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Administrative data

Description of key information

NOAEL (oral) = 1000 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-06-28 to 2013-03-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted under GLP according to OECD TG 422.
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories BV, Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: 290 to 326 g (males), 162 to 222 g (females)
- Fasting period before study: no
- Housing: in groups of three to five animals in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS
- The dose formulations were prepared weekly using the test item as supplied by the Sponsor. The test material was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using a magnetic stirrer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Storage temperature of food: at room temperature (20 ± 5 °C) in glass beakers

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 0, 12.5, 75, 250 mg/mL
- Amount of vehicle: 4 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. To confirm the stability (8 days) samples of about 2 g of each concentration were taken from the middle of each aliquot used on day 7 of the treatment. During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to analytic laboratory and stored there at -20 ± 5 °C until analysis. The samples received were dissolved in TBME by sonication for at least 5 minutes and then diluted to volume with TBME. Sample solutions were further diluted with TBME into the calibration range. The samples were analysed with gas chromatography coupled to an FID.
Duration of treatment / exposure:
The test substance was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 3 post-partum, one day before necropsy.
Frequency of treatment:
daily
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
11
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous non-GLP dose range-finding toxicity study in Han Wistar rats, using dose levels of 0, 50, 200 and 1000 mg/kg/day.
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: In males, it was performed once prior to the first administration of the test item and weekly thereafter. In females, it was prepared once prior to the first administration of the test item, weekly during the pre-pairing and pairing periods and on days 0, 6, 13 and 20 of the gestation period.

BODY WEIGHT: Yes
- Time schedule for examinations: Daily from treatment start to day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Males: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 13; after pairing period weekly.
- Females: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 13; gestation days 0 - 7, 7 - 14 and 14 - 21 and days 1 - 4 of lactation.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 14 of the pre-pairing period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 males and 5 females from each group
- Following parameters were examined: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Leukocyte count (total), Differential leukocyte count, Platelet count, Prothrombin time (= Thromboplastin time), Activated partial Thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day 14 of the pre-pairing period
- Animals fasted: Yes
- How many animals: 5 males and 5 females from each group
- Following parameters were examined: Glucose, Urea, Creatinine, Bilirubin (total), Bile acids, Sodium, Potassium, Chloride, Cholesterol (total), Triglycerides, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Gamma-glutamyl-transferase, Calcium, Phosphorus, Protein (total), Albumin, Globulin, Albumin/Globulin ratio

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: At one time during the study (males shortly before the scheduled sacrifice and females on day 3 post-partum) relevant parameters were performed. This FOB assessment was conducted following the daily dose administration.
- Dose groups that were examined: five P generation males and five P generation females from each group
- Battery of functions tested: hand-held observations, open field observations, reflexes, counts: hind limb, fore limb grip strength
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see attached document Organ Weights and Preservation)

HISTOPATHOLOGY: Yes (see attached document Organ Weights and Preservation)
Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was 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) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At the dose level of 1000 mg/kg bw/day, bedding in mouth and salivation in males starting from day 6 of the pairing period and in females starting from day 1 of the pairing period. At the dose level of 300 mg/kg bw/day, bedding in mouth in females starting from day 1 of the gestation period.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effects on absolute body weight or body weight gain were noted in males and females at any dose level.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At the dose level of 1000 mg/kg bw/day in females, statistically significant reduction in food consumption was noted from day 1 to 4 of the pre-pairing period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
please see Details on results
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
please see Details on results
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Enlarged liver in one female at the dose level of 1000 mg/kg bw/day
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
please see Details on results
Histopathological findings: neoplastic:
not specified
Other effects:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
At the dose level of 1000 mg/kg bw/day, bedding in mouth and salivation in males starting from day 6 of the pairing period and in females starting from day 1 of the pairing period. At the dose level of 300 mg/kg bw/day, bedding in mouth in females starting from day 1 of the gestation period. These observations were considered to be signs of discomfort caused by the treatment with the test item.

BODY WEIGHT AND WEIGHT GAIN
No effects on absolute body weight or body weight gain were noted in males and females at any dose level.

FOOD CONSUMPTION AND COMPOUND INTAKE
No test item-related effects on food consumption were noted in males at any dose level. At the dose level of 1000 mg/kg bw/day in females, statistically significant reduction in food consumption was noted from day 1 to 4 of the pre-pairing period. During the remaining study period, food consumption at the high-dose level was similar to the control values.

HAEMATOLOGY
No test item-related findings were noted in males and females at any dose level.

CLINICAL CHEMISTRY
In males statistically significantly higher creatinine concentration was noted in all dose groups. Mean creatinine concentration was 25.3 μmol/L at the dose level of 1000 mg/kg bw/day and 24.4 μmol/L at dose levels of 300 and 50 mg/kg bw/day versus 19.8 μmol/L in the control group. Further, at the dose levels of 1000 and 300 mg/kg bw/day, statistically significantly reduced concentration of phosphorus was noted. Mean phosphorus concentrations were 1.96 and 1.92 mmol/L at the dose levels of 1000 and 300 mg/kg bw/day, respectively, versus 2.11 mmol/L in the control group. Both findings may indicate renal dysfunction. Histopathological examination revealed lesions in the kidneys in males in the high- and mid-dose levels which were sex- and species-specific for male rats. Therefore, changes in creatinine and phosphorus concentrations were considered to most probably be secondary to these lesions and not relevant for humans. Furthermore, statistically significantly lower concentrations of total bilirubin and bile acids were noted in males at the dose level 1000 mg/kg bw/day. Mean total bilirubin concentration was 1.14 μmol/L at the high-dose level versus 1.88 μmol/L in the control group, mean concentration of bile acids was 14.46 μmol/L at the high-dose level versus 50.41 μmol/L in the control group. Lower total bilirubin and bile acids concentrations were considered to possibly be secondary to the adaptive changes in the liver metabolism caused by the treatment with the test item and not to be adverse. No test item-related effects on biochemistry parameters were noted in females at any dose level.

NEUROBEHAVIOUR
No effects observed.

ORGAN WEIGHTS
At the dose level of 1000 mg/kg bw/day, statistically significantly increased liver weights were noted in males. This finding was considered to be due to adaptive changes in the liver metabolism caused by the treatment with the test item and not as adverse.

GROSS PATHOLOGY
During macroscopically examination enlarged liver was found in one female at the dose level of 1000 mg/kg bw/day. This finding was considered to be related to the treatment with the test item.

HISTOPATHOLOGY: NON-NEOPLASTIC
During microscopic examinations, test item-related findings were noted in the liver of males and females at the dose level of 1000 mg/kg bw/day. These changes consisted of centrilobular hypertrophy of the hepatocytes and were considered to be an adaptive change due to the increased metabolism of the liver. In addition, hypertrophy of the follicular epithelium of the thyroid gland was noted in males at the dose level of 1000 mg/kg bw/day. This finding was considered to be very likely secondary to the increased metabolism in the liver. Further findings related to the treatment were noted in the kidneys of males at the dose levels of 1000 and 300 mg/kg bw/day; hyaline droplets associated with tubular basophilia and presence of granular casts. These findings were considered to be specific for male rats and adverse in this species but not toxicologically relevant for humans.
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects observed
Critical effects observed:
not specified
Conclusions:
Based on the study findings in the rats, the relevant NOAEL for human toxicity is 1000 mg/kg bw/day.
Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of the substance on possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provides information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. The substance was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 3 post-partum, one day before necropsy.

The following dose levels were applied:

Group 1: 0 mg/kg body weight/day (control group)

Group 2: 50 mg/kg body weight/day

Group 3: 300 mg/kg body weight/day

Group 4: 1000 mg/kg body weight/day

A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil).

Mortality and General Tolerability

All animals survived scheduled study period. Bedding in mouth and salivation were noted in males and females at the dose level of 1000 mg/kg bw/day. Bedding in mouth alone was noted in females at the dose level of 300 mg/kg bw/day. These observations were considered to be sign of discomfort caused by the treatment with the test item. No further clinical signs or observations were noted in males or females at any dose level.

Functional Observational Battery

With exception for salivation noted in one male and one female at the dose level of 1000 mg/kg bw/day, no further test item-related findings were noted during the tests of functional observational battery or measurement of locomotor activity.

Food Consumption

At the dose level of 1000 mg/kg bw/day in females, transient reduction in food consumption was noted at the beginning of the treatment. No test item-related effects on food consumption were noted in males at any dose level or females at dose levels of 50 and 300 mg/kg bw/day.

Body Weights

No effects on absolute body weights or body weight gain were noted in males or females at any dose level.

Clinical Laboratory Investigations

In males statistically significantly higher creatinine concentration was noted in all dose groups. This finding was accompanied by statistically significantly reduced concentration of phosphorus in high- and mid-dose groups. These findings were considered to most probably be secondary to lesions in the kidneys which were considered sex- and species-specific for male rats and therefore not relevant for humans. Further findings were statistically significantly lower concentrations of total bilirubin and statistically significantly lower concentration of bile acids in males at the dose level of 1000 mg/kg bw/day. These findings were considered to possibly be secondary to the adaptive changes in the liver metabolism caused by the treatment with the test item and not to be adverse. No further test item-related changes in hematology or biochemistry parameters were noted in males or females at any dose level.

Organ Weights

At the dose level of 1000 mg/kg bw/day, statistically significantly increased liver weights were noted in males. This finding was considered to be due to adaptive changes in the liver metabolism caused by the treatment with the test item and not adverse.

Macroscopical Findings and Histopathological Examinations

During macroscopical examination enlarged liver was found in one female at the dose level of 1000 mg/kg bw/day. This finding was considered to be related to the treatment with the test item.

During microscopic examinations, test item-related findings were noted in the liver of males and females at the dose level of 1000 mg/kg bw/day. These changes consisted of centrilobular hypertrophy of the hepatocytes and were considered to be an adaptive change due to the increased metabolism of the liver. In addition, hypertrophy of the follicular epithelium of the thyroid gland was noted in males at the dose level of 1000 mg/kg bw/day. This finding was considered to be very likely secondary to the increased metabolism in the liver.

Conclusion

Based on the lesions in the kidneys found in males at the dose levels of 1000 and 300 mg/kg bw/day which were considered to be adverse, general NOAEL (No Observed Adverse Effect Level) for males was established at 50 mg/kg bw/day. However, lesions in the kidneys were considered to be specific for male rats and therefore not toxicologically relevant for humans. NOAEL for females was considered to be 1000 mg/kg bw/day, the highest dose level used in the study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study is GLP according to OECD TG 422.

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 toxicity, oral:

The substance is deemed to not show a classifiable specific target organ toxicity. One reliable study conducted according to OECD TG 422, EPA OPPTS 870.3650 and GLP in rats (2013a) using 28-day oral (gavage) at dose levels of 0, 50, 300 and 1000 mg/kg bw/day. The NOAEL for repeated dose toxicity of the substance was 1000 mg/kg bw/day in females and 50 mg/kg bw/day in males, based on hyaline droplet presence at 300 and 1000 mg/kg bw/day doses, which was associated to an increased incidence of tubular basophilia and to the presence of granular casts in one male at the high-dose level. These findings were considered to be sex- and species-specific for male rats and therefore not toxicologically relevant for humans. From regulatory point of view it was concluded, that NOAEL for general toxicity for derivation to humans is 1000 mg/kg bw/day.


Justification for classification or non-classification

-Repeated dose toxicity, oral:

Based on the above stated assessment of the specific target organ toxicity potential after repeated oral exposure to the substance, the compound does not need to be classified according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) or according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.

-Repeated dose toxicity, dermal:

As no data of the specific target organ toxicity potential after repeated dermal exposure of (ethoxymethoxy)cyclododecane is available a classification is not possible according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) or according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.

 

-Repeated dose toxicity, inhalation:

As no data of the specific target organ toxicity potential after repeated dermal exposure of (ethoxymethoxy)cyclododecane is available a classification is not possible according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) or according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.