Cyclopropanecarboxylic acid, 2-[1-(3,3-dimethylcyclohexyl)ethoxy]-2-methylpropyl ester

Endpoint summary

Description of key information

Oral: NOAEL (rat): 30 mg/kg body weight per day ; male/female, OECD TG 407, 2004

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

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13-05-2004 to 26-10-2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines for Screening, Toxicity Testing of Chemicals: Testing Methods for New Substances, (July 13, 1974, amended December 5, 1986)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: US EPA OPPTS 870.3050, Repeated Dose 28-Day Oral Toxicity Study in Rodents (July 2000)

Deviations:

no

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

other: HanBrl:WIST (SPF)

Details on species / strain selection:

The species and strain was selected in accordance with the OECD TG 407 and the other relevant guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Recognised supplier (reported in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: males 132.6 – 154.2 g and females 116.2 – 131.5 g (at acclimatisation); individuals were randomly allocated to treatment groups.
- Fasting period before study: None
- Housing: Makrolon type-4 cages with wire mesh tops and standardized softwood bedding. Cage distribution within the holding rack was randomized.
- Diet: Rodent Pelleted Maintenance Diet (certified supplier), ad libitum (removed overnight before blood sampling for haematology or blood chemistry and during the period of urine collection).
- Water (e.g. ad libitum): ad libitum
- Acclimation period: > 5 days.

DETAILS OF FOOD AND WATER QUALITY: Feed: Rodent Pelleted Maintenance Diet (certified supplier) – batch numbers and certificates of analysis provided in the full study report. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 30-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 2004-05-20 To: 2004-07-01

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

PEG 300

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly. The test item was prepared at the appropriate concentrations in PEG 300 vehicle. The required amounts of test material were weighed out into a suitable container. The formulation was magnetically stirred until all test material was thoroughly mixed. The required volume was made up with vehicle and the formulation returned to the container and mixed using a magnetic stirrer until homogenous.

DIET PREPARATION
- Rate of preparation of diet (frequency): Not applicable.
- Mixing appropriate amounts with (Type of food): Not applicable.
- Storage temperature of food: Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Applicant assessment indicates: Aqueous vehicle was not applicable due to limited solubility. PEG 300 was considered as appropriate based on test item solubility. The stability and homogeneity of the test item formulations were determined during the study. Samples for analysis of concentration and homogeneity were stored deep-frozen (about -20 °C) until analysis. Storage stability samples were collected after storage (2 hours and 7 days under storage conditions) and then delivered to the analytical laboratory where they were stored deep frozen until analysis. Results show the formulations to be homogeneous and stable for at least seven days when stored refrigerated. Stability was confirmed at concentrations of 30, 150 and 750 mg/kg or 6, 30 and 150 mg/mL following storage. Formulations were therefore prepared weekly during the treatment period, divided into daily aliquots and stored.
- Concentration in vehicle: Samples of the test item formulations were taken and analysed for concentration of test item (method of analysis provided in full study report). The results indicate that the prepared formulations were within ±6% of the nominal concentration. Formulations was assessed and confirmed at nominal concentrations, during refrigerated storage. The test item concentrations for each group are indicated in table 1.
- Amount of vehicle (if gavage): Treatment volume was 5 mL/kg for control (negative, untreated group) and all treatment groups with applicable test item concentrations per group. For further information see 'Doses / concentrations'.
- Other: Dose-formulations were analysed during the study and were reported as with ± 10 % applied limits.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The stability and homogeneity of the test item formulations were determined during the study. Samples for analysis of concentration and homogeneity were stored deep-frozen (about -20 °C) until analysis. Storage stability samples were collected after storage (2 hours and 7 days under storage conditions) and then delivered to the analytical laboratory where they were stored deep frozen until analysis. Results show the formulations to be homogeneous and stable for at least seven days when stored refrigerated. Stability was confirmed at concentrations of 30, 150 and 750 mg/kg or 6, 30 and 150 mg/mL following storage. Formulations were therefore prepared weekly during the treatment period, divided into daily aliquots and stored.
- The analysis consisted of GC FID analysis with internal calibration (within a dedicated formulation analysis report attached to the full study report). Delivered samples were dissolved in an appropriate volume (about 3/4 of target volume) of cyclohexane using an ultrasonic bath. Then, the 100-mL volumetric flasks were filled to the
mark with cyclohexane. Depending on the dose group, the latter sample solutions were further diluted with cyclohexane to yield concentrations within the calibration range. The concentration of test item in the final solution was quantified by GC using FID detection as detailed in the chromatographic section. The analytical method was validated (details available within the full study report).
- Mean concentrations of dose-formulations analysed during the study were within ± 10% applied limits and % difference from mean were within 6% nominal confirming accurate test item/vehicle formulation.

Duration of treatment / exposure:

Minimum period 28 days followed by a 14 day recovery period (treatment free). The last dose was administered on Day 28.

Frequency of treatment:

Once daily at approximately the same time each day.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Recovery control group

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Low - Group

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Intermediate - Group

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

High - Group

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

Recovery High – Group

No. of animals per sex per dose:

5 per sex per dose (5 male / 5 female); 5 per sex per dose for recovery phase groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the results of a previously conducted 5-day sighting study (Report number attached to and cited in the full study report). Basis: other: nominal in vehicle (PEG 300).
- Rationale for animal assignment (if not random): Randomly assigned
- Post-exposure recovery period in satellite groups: No satellite groups.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily up to day 3, then once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All individuals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed in random sequence once before commencement of administration and once weekly (weeks 1-3) thereafter. Additional functional observations were made as ‘additional evaluations’. During week 4, relevant Functional Observational parameters from a modified lrwin screen test, grip strength, locomotor activity and of functional/behavioural toxicity were evaluated in all individuals.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded prior to dosing on Day 1 and at weekly intervals thereafter. Body weights were also performed prior to termination.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not applicable.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable.
- Other: Food consumption was recorded for each cage group at weekly intervals throughout the study.

FOOD EFFICIENCY: No.

WATER CONSUMPTION: No.
- Time schedule for examinations: Typically, daily. Water intake can be observed daily, for each cage group, by visual inspection. If any abnormalities are noted then further quantitative investigation is performed.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of treatment period (day 29) for all test and control group individuals. End of recovery period (day 15; recovery phase) for all recovery group individuals.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight/18 hours.
- How many animals: All main study and recovery.
- Parameters checked: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular haemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Platelet (thrombocyte) count, Reticulocyte count, Reticulocyte maturity index, Methemoglobin, Totalleukocyte count, Differential leukocyte count, Coagulation: Thromboplastin time, Activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of treatment period (day 29) for all test and control group individuals. End of recovery period (day 15; recovery phase) for all recovery group individuals
- Animals fasted: Yes, overnight/18 hours.
- How many animals: All main study and recovery.
- Parameters checked: Glucose, Urea, Creatinine, Bilirubin, total, Cholesterol, total, Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Glutamate dehydrogenase, Creatine kinase, Alkaline phosphatase, Gamma-glutamyl-transferase, Sodium, Potassium, Chloride, Calcium, Phosphorus inorganic, Protein, total, Albumin, Globulin, Albumin/Globulin ratio

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalytical investigations were performed on all test and control group animals during day 29 fasting period and on all during fasting in the recovery.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (food withheld during time of urine collection; overnight/18 hours)
- Parameters checked: Volume (18 hours), Specific gravity (relative density), Colour, Appearance, pH, Nitrite, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Erythrocytes, Leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes. Was conducted as part of ‘special evaluations’
- Time schedule for examinations: Additional functional observations were made as ‘additional evaluations’. During week 4, relevant Functional Observational parameters from a modified lrwin screen test, grip strength, locomotor activity and of functional/behavioural toxicity were evaluated in all individuals.
- Dose groups that were examined: All.
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

OTHER: Additional post-termination observations were made at necropsy.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- organs weighed: Adrenal glands
Aorta, Bone (sternum, femur including joint), Bone marrow (femur), Brain (4 levels), Cecum, Colon, Duodenum, Epididymides (fixed in Bouin's solution), Esophagus, Eyes with optic nerve (fixed in Davidson's solution), Harderian gland (fixed in Davidson's solution), Heart, Ileum, with Peyer's patches, Jejunum with Peyer's patches, Kidneys, Larynx, Lacrimal gland (exorbital), Liver, Lungs (infused with formalin at necropsy), Lymph nodes (mesenteric, mandibular), Mammary gland area, Nasal cavity, Ovaries, Pancreas, Pituitary gland, Prostate gland (incl. coagulating gland), Rectum, Salivary glands (mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, midthoracic, lumbar), Spleen, Stomach, Testes (fixed in Bouin's solution), Thymus, Thyroid (incl. parathyroid gland), Tongue, Trachea, Urinary bladder (infused with formalin at necropsy), Uterus, Vagina, Gross lesions

HISTOPATHOLOGY: Yes
- Organs and tissues preserved in neutral buffered 4% formalin (unless otherwise indicated):
Adrenal glands, Bone marrow (femur), Brain (4 levels), Cecum, Colon, Duodenum, Epididymides (fixed in Bouin's solution), Heart, Ileum, with Peyer's patches, Jejunum with Peyer's patches, Kidneys, Liver, Lungs (infused with formalin at necropsy), Lymph nodes (mesenteric, mandibular), Ovaries, Prostate gland (incl. coagulating gland), Rectum, Sciatic nerve, Seminal vesicles, Spinal cord (cervical, midthoracic, lumbar), Spleen, Stomach, Testes (fixed in Bouin's solution), Thymus, Thyroid (incl. parathyroid gland), Trachea, Urinary bladder (infused with formalin at necropsy), Uterus, Vagina, Gross lesions. Microscopic analysis was conducted thereof. Any macroscopically observed lesions were also processed.

Statistics:

The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, organ weights and ratios:
The Dunnett-test (many to one t-test) based on a pooled variance estimate were applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex. The Steel-test (many-one rank test) were applied instead of the Dunnett-test when the data cannot be assumed to follow a normal distribution. Fisher's exact-test were applied to the macroscopic findings.

The following statistical methods were used for statistical analysis of clinical laboratory data:
Quantitative data were analyzed by a one-way analysis of variance (ANOVA) when the variances are considered homogeneous according to Bartlett. Alternatively, if the variances are considered to be heterogenous (p < or = 0.05), a non-parametric Kruskai-Wallis test was used. Treated groups were compared to the control groups using Dunnett's test if the ANOVA was significant at the 5% level and by Dunn's test in the case of a significant Kruskai-Wallis test (p < or = 0.05).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 750 mg/kg bw/day: episodes of sedation were noted intermittently in males (days 17-18 and 24-26) and persistently in females (days 16-28). Emaciation was recorded in one female treated with 750 mg/kg bw/day and in two females during the first week of recovery.

No other findings were considered of toxicological relevance.

Mortality:

no mortality observed

Description (incidence):

There was no test item related mortality.

One control male (#10) was found dead on treatment day 12.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 750 mg/kg bw/day: mean body weights of females were slightly lower (-5%) during the treatment period than those of the control females and were considered to be test item related. During recovery no effects attributed to treatment were noted.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 750 mg/kg bw/day: females treated indicated mean daily food consumption values were slightly lower than those of the controls. During recovery no differences were noted.

Food efficiency:

no effects observed

Description (incidence and severity):

See body weight and weight changes sections.

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

not examined

Description (incidence and severity):

No effects on water consumption were reported nor in related clinical signs/functional observations or urinalysis investigations were anything significant noted.

Ophthalmological findings:

not examined

Description (incidence and severity):

There were no toxicologically significant reported effects to the eyes (in life or post termination) in the parameters examined.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At 750 mg/kg bw/day: mean absolute and relative reticulocyte counts of the females were reduced, after four weeks of treatment. Both values exceeded the lower limit of the historical control values. The mean reticulocyte maturity indices were shifted towards cells of low fluorescence (increased) from middle fluorescence (reduced) and high fluorescence (reduced), which generally indicates retention of older cells and slower/impaired replacement of younger cells. This finding was considered to be test item related, but was largely reversible after 2 weeks of recovery.

After two weeks recovery all other differences were within the ranges of historical controls.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 750 mg/kg bw/day: Aspartate aminotransferase activity was increased in males/females and lactate dehydrogenase activity was elevated in females when compared with the controls. These values exceeded the ranges of the historical control data and were considered related to metabolic activation caused by the test item. These findings were, however, reversible during the recovery period and therefore considered to be non-adverse. After two weeks recovery all differences were within the ranges of historical controls.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

At 750 mg/kg bw/day: Elevated ketone noted in the urinalysis parameters of males/females after 4 weeks of treatment, was considered possibly related to metabolism of the test item. After 2 weeks of recovery, this finding was no longer observed. Indicating recovery.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

No functional observation findings were considered of toxicological relevance.

At 750 mg/kg bw/day: reduced mean hindlimb grip strength was noted in males whereas females had reduced mean fore- and hind-limb grip strength. These differences were considered to be test item related. Reductions of locomotor activity were noted in males and females.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 750 mg/kg bw/day: males had elevated absolute and relative liver weights, as well as elevated kidney to body weight ratios. Females had elevated absolute and relative liver weights, elevated absolute and relative kidney weights and reduced absolute and relative spleen weights. Additionally, elevated heart-to-body weight ratio when compared with controls. After two weeks recovery, no test item-related changes were noted in males or females previously treated with 750 mg/kg bw/day indicating recovery.

At 150 mg/kg bw/day: males had elevated liver-to-body weight ratios and elevated kidney-to-body weight ratios noted after four weeks of treatment. Females had elevated absolute and relative liver weights, and reduced absolute spleen weights.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 750 mg/kg bw/day: item-related macroscopic findings were recorded in the Harderian Glands (black foci: one female after treatment, and five recovery females).

At 150 mg/kg bw/day: item-related macroscopic findings were recorded at the liver (clay-coloured: 4 females).

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At 750 mg/kg bw/day: The test item induced histopathological changes in the heart, skeletal muscle, kidneys, Harderian gland, liver, and thyroid.
HEART : minimal to severe sarcoplasmic vacuolation, minimal to moderate single cell necrosis, increased incidence and mean grade of mononuclear foci, and minimal myocardial (interstitial) fibrosis were recorded in main study males/females treated with 750 mg/kg/day or 150 mg/kg/day, reflecting a cardiotoxic potential of the test item. After a 14-day treatment-free recovery period, minimal myocardial fibrosis was recorded in two females previously treated with 750 mg/kg/day. These changes were considered adverse effects.
SKELETAL MUSCLE: minimal to slight mixed-cellular, interstitial inflammation, accompanied by minimal to slight interstitial edema, minimal single fiber necrosis, and partly minimal muscle cell regeneration was recorded in some main study females treated with 750 mg/kg/day. These changes were considered adverse effects.
KIDNEYS: increased incidence and mean grade of hyaline droplets and of tubular basophilia was recorded in males treated with 750 mg/kg/day and 150 mg/kg/day.
The incidence and mean grade of tubular basophilia was still increased in recovery males. In rats, these changes were adverse effects, whose relevance for human health risk assessment is unclear. Applicant assessment indicates that this may be: cortical hyaline droplets that could be considered to represent alpha2uglobulin, which is a male rat-specific lesion, supported by absence of these kidney related effects in females. This is suggestive of this effect not being relevant to human health. However, further evidence may be required to support that hypothesis.
HARDERIAN GLAND: minimal acinar degeneration, minimal to slight acinar hyperplasia, and increased porphyrin deposition was recorded in females treated with 750 mg/kg/day or 150 mg/kg/day. Although these findings were considered adverse effects in the rat, their relevance for human health risk assessment are unclear.
LIVER: increased incidence and mean grade of liver fatty change was recorded in main study females treated with 750 mg/kg/day or 150 mg/kg/day. After recovery this finding showed tendency to regression. The toxicological relevance of this finding is unclear. Additionally an increased incidence and mean grade of hepatocellular hypertrophy was recorded. This was absent from recovery males/females. This finding was not an adverse effect.
THYROID: an increased incidence of minimal follicular hypertrophy in males. This was absent from recovery males/females. This finding was not considered an adverse effect.

At 150 mg/kg bw/day: see mentioned sections above (HEART, KIDNEYS, HARDERIAN GLAND and LIVER)

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related abnormalities detected.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

gross pathology

histopathology: non-neoplastic

Critical effects observed:

no

Conclusions:

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for males and females is defined as 30 mg/kg body weight per day in males and females.

Executive summary:

The study was performed according the requirements of OECD TG 407, EU method B.7 and Japanese Guidelines for Screening, Toxicity Testing of Chemicals: Testing Methods for New Substances guidelines under GLP conditions. Following a previously conducted 5-day sighting study, the systemic toxic potential of the test item was assessed orally in a 28 day gavage study in HanBrl:WIST (SPF) rats. Recovery from any effects was evaluated during a subsequent 14 day recovery period. Three groups, each comprising five male and five female rats, received test item at doses of 30, 150 or 750 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (PEG 300) at a dose volume of 5 mL/kg. Two recovery groups, each of five males and five females, were treated with the high dose (750 mg/kg bw/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days. Clinical signs, functional observations including sensory reactivity, grip strength and motor activity were performed, body weight change, food consumption were monitored during the study. Hematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment free period. All individuals were subjected to gross necropsy examination and at termination. Histopathological examination of selected tissues was performed. There was no test item related effect on mortalities. At 750 mg/kg bw/day: episodes of sedation were noted intermittently in males (days 17-18 and 24-26) and persistently in females (days 16-28). Emaciation was recorded in one female treated with 750 mg/kg bw/day and in two females during the first week of recovery.No other findings were considered of toxicological relevance. In functional observations, reduced mean hindlimb grip strength was noted in males whereas females had reduced mean fore- and hind-limb grip strength. These differences were considered to be test item related. Reductions of locomotor activity were noted in both sexes. Females treated indicated mean daily food consumption values were slightly lower than those of the controls. During recovery no differences were noted.Females treated indicated mean daily food consumption values were slightly lower than those of the controls. During recovery no differences were noted.Aspartate aminotransferase activity was increased in males/females and lactate dehydrogenase activity was elevated in females when compared with the controls. These values exceeded the ranges of the historical control data and were considered related to metabolic activation caused by the test item. These findings were, however, reversible during the recovery period and therefore considered to be non-adverse. After two weeks recovery all differences were within the ranges of historical controls.In urinalysis at 750 mg/kg bw/day: elevated ketone noted in males/females after 4 weeks of treatment, was considered possibly related to metabolism of the test item. After 2 weeks of recovery, this finding was no longer observed. Indicating recovery. Males had elevated absolute and relative liver weights, as well as elevated kidney to body weight ratios. Females had elevated absolute and relative liver weights, elevated absolute and relative kidney weights and reduced absolute and relative spleen weights. Additionally, elevated heart-to-body weight ratio when compared with controls. After two weeks recovery, no test item-related changes were noted in males or females previously treated with 750 mg/kg bw/day indicating recovery. At 150 mg/kg bw/day: males had elevated liver-to-body weight ratios and elevated kidney-to-body weight ratios noted after four weeks of treatment. Females had elevated absolute and relative liver weights, and reduced absolute spleen weights.At 750 mg/kg bw/day: item-related macroscopic findings were recorded in the Harderian Glands (black foci: one female after treatment, and five recovery females, whereas at 150 mg/kg bw/day: item-related macroscopic findings were recorded at the liver (clay-coloured: 4 females).At 750 mg/kg bw/day: The test item induced histopathological changes in the heart, skeletal muscle, kidneys, Harderian gland, liver, and thyroid. In the heart : minimal to severe sarcoplasmic vacuolation, minimal to moderate single cell necrosis, increased incidence and mean grade of mononuclear foci, and minimal myocardial (interstitial) fibrosis were recorded in main study males/females treated with 750 mg/kg/day or 150 mg/kg/day, reflecting a cardiotoxic potential of the test item. After a 14-day treatment-free recovery period, minimal myocardial fibrosis was recorded in two females previously treated with 750 mg/kg/day. These changes were considered adverse effects. In skeletal muscle: minimal to slight mixed-cellular, interstitial inflammation, accompanied by minimal to slight interstitial edema, minimal single fiber necrosis, and partly minimal muscle cell regeneration was recorded in some main study females treated with 750 mg/kg/day. These changes were considered adverse effects. In kidneys: increased incidence and mean grade of hyaline droplets and of tubular basophilia was recorded in males treated with 750 mg/kg/day and 150 mg/kg/day. The incidence and mean grade of tubular basophilia was still increased in recovery males. In the Harderian gland: minimal acinar degeneration, minimal to slight acinar hyperplasia, and increased porphyrin deposition was recorded in females treated with 750 mg/kg/day or 150 mg/kg/day. Although these findings were considered adverse effects in the rat, their relevance for human health risk assessment are unclear. In the liver: increased incidence and mean grade of liver fatty change was recorded in main study females treated with 750 mg/kg/day or 150 mg/kg/day. After recovery this finding showed tendency to regression. The toxicological relevance of this finding is unclear. Additionally an increased incidence and mean grade of hepatocellular hypertrophy was recorded. This was absent from recovery males/females. This finding was not an adverse effect. In the thyroid: an increased incidence of minimal follicular hypertrophy in males. This was absent from recovery males/females. This finding was not considered an adverse effect. The oral (gavage) administration of the test item to males/females at dose levels of 30, 150 or 750 mg/kg bw/day resulted indicated that, the No-Observed-Effect-Leve (NOEL) was 30 mg/kg bw/day and the No-Observed-Adverse-Effect-Level (NOAEL) was regarded to be 30 mg/kg bw/day for males/females.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

30 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study is GLP compliant and of a high quality (Klimisch 1); The available information as a whole meets the tonnage driven information requirements of REACH.

System:

cardiovascular

Organ:

heart

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

Repeated dose - Oral:

Key study: OECD TG 407, 2004 : The study was performed according the requirements of OECD TG 407, EU method B.7 and Japanese Guidelines for Screening, Toxicity Testing of Chemicals: Testing Methods for New Substances guidelines under GLP conditions. Following a previously conducted 5-day sighting study, the systemic toxic potential of the test item was assessed orally in a 28 day gavage study in HanBrl:WIST (SPF) rats. Recovery from any effects was evaluated during a subsequent 14 day recovery period. Three groups, each comprising five male and five female rats, received test item at doses of 30, 150 or 750 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (PEG 300) at a dose volume of 5 mL/kg. Two recovery groups, each of five males and five females, were treated with the high dose (750 mg/kg bw/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days. Clinical signs, functional observations including sensory reactivity, grip strength and motor activity were performed, body weight change, food consumption were monitored during the study. Hematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment free period. All individuals were subjected to gross necropsy examination and at termination. Histopathological examination of selected tissues was performed. There was no test item related effect on mortalities. At 750 mg/kg bw/day: episodes of sedation were noted intermittently in males (days 17-18 and 24-26) and persistently in females (days 16-28). Emaciation was recorded in one female treated with 750 mg/kg bw/day and in two females during the first week of recovery.No other findings were considered of toxicological relevance. In functional observations, reduced mean hindlimb grip strength was noted in males whereas females had reduced mean fore- and hind-limb grip strength. These differences were considered to be test item related. Reductions of locomotor activity were noted in both sexes. Females treated indicated mean daily food consumption values were slightly lower than those of the controls. During recovery no differences were noted.Females treated indicated mean daily food consumption values were slightly lower than those of the controls. During recovery no differences were noted.Aspartate aminotransferase activity was increased in males/females and lactate dehydrogenase activity was elevated in females when compared with the controls. These values exceeded the ranges of the historical control data and were considered related to metabolic activation caused by the test item. These findings were, however, reversible during the recovery period and therefore considered to be non-adverse. After two weeks recovery all differences were within the ranges of historical controls.In urinalysis at 750 mg/kg bw/day: elevated ketone noted in males/females after 4 weeks of treatment, was considered possibly related to metabolism of the test item. After 2 weeks of recovery, this finding was no longer observed. Indicating recovery. Males had elevated absolute and relative liver weights, as well as elevated kidney to body weight ratios. Females had elevated absolute and relative liver weights, elevated absolute and relative kidney weights and reduced absolute and relative spleen weights. Additionally, elevated heart-to-body weight ratio when compared with controls. After two weeks recovery, no test item-related changes were noted in males or females previously treated with 750 mg/kg bw/day indicating recovery. At 150 mg/kg bw/day: males had elevated liver-to-body weight ratios and elevated kidney-to-body weight ratios noted after four weeks of treatment. Females had elevated absolute and relative liver weights, and reduced absolute spleen weights.At 750 mg/kg bw/day: item-related macroscopic findings were recorded in the Harderian Glands (black foci: one female after treatment, and five recovery females, whereas at 150 mg/kg bw/day: item-related macroscopic findings were recorded at the liver (clay-coloured: 4 females).At 750 mg/kg bw/day: The test item induced histopathological changes in the heart, skeletal muscle, kidneys, Harderian gland, liver, and thyroid. In the heart : minimal to severe sarcoplasmic vacuolation, minimal to moderate single cell necrosis, increased incidence and mean grade of mononuclear foci, and minimal myocardial (interstitial) fibrosis were recorded in main study males/females treated with 750 mg/kg/day or 150 mg/kg/day, reflecting a cardiotoxic potential of the test item. After a 14-day treatment-free recovery period, minimal myocardial fibrosis was recorded in two females previously treated with 750 mg/kg/day. These changes were considered adverse effects. In skeletal muscle: minimal to slight mixed-cellular, interstitial inflammation, accompanied by minimal to slight interstitial edema, minimal single fiber necrosis, and partly minimal muscle cell regeneration was recorded in some main study females treated with 750 mg/kg/day. These changes were considered adverse effects. In kidneys: increased incidence and mean grade of hyaline droplets and of tubular basophilia was recorded in males treated with 750 mg/kg/day and 150 mg/kg/day. The incidence and mean grade of tubular basophilia was still increased in recovery males. In the Harderian gland: minimal acinar degeneration, minimal to slight acinar hyperplasia, and increased porphyrin deposition was recorded in females treated with 750 mg/kg/day or 150 mg/kg/day. Although these findings were considered adverse effects in the rat, their relevance for human health risk assessment are unclear. In the liver: increased incidence and mean grade of liver fatty change was recorded in main study females treated with 750 mg/kg/day or 150 mg/kg/day. After recovery this finding showed tendency to regression. The toxicological relevance of this finding is unclear. Additionally an increased incidence and mean grade of hepatocellular hypertrophy was recorded. This was absent from recovery males/females. This finding was not an adverse effect. In the thyroid: an increased incidence of minimal follicular hypertrophy in males. This was absent from recovery males/females. This finding was not considered an adverse effect. The oral (gavage) administration of the test item to males/females at dose levels of 30, 150 or 750 mg/kg bw/day resulted indicated that, the No-Observed-Effect-Leve (NOEL) was 30 mg/kg bw/day and the No-Observed-Adverse-Effect-Level (NOAEL) was regarded to be 30 mg/kg bw/day for males/females.

Justification for classification or non-classification

The substance meets classification criteria under Regulation (EC) No 1272/2008 for specific organ toxicity repeated exposure STOT-RE: category 2: H373: May cause damage to organs : Heart

Since there was reported significant effects relevant to humans reported at guidance related levels (ORAL ≤ 300 mg/kg bw/day) then there is requirement to classify STOT-RE: category 2.

References:

1. ECHA Guidance on Application on the CLP Criteria, (v5.0, July 2017), Section 3.9.2 : Table 3.16 - Equivalent guidance values for 28-day and 90-day studies