance immediately after dosing it was concluded that plough nose-first into bedding and salivation were induced by a bad taste of the test substance or local affection of the upper digestive tract.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight of male animals of test group 3 (500 mg/kg bw/d) was significantly reduced on mating day 8 (-5%). Mean body weight change values in male animals of test group 3 (500 mg/kg bw/d) were significantly lower between premating days 0-7 (- 53%) and 0-13 (-41%). These changes were assessed to be related to treatment and adverse.
No significant changes were observed in female animals of test group 3 (500 mg/kg bw/d) as well as in male and female animals of test groups 1 and 2 (50 and 150 mg/kg bw/d).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During the premating period food consumption was significantly decreased in males of test group 3 (500 mg/kg bw/d) between study days 0-7 and 0-13 as well as in females of the same test group between study days 0-7, 7-13 and 0-13. The changes were assessed to be related to treatment. No comparable observation were made in any other test group of the study. No impairment of food consumption was observed for female animals of any test group during the gestation and lactation periods.

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

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related, adverse changes among hematological parameters were observed. In male rats of test group 3 (500 mg/kg bw/d) absolute neutrophil cell counts and in females of the same test group absolute monocyte cell counts were higher compared to controls. In males the absolute neutrophil counts were within the historical control range and in females the absolute monocyte counts were marginally above the range (males absolute neutrophils 0.70-1.35 Giga/L; females absolute monocytes 0.05-0.09 Giga/L; PART III, Supplement). No other changes in the differential blood cell counts occurred. Therefore, the change in males was regarded as incidental and not treatment-related, and the marginally higher monocyte counts in females were regarded as maybe treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002).
In males of test group 2 (150 mg/kg bw/d) relative neutrophil cell counts were decreased and relative lymphocyte cell counts were increased. Both parameters were not dose-dependently changed and therefore this alteration was regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The only treatment related effect were increased urea levels and decreased glucose levels in high dose males. These changes might be related to the a2µ nephropathy observed in all dose groups, which does not represent a human relevant hazard.

In females of test groups 2 and 3 (150 and 500 mg/kg bw/d) inorganic phosphate and cholesterol levels were increased. However, cholesterol levels in both mentioned test groups and inorganic phosphate values at least in test group 2 were within the historical control ranges (cholesterol 1.13-1.77 mmol/L; inorganic phosphate 1.14-1.55 mmol/L; PART III, Supplement). Therefore, the changes in the mentioned test groups were regarded as incidental and not treatment-related. Higher inorganic phosphate levels in females of test group 3 (500 mg/kg bw/d) were the only altered parameter in these individuals and therefore this increase was regarded as maybe treatment-related but not adverse (ECETOC Technical Report No. 85, 2002). In males of test groups 1 and 2 (50 and 150 mg/kg bw/d) total bile acid levels were lower compared to controls, but this change was not dose-dependent. Therefore, it was regarded as incidental and not treatment-related.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related, adverse changes among urinalysis parameters were observed.

In males of test group 3 (500 mg/kg bw/d) urine pH value was lower and in females of the same test group urine volume was higher compared to controls. Both parameter changes without any other changes in the kidneys were regarded as treatment-related but not adverse.
In males of test groups 1, 2 and 3 (50, 150 and 500 mg/kg bw/d) higher incidences of transitional epithelial cells and granulated and epithelial casts were found in the urine sediment. This observation correlates well with the histopathological finding of alpha2u-Globulinurie, which was regarded as a rat specific effect with no relevance to humans (Hard et al., 1993).

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Home cage observations: Twitching was observed in 2 females (Nos. 137 and 138) of test group 3 (500 mg/kg bw/d).
Open field observations: Twitching was observed in 2 females (Nos. 137 and 138) of test group 3 (500 mg/kg bw/d).
Sensorimotor tests/reflexes: No test substance-related effects were observed.
Quantitative Parameters: No test substance-related effects were observed.
Motor activity measurement: A significant deviation concerning the overall motor activity (summation of all intervals) was observed in female animals of test group 1 (50 mg/kg bw/d) in comparison to the concurrent control group. Regarding single intervals in male animals of test group 1 (50 mg/kg bw/d) a significantly reduced activity was observed in interval 6 as well as in female animals of test group 1 (50 mg/kg bw/d) in interval 11. As no other single intervals as well as the overall motor activity showed significant deviations to the control values and a dose-response relationship was not observed, the finding was assessed to be incidental and not related to treatment.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

All mean absolute weight parameters showed no significant differences when compared to the control group.
The significant increase of the relative kidney weight in high dose males (119%) correlated with histopathological findings and was considered treatment-related, but not relevant for humans.

In males of test group 2 (150 mg/kg bw/d), the significant increase of the liver weight (2.507%) was within the historical control range (2.126-2.533%). In males of test group 3 (500 mg/kg bw/d), the weight was marginally beyond the control range but the difference was not statistically significant and had no histopathological correlate. In females of test group 2 and 3, the significant increase of the liver weight (2.55% and 2.673%, respectively) was within the historical control range (2.167-2.814%). Thus, liver weight changes in both sexes were assessed as not treatment-related. All other mean relative weight parameters showed no significant differences when compared to the control group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The kidneys of male animals in test groups 2 and 3 (150 and 500 mg/kg bw/d) showed a light brown discoloration, which was most likely consistent with increased tubular accumulation of eosinophilic droplets and was regarded as treatment-related. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in the kidneys of male animals. The number and pattern of eosinophilic droplets in the proximal convoluted tubules was highly discerned in the H&E stained tissues. Thus, a dose-dependent increase of eosinophilic droplets was noted. This finding was characterized not only by an increase in the number of droplets in the tubular epithelium cells but also by an increase in the number of affected tubules. With increasing dose, the form of the eosinophilic droplets changed to more angular and crystal-shaped eosinophilic aggregates. Concomitantly, tubular degeneration and regeneration in the cortex and outer stripe of the outer medulla (OSOM) and granular casts at the junction of the OSOM and the inner stripe of the outer medulla (ISOM) accompanied this process. The immunostaining with an antibody to α2u-globulin correlated partially with the distribution pattern of eosinophilic droplets. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Key result

Dose descriptor:

NOAEL

Remarks:

human relevant

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

Remarks on result:

other: tubular damage in the kidneys of male animals of all test groups as a consequence of an α2µ nephropathy does not represent a risk for humans

Key result

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

7

Species:

rat

Quality of whole database:

OECD 422, GLP (RL1)

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

Additional information

OECD 422

Laromer TBCH was given daily by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0, 50, 150 and 500 mg/kg bw/day. Control animals were dosed daily with the vehicle only (corn oil). The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and 4 days of lactation period in females up to one day prior to the day of schedule sacrifice of the animals. The animals' health was checked daily. A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals. Food consumption was determined regularly once weekly before and after the mating period, as well as in dams during gestation (days 0-7, 7-14, 14-20) and lactation (days 1-4). In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, and on postnatal days (PND) 0 and 4. Towards the end of the administration period a functional observational battery was performed and motor activity was measured in 5 animals per sex and test group. Clinicochemical and hematological examinations as well as urinalyses were performed in 5 animals per sex and group towards the end of the administration period. All parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

For the animals of the test group with 500 mg/kg bw/day following findings were observed: Twitching (mostly slight, sometimes moderate) was observed during the entire administration period in 9 male and all female animals from premating day 8 onwards on different study days (mostly 0-2 hours, partly 2-5 hours after treatment). Tonic-clonic convulsions were observed in up to 6 female animals during gestation and lactation periods. Piloerection was observed in 2 male animals from mating day 2 (study day 15) onwards on different study days. Reduced food consumption was observed for male and female animals during the premating phase. Mean body weight of male animals was significantly reduced on mating day 8 (-5%). Mean body weight change values of male animals were significantly lower between premating days 0-7 (-53%) and 0-13 (-41%). Increased urea levels and decreased glucose levels were determined in male animals, likely indicative of reduced renal function. α2µ-globulin was detected in kidney tubules of all male animals (minimal to severe) as well as tubular degeneration/regeneration (minimal to severe). Granular casts occured in the kidneys of 8 out of 10 male animals (minimal to severe). In mid-dose males α2µ-globulin was also detected in the kidney tubules of all male animals (slight to severe) as well as tubular degeneration/regeneration (minimal to moderate). Granular casts occured in the kidneys of 6 out of 10 male animals (minimal to slight). Low dose males showed the same alterations in the kidney, but with lower incidence and severity.

Under the conditions of the present Combined Repeated-Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test the oral administration of Laromer TBCH by gavage to male and female Wistar rats revealed signs of systemic toxicity at a dose level of 500 mg/kg bw/day. The no observed adverse effect level (NOAEL) for general systemic toxicity was 150 mg/kg bw/day for male and female Wistar rats taking into account that tubular damage in the kidneys of male animals of all test groups as a consequence of an α2u-nephropathy does not represent a risk for humans (Durham and Swenberg, 2013).

OECD 408

In a subchronic (90-day) study according to OECD 408, 10 males and females per group were exposed orally via gavage to Laromer TBCH in corn oil. Dose levels were selected based on the previous OECD 422 as 50, 150, and 500 mg/kg bw/day.

Males in all dose groups showed alterations in the kidney. These adverse changes consisted of an increased incidence and severity of hyaline droplet accumulation which correlated with pale discoloration seen at 500 mg/kg bw/day and with higher kidney weight starting at 150 mg/kg bw/day, granular casts, tubular degeneration and an increased incidence and severity of tubular basophilia, consistent with alpha 2µ-globulin nephropathy.

At 500 mg/kg bw/day, non-adverse transient tremors of the forelimbs, head and shoulder muscles and closed eyes were observed in males and females, as well as clonic movements in one male during functional observations. Non-adverse higher body weight (gain) and food consumption were observed in females, as well as non-adverse clinical pathology changes consisting of higher white blood cell, neutrophil, lymphocyte, monocyte, eosinophil, large unstained cell, and reticulocyte counts. The higher reticulocyte count correlated with the nonadverse increased incidence and severity of extramedullary hematopoiesis and increased mean severity of pigment in the spleen. In addition, non-adverse lower albumin, total protein, and glucose concentrations, and higher urea and inorganic phosphate concentrations were observed in males. Whereas females, showed non-adverse higher cholesterol, HDL cholesterol and potassium concentrations. In the liver, hepatocellular hypertrophy was noted in males and females which generally correlated with higher liver weights in both sexes. In the thyroid gland, an increased incidence of follicular cell hypertrophy was noted in males. Both histopathological observation were minor and considered non-adverse.

The human relevant NOAEL (excluding a2µ-globulin nephropathy) was set at 500 mg/kg bw/day.

Other than in the OECD 422, twitching was only observed on very few days in the OECD 408 despite longer treatment with the same doses. This difference is likely due to differences in observation times. In the OECD 422, it was stated that twitching occured mostly between 0-2h, partly between 2-5h after dosing. According to the lab, the technicians leave the room after dosing and return for the 0-2h observation close to the end of the interval. In the OECD 408, cage side observations were performed within 1h after dosing. No later time points were checked, except on the days of the FOB examinations, when the tremors were observed. In addition, in the concurrently performed OECD 414 (same lab as the OECD 408), the rats were observed 2h after dosing and twitching was observed comparable to the OECD 422.

All studies agree that the effect is transient and histopathology revealed no neural damage. In addition to a LOAEL of 500 mg/kg bw/day, no classification for STOT RE is warranted.

For human risk assessment (DNEL derivation) the lower NOAEL of 150 mg/kg bw/day will be used as a starting point, since the relevant effect was likely missed in the OECD 408.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Effects were observed at 500 mg/kg bw/day in a subacute study. This is above the proposed limit of 300 mg/kg bw/day according to Regulation (EC) No 1272/2008 (CLP). Consequently, the registered substance does not need to be classified for specific target organ toxicity and the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.