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Description of key information

A No Observed Adverse Effect Level (NOAEL) of 50 mg/kg body weight/day was established from a repeated dose study, based on adrenals effects in females.

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:
September 2012 - May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks
- Weight at study initiation: Males: 339 to 385 g, Females: 196 to 223 g
- Housing: Group-housed in Makrolon type-4 cages during acclimatization, individually during pre-pairing, and group-housed during pairing period or individually thereafter in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet (e.g. ad libitum): Pelleted standard Harlan Teklad 2914C (batch no. 20/12) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water (e.g. ad libitum): Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: At least 5 days under test conditions after health examination. Only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%):30 - 70%)
- Air changes (per hr): 10 - 15 air changes per hour,
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Method: Oral, by gavage
Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type studies.
Frequency of Administration: Once daily
Daily Target Dose Level:
Group 1: 0 mg/kg/day
Group 2: 50 mg/kg/day
Group 3: 250 mg/kg/day
Group 4: 1000 mg/kg/day (day 1 of treatment), 500 mg/kg/day (day 2 of treatment onwards)

Rationale for Dose Level Selection: The dose levels were selected based on a previous non-GLP dose range-finding toxicity study in Wistar rats, Harlan Laboratories study D61183.
Dose Volume: 5 mL/kg body weight
Dose Concentrations:
Group 1: 0 mg /mL/day
Group 2: 10 mg /mL/day
Group 3: 50 mg /mL/day
Group 4: 200 mg /mL/day (day 1 of treatment), 100 mg /mL/day (day 2 of treatment onwards)
Duration of Acclimatization Period: 7 days
Duration of Treatment Period: Males: Minimum 4 weeks
Females: Approximately 7 weeks
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of Dose Formulations
The dose formulations were analyzed using a GCMS method provided by the Sponsor. The samples (generally 2 g each) were delivered to the analytical laboratory.
Transport of Dose Formulations to Analytical Laboratory: At ambient temperature (20 ± 5 °C)
Storage of Dose Formulations in Analytical Laboratory (if Needed): Frozen (ca. -20 ± 5 °C)

The linearity of the analytical systems (GCMS) used for sample analyses was demonstrated with a good relationship between peak areas measured and working standard concentrations. All calibration points used met the acceptance limit of ±20% variation from the calibration curve derived by linear regression analysis. The regression coefficients (r²) calculated were found to be better than 0.99. The 2-ethylhexyl 3,5,5-trimethylhexanoate peak was assigned in sample chromatograms by comparison to that of working standards. In blank sample chromatograms no peak appeared at the retention time of 2-ethylhexyl 3,5,5-trimethylhexanoate and, therefore, the absence of the test item in the vehicle control samples (corn oil) was confirmed. The 2-ethylhexyl 3,5,5-trimethylhexanoate concentrations in the dose formulations ranged from 84.0% to 103.2% with reference to the nominal and were within the accepted range of ±20%. The homogeneous distribution of 2-ethylhexyl 3,5,5-trimethylhexanoate in the preparations was approved because single results found did not deviate more than 7.8% from the corresponding mean and met the specified acceptance criterion of =15%. In addition, the test item was found to be stable in application formulations when kept up to eight days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean. In conclusion, the results indicate the accurate preparation and storage of the test item 2-ethylhexyl 3,5,5-trimethylhexanoate in vehicle during this study.
Duration of treatment / exposure:
Males: Minimum 4 weeks and Females: Approximately 7 weeks
Frequency of treatment:
Once daily by gavage
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
500 mg/kg bw/day as of day 2 onwards
No. of animals per sex per dose:
12 males and 12 females
Control animals:
yes, concurrent vehicle
Details on study design:
Method: Oral, by gavage
Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type studies.
Frequency of Administration: Once daily
Daily Target Dose Level:
Group 1: 0 mg/kg/day
Group 2: 50 mg/kg/day
Group 3: 250 mg/kg/day
Group 4: 1000 mg/kg/day (day 1 of treatment), 500 mg/kg/day (day 2 of treatment onwards)
Rationale for Dose Level Selection: The dose levels were selected based on a previous non-GLP dose range-finding toxicity study in Wistar rats, Harlan Laboratories study D61183.
Dose Volume: 5 mL/kg body weight
Dose Concentrations:
Group 1: 0 mg /mL/day
Group 2: 10 mg /mL/day
Group 3: 50 mg /mL/day
Group 4: 200 mg /mL/day (day 1 of treatment), 100 mg /mL/day (day 2 of treatment onwards)
Duration of Acclimatization Period: 7 days
Duration of Treatment Period: Males: Minimum 4 weeks
Females: Approximately 7 weeks

Mating, Gestation and Lactation: During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if:
- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.
The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.
If a female did not mate during the 14-day pairing period, a second pairing of this female with a male in the same group, which had already mated successfully, was considered. If mating was not recorded during this additional pairing period of a maximum of 14 days, the female was sacrificed and, if indicated, the reproductive organs examined histopathologically in order to ascertain the reason for the infertility.
All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.
Positive control:
none
Observations and examinations performed and frequency:
Observations
The following observations were recorded as follows:
Viability / Mortality: Twice daily

Clinical Signs: Daily cage-side clinical observations (once daily, 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.

Food Consumption: Males: Weekly during pre-pairing, Females: Pre-pairing period days 1 - 8 and 8 - 13; gestation days 0 - 7, 7 - 14 and 14 - 21 post coitum, and days 1 - 4 post partum. No food consumption was recorded during the pairing period.

Body Weights: Recorded daily from treatment start to day of necropsy.

Pup Data: The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.

Detailed Clinical Observations (Weekly)
Once prior to the first administration of the test item and weekly thereafter, detailed clinical observations were performed outside the home cage. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

Functional Observation Battery
Grip Strength: At one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 post partum), relevant parameters from a modified Irwin screen test were performed with five P generation males from each group and five P generation females from groups 1, 2 and 4 and four P generation females from group 3 in place of the usual weekly behavioral observation. This FOB assessment was conducted following the daily dose administration. Any abnormal findings were recorded and, where appropriate, graded in severity.

Locomotor Activity: Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 10-minute intervals over a period of 60 minutes. These data and the total activity over 60 minutes were reported.
Sacrifice and pathology:
Pathology: Males were sacrificed after treatment for at least 28 days, when no longer needed for the assessment of reproductive effects. Dams and pups were sacrificed on day 4 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

Necropsy: All animals sacrificed or found dead were weighed and subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. At the scheduled sacrifice, all animals were sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated. Dead pups, except those excessively cannibalized, were examined macroscopically. All parent animals and pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred. For the parent animals, special attention was directed at the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

Organ Weights: At the scheduled sacrifice, the testes and epididymides of all parental males were weighed separately. In addition, from 5 males and females killed at the end of the study which were selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken: Adrenal glands (weighed as pairs), Brain, Heart, Kidneys (weighed as pairs), Liver, Thymus, and Spleen.

Tissue Preservation: The following tissues from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution: Prostate, Seminal vesicles with coagulating gland, Testes (in Bouin’s fixative), Epididymides (in Bouin’s fixative).
The following tissues from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution: Ovaries.
In addition, from the five males and females per group selected for organ weights and from all animals found dead or killed in extremis, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution: Gross lesions, Brain, Spinal cord, Small and large intestines (incl. Peyer’s patches), Stomach, Liver, Kidneys, Adrenals, Spleen, Heart, Thymus, Thyroids, and parathyroids if possible, Trachea and lungs (preserved by inflation, with fixative and then immersion), Uterus (with vagina), Urinary bladder, Lymph nodes (mesenterial, mandibular), Peripheral nerve (sciatic), Bone marrow.
Histotechnique: All organ and tissue samples to be examined by the prinicipal investigator for histopathology were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin.

Histopathology: Slides of all organs and tissues listed collected at terminal sacrifice from the animals of the groups 1 to 4 were examined by the principal investigator for histopathology. The same applied to all occurring gross lesions and to all animals, which died spontaneously or had to be terminated in extremis.
Special emphasis was made on the stages of spermatogenesis (qualitative assessment) and histopathology of interstitial cell structure.
If test item-related morphologic changes were detected in organs of any high-dose animal, those same organs from the mid- and low-dose group were examined to establish a no-effect level, if possible.

Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, if necessary.
Other examinations:
Clinical Laboratory Investigations
Blood samples were obtained at the end of the pre-pairing period from 5 males and 5 females from each group. Blood samples were drawn sublingually from all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

Clinical laboratory data are expressed, with a few exceptions, in general accordance with the International System of Units (SI).

Hematology : The following hematology parameters were determined: Complete Blood Cell Count
Erythrocyte count (Mean corpuscular haemoglobin), Hemoglobin (Hemoglobin concentration distribution width), Hematocrit (Leukocyte count, total), Mean corpuscular volume (Differential leukocyte count), Red cell volume distribution width (Platelet count), Mean corpuscular hemoglobin concentration.

Coagulation: Prothrombin time (= Thromboplastin time) (Activated partial Thromboplastin time)

Clinical Biochemistry: The following clinical biochemistry parameters were determined: Glucose, Urea, Creatinine, total Bilirubin, total Cholesterol, Triglycerides, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Gamma-glutamyl-transferase, Bile acids, Sodium, Potassium, Chloride, Calcium, Phosphorus, total Protein, Albumin, Globulin, Albumin/Globulin ratio
Statistics:
Data Compilation: The following data were recorded on-line: clinical signs and observations, food consumption, body weights, necropsy data, organ weights (TOX CONTROL), reproduction and litter data (RCC-TOX LIMS or TOX CONTROL, as appropriate). Microscopic data were entered into the PathData System. All other data were recorded on data sheets and compiled manually.

From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, post-implantation losses, mean litter size, pup sex ratios and viability indices.

For reproduction data, group mean values were calculated both on a litter basis and on a percentage per group basis. Mean pup weights were calculated from the individual weights both on a per group and on a per litter basis.

Computer-generated values in the tables represent the rounded-off results of calculations which used the exact raw data values.
Statistical Analysis: 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 [see References (2)] (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 [see References (3)] (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 [see References (4)] was applied if the variables could be dichotomized without loss of information.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Males:
- During days five to eight of pre-pairing period at 500 mg/kg bw/day, 2 of 12 males showed ruffled fur.
Females:
- At the beginning of dosing in the pre-pairing period at 1000 or 500 mg/kg bw/day, respectively, decreased activity, abnormal or hunched posture, ruffled fur, weakened condition, ataxia and/or prostrating was noted in 5 of 12 females. Thereafter, no specific clinical signs were detected until the end of gestation period.
- Towards the end of gestation period (starting predominantly on days 20 or 21), 8 of originally 12 females at 250 mg/kg bw/day, showed clinical signs such as ruffled fur, decreased activity, hunched posture, ptosis, weakened condition, diarrhea, reddish nasal secretion and/or were prostrating and at 500 mg/kg bw/day, 6 of 11 remaining females showed clinical signs such as ruffled fur, weakened condition, decreased activity, prostrating, chromodacryorrhea of eyes, diarrhea, ptosis and/or hunched posture.
- During lactation period at 250 mg/kg bw/day, 4 of 5 remaining females showed decreased activity, hunched posture, ruffled fur, weakened condition, and/or chromodacryorrhea of eyes and at 500 mg/kg bw/day, 3 of 5 remaining females showed ruffled fur.

In summary, major clinical signs such as ruffled fur, decreased activity, hunched posture, weakened condition, diarrhea, ptosis, reddish nasal secretion, chromodacryorrhea and/or prostration occurred in females at 250 and 500 mg/kg bw/day with sudden onset at the end of gestation period when females went into labour during giving birth. At the same time increased numbers of mortalities occurred. These findings were considered to be test item-related.
Mortality:
mortality observed, treatment-related
Description (incidence):
At a high dose level of 1000 mg/kg bw/day, three premature mortalities in females occurred after the first application. Two females were found dead on day 2 of the pre-pairing period and one female was killed for ethical reasons on day 2. Due to these deaths, the dose level of 1000 mg/kg bw/day was considered to be too high and therefore reduced to 500 mg/kg bw/day from day 2 of dosing onwards.

During the gestation period at the time of giving birth (approx. days 20 to 21) or at day 1 of lactation period, 7 of 12 females spontaneously died or were killed in extremis at 250 mg/kg bw/day and 6 of 11 remaining females at 500 mg/kg bw/day.

In summary, 7 of 12 females at the mid dose of 250 mg/kg bw/day and 9 of 14 females at the high dose of 500 mg/kg bw/day died prematurely. These premature mortalities in females were considered to be test item-related. Additionally, a single male at 250 mg/kg bw/day was found dead on day 5 of pre-pairing period which was considered to be due to vagus reflex following gavage and not test item-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
Pre-Pairing and Pairing Periods: At 500 mg/kg bw/day, the mean body weight and mean body weight gains were markedly reduced throughout the pre-pairing. Body weights remained slightly but not statistically significantly lower during the pairing period. Body weight gains were similar or slightly higher after pairing period.

Females:
Pre-Pairing, Pairing, Gestation and Lactation Periods: At 250 and 500 mg/kg bw/day, the mean absolute body weights and often additionally the mean body weight gains were reduced throughout the study period. Increased body weight gains at 250 and 500 mg/kg bw/day during days 3 to 16 of gestation period did not compensate for an overall reduction in absolute body weights at the end of the respective study periods.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males:
Pre-Pairing Period: At 500 mg/kg bw/day, the mean food consumption was markedly reduced during days 1 to 8 of pre-pairing and recovered during days 8 to 13.

Females:
At 250 and 500 mg/kg bw/day, the mean food consumption was sporadically reduced during prepairing and at the end of gestation period and markedly reduced during days 1 to 4 of lactation period.
An increase of food consumption in group 3 and 4 females during days 7 to 17 (p<0.01) did not fully compensate the general reduction noted over all study periods.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males:
The following statistically significant findings were noted in males at the end of pre-pairing period:
- Decreased hemoglobin (p<0.05), decreased hematocrit (p<0.01), increased hemoglobin distribution width (p<0.05), shortened activated partial thromboplastin time (p<0.05) in males at 500 mg/kg bw/day. Values showed statistical significance of mainly 5%, only and stayed within ranges of historical control data. Nevertheless, findings were considered to be test item-related since they were only noted in the high dose group.

- Decreased white blood cell count (p<0.01), decreased absolute neutrophils (p<0.01) and decreased absolute lymphocytes (p<0.01) were noted in group 2 and 4 males at 50 and 500 mg/kg bw/day. Since findings were not distributed dose-dependently and values stayed well within ranges of historical control data, these findings were considered to stay within ranges of normal biological variation.

Females:
The following statistically significant findings were noted in females at the end of pre-pairing period:
- Increased red blood cell count (p<0.05) in high dose females at 500 mg/kg bw/day. In absence of any other alterations in the red blood cell parameters and values well within ranges of historical control data, this single finding was considered to be not test item-related.

Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males and Females:
The following statistically significant findings were noted in males and/or females at the end of pre-pairing period:
- Increased creatinine in males at 250 mg/kg bw/day (p<0.05) and 500 mg/kg bw/day (no statistical significance)
- Decreased total bilirubin in both sexes at 250 mg/kg bw/day (no statistical significance in males, p<0.01 in females) and 500 mg/kg bw/day (both p<0.01). Even though a decrease in bilirubin was considered to be not clinically relevant, the distribution of this finding was considered to indicate a test item-related effect.
- Decreased protein and albumin in females at 250 and 500 mg/kg bw/day (with p<0.01 and p<0.05)
- Increased phosphorus in males at 500 mg/kg bw/day (p<0.05) with mean values above ranges of historical control data and increased chloride in females at 500 mg/kg bw/day (p<0.05)
- Increased mean alkaline phosphatase in females at 250 and 500 mg/kg bw/day (p<0.05 and no statistical significance, respectively)

These findings were considered to be test item-related and to correlate to histopathologic findings in the liver (periportal fatty change, hypertrophy, necrosis) and in the kidneys (hyaline droplets, tubular basophilia and vacuolation, casts) most likely indicating changes in liver and kidney metabolism.
The remainder of findings were not distributed dose-dependently and therefore considered to within ranges of normal biological variation.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional Observational Battery:
In males, none of the parameters under investigation during the functional observational battery shortly before scheduled necropsy gave an indication of a test item-related effect.

In females (day 3 post partum), observation in the home cage of females at 250 and 500 mg/kg bw/day revealed the clinical signs of ruffled fur in single animals.
Grip Strength:
Mean values of grip strength (fore- and hind paws) gave no indication of test item-related effects in both sexes.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant findings were noted:
- Increased absolute liver weights and liver to body weight ratios and/or liver to brain weight ratios in males and females at 250 and 500 mg/kg bw/day (mainly p<0.01)
- Increased absolute kidney weights, kidney to body weight ratios and/or kidney to brain weight ratios in males at 500 mg/kg bw/day (p<0.01) and in females at 250 and 500 mg/kg bw/day (p<0.01 and p<0.05)
- Increased absolute adrenal weights in females at 250 and 500 mg/kg bw/day (p<0.01 and p<0.05) and increased adrenal to body weight ratios at 250 mg/kg bw/day (p<0.01)

Increased absolute and relative liver weights in both sexes at 250 and 500 mg/kg bw/day and increased absolute and/or relative kidney weights in both sexes at 500 and additionally in females at 250 mg/kg bw/day were considered to be test item-related. These changes in organ weights correlated to histological findings. Increased absolute and relative adrenal weights in females at 250 and 500 mg/kg bw/day were considered to be stress-related. The remainder of findings in females, such as increased brain or heart weights was considered to be of not toxicological relevance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Males
The following test item-related findings were noted at 250 and 500 mg/kg bw/day:
- Liver enlargement (2/12 males at 250 mg and 5/12 males at 500 mg/kg bw/day)
- Pelvic dilation of the kidneys (1/12 males at 250 and 500 mg/kg bw/day)
The remainder of findings was within ranges of normal background findings.

Females
The following main findings were noted at 500 mg/kg bw/day:
- Findings predominantly noted in spontaneous decedents or animals killed in extremis compared to planned necropsies
- In premature decedents uterus horns often containing foetuses; Discolored tan liver often with accentuated lobular pattern; Discolored greenish, tan or light brown kidneys; Enlarged adrenal glands; Reduced spleen and thymus size; Discolored reddish lungs; The remainder of findings within ranges of normal background findings.

Females at planned necropsy with no specific macroscopic findings
The following main findings were noted at 250 mg/kg bw/day:
- Findings predominantly noted in spontaneous decedents or animals killed in extremis compared to planned necropsies; In premature decedents uterus horns often containing foetuses; Discolored tan or clay-colored liver or grey white liver foci, sporadically liver with accentuated lobular pattern or liver enlargement ; Discolored greenish or light brown kidneys; Enlarged adrenal glands; Reduced spleen and thymus size.
- The remainder of findings within ranges of normal background findings
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver: In females, minimal to slight single cell necrosis/apoptosis in the liver of three females group 4 which died or were killed in moribund conditions after the first application (indicative for possible liver failure). This finding was considered to be adverse. In females, further findings consisted of higher degrees of periportal fatty change and hepatocellular hypertrophy in females of groups 2, 3 and 4. In males, increased incidence of minimal centrilobular hepatocellular hypertrophy in group 4.

Kidneys: hyaline droplet nephropathy characterized by increased severity of hyaline droplets, increased incidence and severity of tubular basophilia, hyaline and granular casts, mononuclear infiltrates and tubular dilation in males groups 2, 3 and 4. These findings were considered to be characteristic for hyaline droplet nephropathy. The nephropathy is deemed to be related to treatment and is considered to represent an adverse change for the rat only. In females, higher degrees of tubular vacuolation in the renal cortex in animals groups 3 and 4 (likely osmonephrotic effect leading to renal failure).

Adrenal glands: pronounced vacuolation of the Zona fasciculata along with diffuse cortical cells hypertrophy regarded as pre-stages of the up to massive hemorrhagic necrosis (leading to adrenal failure) recorded in females groups 3 and 4. The hemorrhagic necrosis was considered to be of adverse character.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: For general toxicity in females based on the gross and microscopic findings in the adrenal glands
Critical effects observed:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (actual dose received)
System:
endocrine system
Organ:
adrenal glands
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Conclusions:
A NOAEL (No Observed Adverse Effect Level) of 50 mg/kg body weight/day based on adrenal gland effects in females was established.
Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of test substance on the 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 behavior, conception,

development of the conceptus and parturition.

This study provides information to assess the need to conduct further investigations and provides guidance in the design of subsequent studies. The test susbtance was administered to male rats for at least 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached

day 4 post partum.

Test item-related findings in males at 250 and 500 mg/kg bw/day were liver enlargement, pelvic dilation of the kidneys and reddish foci of the thymus.

In females at 250 and 500 mg/kg bw/day, test item-related macroscopic findings were predominantly noted in spontaneous decedent females or females killed in extremis. Main findings were

discolored tan livers often with accentuated lobular pattern, discolored greenish, tan or light brown kidneys, enlarged adrenal glands and/or reduced spleen and thymus size. In premature

decedents uterus horns often contained fetuses. The remainder of findings was within ranges of normal background findings.

Primary target organs were liver, kidneys and adrenal glands. In the liver, minimal to slight single cell necrosis/apoptosis was noted in three females group 4 which died or were killed in moribund conditions after the first application (indicative for possible liver failure). Additionally, higher degrees of periportal fatty change and hepatocellular hypertrophy were noted in females of groups 2, 3 and 4. In males group 4, increased incidence of minimal centrilobular hepatocellular hypertrophy occurred. Due to a low severity grade and in the absence of any further alteration of toxicologically relevant parameters, the finding of fatty change in the liver of females in group 2 was considered to be not adverse.

In the kidneys, increased severity of hyaline droplet nephropathy was noted, characterized by increased severity of hyaline droplets, increased incidence and severity of tubular basophilia,

hyaline and granular casts, mononuclear infiltrates and tubular dilation in males groups 2, 3 and 4. In females groups 3 and 4, higher degrees of tubular vacuolation in the renal cortex were noted.

Hyaline droplet nephrophathy recorded in males was considered to be related to accumulation of alpha2-micoglobulin and therefore an adverse change for the male rat only with limited

toxicological relevance for other species including man.

In the adrenal glands, pronounced vacuolation of the Zona fasciculata along with diffuse cortical cells hypertrophy regarded as pre-stages of the up to massive hemorrhagic necrosis (leading to

adrenal failure) were recorded in females groups 3 and 4.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Key study was of good quality, hence was assigned 1 (reliable without restriction) and was carried out uinder GLP.
System:
endocrine system
Organ:
adrenal glands

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

Justification for classification or non-classification

There were weak, but dose-depended adverse effects on the adrenals of female rats after repeat oral dosing. These effects meet the criteria for a STOT Rep. Exp. 2 (Specific Target Organ Toxicant by Repeat Exposure) classification by the oral route with an H373 Hazard Statement “Causes damage to organs through prolonged or repeated oral exposure.”  No STOT RE classification for dermal and inhalation route. For futher discussion, please refer to the attached document (Justification for Classification (STOT RE: oral)) in IUCLID section 13 (STOT RE justification).