2-ethylhexyl 3,5,5-trimethylhexanoate

Repeated dose toxicity: oral

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

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:

Animals: Rat, RccHan TM : WIST(SPF)
Rationale: Recognized by international guidelines as a recommended test system.
Breeder: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
Number of Animals:
Group 1: 12 males and 12 females
Group 2: 12 males and 12 females
Group 3: 12 males and 12 females
Group 4: 12 males and 14 females
Group 10: 2 males and 0 females
Total Number of Animals: 50 males and 50 females
Age (at Delivery): 10 weeks
Body Weight Range (at Start of Treatment): Males: 339 to 385 g, Females: 196 to 223 g
Identification: Cage card and individual animal number (ear tattoo).
Pups: On day 1 post partum, pups were individually tattooed with Indian ink.
Randomization: Performed after at least three days of acclimatization, using a computed-generated random algorithm. Body weights (recorded on the day of allocation) were taken into consideration.
Acclimatization: At least 5 days under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
The group identification and animal numbers assigned to treatment are stated in the following table:
Allocation and Dose Levels [mg/kg bw/day]
Group 1 (Control, 0 mg/kg bw/day): Males 1 – 12 and Females 49 - 60
Group 2 (50 mg/kg bw/day): Males 13 - 24 and Females 61 - 72
Group 3 (250 mg/kg bw/day): Males 25 - 36 and Females 73 - 84
Group 4 (1000 mg/kg bw/day on day 1 and 500 mg/kg bw/day as of day 2 onwards): Males 37 - 48 and Females 85 – 96 & 200 - 201
Group 10 (Reserve Animals**): Males 100 - 101
** Reserve females were used for the replacement of two females in group 4 which died at the start of the treatment, reserve males were removed from the study. Any raw data collected during the acclimatization period on reserve animals was not reported but retained in the raw data.
Husbandry
Room Number, Itingen: E19
Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). Values outside of these ranges occasionally occurred, usually following room cleaning, which was considered not to have any influence on the study. These data were not reported but were retained in the raw data. There was 12-hour fluorescent light / 12-hour dark cycle with music during the light period.
Accommodation: 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: Pelleted standard Harlan Teklad 2914C (batch no. 20/12) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. Results of respective analyses for contaminants are included in study report.
Water: Community tap-water from Itingen was available ad libitum in water bottles. Results of bacteriological assay, chemical and contaminant analyses of respective samples are included in study report.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on 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

Details on mating procedure:

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.

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)
Samples of dose formulations were not discarded without the written authorization of the study director.
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 DRAGOXAT® 89 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 DRAGOXAT® 89 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:

Duration of Treatment Period: Males: Minimum 4 weeks and Females: Approximately 7 weeks

Frequency of treatment:

once daily by gavage

Details on study schedule:

Study Schedule
Study Sequence Females
Acclimatization: 5 days minimum
First Test Item Administration: Day 1 of pre-pairing, Day 2 of pre-pairing (nos. 200 and 201)
Pre-Pairing: 14 days / 13 days (nos. 200 and 201)
Blood Sampling: End of pre-pairing
Pairing: 14 days maximum
Gestation: Approximately 21 days
Treatment Ends: On day 3 post partum (day of birth = day 0 pp)
Necropsy: On day 4 post partum (females and pups)
Study Sequence Males
Acclimatization: 5 days minimum
First Test Item Administration: Day 1 of pre-pairing
Pre-Pairing: 14 days / 13 days (mates for nos. 200 and 201)
Blood Sampling: End of pre-pairing
Pairing: 14 days maximum
Treatment Ends: On day before sacrifice
Necropsy: After treatment of at least 28 days, when no longer needed for assessment of reproductive effects

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 was used as the top dose from day 2 onwards

No. of animals per sex per dose:

12 males and 12 females

Control animals:

yes, concurrent vehicle

Positive control:

not applicable

Parental animals: Observations and examinations:

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.

Sperm parameters (parental animals):

PAS-stained sections of the testes were used for a detailed qualitative examination (sperm staging), taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings were noted.

Litter observations:

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.

Postmortem examinations (parental animals):

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. A histopathology peer review was performed by Dr. K. Weber at Anapath GmbH.

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 (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):

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.
- Towards the end of gestation period (starting predominantly on days 20 or 21), 6 of 11 remaining females at 500 mg/kg bw/day, 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.
- During lactation period at 500 mg/kg bw/day, 3 of 5 remaining females showed ruffled fur.
- Additional other sporadic findings such as scabs or hair loss were considered to be background findings.
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):

A high dose level of 1000 mg/kg bw/day, three premature mortalities in females occurred after first application. Two females were found dead on day 2 of the pre-pairing period and one female (no. 92) 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 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.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg bw/day, the mean body weight and mean body weight gains of males 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 the pairing period.
In females, 250 and 500 mg/kg bw/day caused a reduction in the mean absolute body weights and often additionally the mean body weight gains throughout study periods. 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):

In males, the mean food consumption was markedly reduced at 500 mg/kg bw/day during days 1 to 8 of pre-pairing and recovered during days 8 to 13.
In 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):

In males, the following statistically significant findings were noted 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 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.
- The remainder of findings (decreased absolute basophils in group 3 and 4 with p<0.01 and decreased monocytes in group 2 with p<0.05) was considered to be within ranges of normal biological variation and values stayed well within ranges of historical control data.
In 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):

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)
- Decreased total bilirubin in females at 250 mg/kg bw/day and in both sexes at 500 mg/kg bw/day. 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
- Increased phosphorus in males at 500 mg/kg bw/day with mean values above ranges of historical control data and increased chloride in females at 500 mg/kg bw/day
- Increased mean alkaline phosphatase in females at 250
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 be within ranges of normal biological variation.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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.
During the functional observational battery 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.
Locomotor Activity: No statistically significant changes were noted in the total level of locomotor activity in both sexes. Slightly increased locomotor activity in males group 4 with single statistical significance of p<0.05 at the time measurement interval 10 to 20 minutes was considered to be within ranges of normal biological variance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Liver: In females, minimal to slight single cell necrosis/apoptosis was seen in the liver of three females exposed to 500 mg/kg bw/day 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 50, 250 and 5000 mg/kg bw/day. In males, increased incidence of minimal centrilobular hepatocellular hypertrophy was seen in the highest dose group of 5000 mg/kg bw/day.
- 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 50, 250 and 5000 mg/kg bw/day. These findings were considered to be characteristic for hyaline droplet nephropathy. The nephropathy is deemed to be related to treatment but is considered to represent an adverse change only in the rat only hence is not toxicologically relevant to humans. In females, higher degrees of tubular vacuolation in the renal cortex in animals administered 250 and 5000 mg/kg bw/day (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 in 250 and 5000 mg/kg bw/day dose groups. The hemorrhagic necrosis was considered to be of adverse character.
Lesions Secondary to Treatment
- Gastrointestinal tract: Erosions and/or ulcerations in the stomach, duodenum and cecum of females in groups 250 and 5000 mg/kg bw/day. In the stomach, these findings were sometimes along with focal inflammation and submucosal edema.
- Lymphatic organs: Lymphoid depletion in the spleen, increased severity of lymphoid depletion in the thymus as well as minimal increased tingible body macrophages in thymus, mesenteric and mandibular lymph nodes in females in 250 and 5000 mg/kg bw/day dose groups.

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis. All findings recorded were within the range of normal background alterations.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Mating Performance and Fertility
All paired females mated. In the control group, 11/12 and 1/12 females mated within the first and second pairing period respectively. In the 50 mg/kg bw/day dose group, 11/12 and 1/12 females mated within the first and second pairing period respectively. In the 250 mg/kg bw/day dose group, 12/12 females mated within the first pairing period and in the 500 mg/kg bw/day dose group, all 11 surviving females (of 14) mated within the first pairing period.
The median and mean precoital times were unaffected by treatment. Mean (and median) precoital times were 3.6 (3), 2.6 (2), 3.9 (4) and 3.0 (3) days in dose groups 0, 50, 250 and 500 mg/kg bw/day, respectively. Four females were not pregnant; one in the control group, two in the 50 mg/kg bw/day dose groups and one in the 250 mg/kg bw/day dose group. Consequently, fertility indices and conception rates were 91.7%, 83.3%, 91.7% and 100% in dose groups 0, 50, 250 and 500 mg/kg bw/day, respectively.
The gestation index was 100%, 100%, 36.4% and 27.3% in dose groups 0, 50, 250 and 500 mg/kg bw/day, respectively. In the 250 mg/kg bw/day dose group, the reduction of the gestation index was due to six pregnant females that died spontaneously or were killed in extremis at the time of giving birth; no living pups were found by these females, but uteri contained fetuses at necropsy. In one female, beginning parturition was observed at the end of the day 21 of the gestation period but no pups were found on the next day at first litter check. The female exhibited a body weight loss of ca. 20% on that day, therefore indicating that the animal most probably gave birth to pups but cannibalized them before the first litter check. In the 500 mg/kg bw/day dose group, six pregnant females died spontaneously or were terminated because of a bad condition at the end of gestation period. In five of these females uteri contained fetuses at necropsy. Two further females in this group had only dead pups at first litter check, which led to the reduction of the gestation index.
In summary, no dose-related changes were noted in the mating performance or fertility (percentage of mating, fertility index or conception rate), whereas the gestation index (the number of females with living pups in regard to pregnant females) was markedly reduced in the 250 and 500 mg/kg bw/day dose groups. This reduction of gestation indices was considered to be due to maternal toxicity.
Corpora Lutea Count
Mean number of corpora lutea per dam (determined at necropsy) was similar in the first three dose groups (14.4, 13.7, 14.0 in order of ascending dose levels) and slightly, but not statistically significantly, reduced in the top dose group (11.4). This reduction was caused by one (out of five) female that showed two corpora lutea only and was considered to be incidental, since the corpora lutea counts of the remaining four females which gave birth, as well as of the six females, which died without recorded births were in a normal range.
Duration of Gestation
The mean duration of gestation was unaffected by exposure to the test item. Mean duration of gestation was 21.9, 21.8, 21.2 and 21.6 days, in 0, 50, 250 and 500 mg/kg bw/day dose groups. .
Implantation Rate and Post-Implantation Loss
The mean number of implantation sites per dam was similar in the first three dose groups (mean of 13.3, 11.8, 12.0 in order of ascending dose levels) and slightly, not statistically significantly, reduced in the top dose group (11.0). This reduction was caused by one (out of five) female that had one implantation site only and was considered to be incidental, since the number of implantation sites of the remaining four females which gave birth, as well as of the six females, which died without recorded birth, were in a normal range.
In the 250 and 500 mg/kg bw/day dose groups, the total and mean post-implantation loss, as well as the mean incidence of post-implantation loss as a percentage of total implantations were markedly increased. This was considered to be a test item-related effect.

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 microscopic findings recorded in the adrenal glands

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

other: for reproduction/developmental toxicity, based on a higher post-implantation and post natal loss in the mid and high dose group.

Dose descriptor:

NOAEL

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Due to the findings of the male kidneys a histopathological NOAEL (No Observed Adverse Effect Level) for males could not be established.

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

see details on results

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

see details on results

Gross pathological findings:

no effects observed

Description (incidence and severity):

see details on results

Histopathological findings:

not examined

Litter Size at First Litter Check
In group 3 and 4, due to increased incidence of post implantation loss, the birth index (number of pups born alive/ number of implantations) resulted to be statistically significantly reduced (56.3% and 63.6% compared to 89.0% in the control group). Consequently, the mean litter size was lower (mean number of living pups per dam at first litter check was 6.8 and 7.0 in groups 3 and 4 compared to 11.8 in the control group). In group 3, female no. 82 had living pups, but was killed in extremis on day 1 of lactation period and therefore excluded from the statistics on summary breeding data per group.
Postnatal Loss Days 0 - 4 Post Partum
Mean postnatal loss was markedly increased in group 3 and 4, respectively. Correspondingly, in group 3 and 4, the viability index was statistically significantly decreased (51.9% and 62.9% compared to 97.7% in controls).
Observations (Litter Data - F1 Pups)
External Examination at First Litter Check and during Lactation
In group 3 and 4, examination of the pups revealed an increased incidence of no milk in the stomach and pups that were cold when touched. Additionally, in group 4, three pups from litter 96 showed hematoma and one pup from litter 88 an abscess at the abdomen which was considered to show increased cannibalism in dams. The findings were considered to be likely in consequence of poor clinical condition of the dams due to systemic toxicity and might be a secondary effect to the maternal toxicity.
Sex Ratios
Sex ratios at first litter check and on day 4 post partum were unaffected by exposure to the test item. In group 4, more living female than male pups were noted (63% female versus 37% male pups). Due to a low number of litters at this dose level, the statistical significance of this effect was not conclusive.
Body Weights to Day 4 Post Partum
Mean pup weights on day 1 and 4 post partum were statistically significantly reduced in group 3 and 4.
Macroscopic Findings
No findings were noted at macroscopic examination of F1 pups in all groups 1 to 4.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

viability

mortality

body weight and weight gain

Reproductive effects observed:

not specified

P Animals Breeding for F1 Litters

Group	1	2	3	4
(mg/kg/day)	0	50	250	500
Number of females paired	12	12	12	11
Number of females mated	12	12	12	11
Number of females pregnant (A)	11	10	11	11
Number of pregnant females, which died without giving birth (B)	0	0	6	6
Number of pregnant females, which had only dead pups at FLC (C)	0	0	0	2
Number of pregnant females, which had no pups at first litter check (D)	0	0	1	0
Number of females with living pups at first litter check	11	10	4	3
Numbers of females which lost their litters during lactation (E)	0	0	1	0
Number of females terminated during lactation (F)	0	0	1	0
Number of females which reared their pups until day 4 post partum	11	10	2	3

(A) Female nos. 57, 63, 64 and 73 were not pregnant

(B) Female nos. 74, 76, 77, 78, 79, 83, 85, 86, 89, 94, 95 and 201

(C) Female nos. 90 and 200

(D) Female no. 75

(E) Female no. 80

(F) Female no. 82

Conclusions:

Administrations at 250 and 500 mg/kg/day caused an increased mortality in females, adverse clinical signs in females, a reduction of food consumption and body weights in both sexes at 500 mg/kg and also in females at 250 mg/kg bw/day, changes in hematology parameters in males at 500 mg/kg and in biochemistry parameters in both sexes at 250 and 500 mg/kg bw/day.
At 250 and 500 mg/kg bw/day, the gestation index and birth index was markedly reduced and the post implantation and post natal loss was markedly increased. The pup body weight development was significantly reduced in groups 3 and 4.
At necropsy, an increase of the liver weights or ratios in both sexes at 250 and 500 mg/kg bw/day, of kidney weights or ratios in males at 500 and additionally in females at 250 mg/kg bw/day and of adrenal weights in females at 250 and 500 mg/kg bw/day was observed. These findings correlated with findings noted during the histopathology examination: single cell necrosis in the liver of decedent females in group 4, periportal fatty change or hypertrophy in the liver of females in groups 2, 3 and in both sexes in group 4, hyaline droplet nephropathy in males in groups 2, 3 and 4, tubular vacuolation in the kidneys in females in groups 3 and 4 and vacuolation, cortical cell hypertrophy and hemorrhagic necrosis in the adrenal glands in females in groups 3 and 4.

Due to a low severity grade and in the absence of any altered parameters of toxicological relevance in females of group 2, such as changes in biochemistry, clinical signs or mortality, the finding of fatty change in this dose group were considered to be not adverse. Hyaline droplet nephrophathy recorded in males is deemed to be related to treatment and is considered to represent an adverse change for the male rat only. Since the hyaline droplets in the male rat relate to accumulation of alpha2-micoglobulin, and little or none of this protein is present in man, a nephropathy in man that involves the same mechanism is unlikely to occur with the test item and has limited relevance for other species including man.

Because of hyaline droplet nephrophathy noted in males down to the low dose level, no NOEL (No Observed Effect Level) for general toxicity was established in males. NOAEL (No Observed Adverse Effect Level) for general toxicity in females was established at 50 mg/kg
body weight/day.
The NOEL (No Observed Effect Level) and NOAEL (No Observed Adverse Effect Level) for
reproduction/developmental toxicity was considered to be 50 mg/kg/day, based on a higher postimplantation
and post natal loss in the mid and high dose group.

Executive summary:

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item to rats over approximately 28 days. The test item was administered in corn oil as vehicle at dosages of 50, 250, and 500 (respectively 1000 on day 1) mg/kg body weight/day, and controls received the vehicle only. Males were administered the test item for at least 28 days and female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

Administrations at 250 and 500 mg/kg/day caused an increased mortality in females, adverse clinical signs in females, a reduction of food consumption and body weights in both sexes at 500 mg/kg and also in females at 250 mg/kg bw/day, changes in hematology parameters in males at 500 mg/kg and in biochemistry parameters in both sexes at 250 and 500 mg/kg bw/day.

At 250 and 500 mg/kg bw/day, the gestation index and birth index was markedly reduced and the post implantation and post natal loss was markedly increased. The pup body weight development was significantly reduced in groups 3 and 4.

At necropsy, an increase of the liver weights or ratios in both sexes at 250 and 500 mg/kg bw/day, of kidney weights or ratios in males at 500 and additionally in females at 250 mg/kg bw/day and of adrenal weights in females at 250 and 500 mg/kg bw/day was observed.

These findings correlated with findings noted during the histopathology examination: Single cell necrosis in the liver of decedent females in group 4, periportal fatty change or hypertrophy in the liver of females in groups 2, 3 and in both sexes in group 4, hyaline droplet nephropathy in males in groups 2, 3 and 4, tubular vacuolation in the kidneys in females in groups 3 and 4 and vacuolation, cortical cell hypertrophy and hemorrhagic necrosis in the adrenal glands in females in groups 3 and 4.

Due to a low severity grade and in the absence of any altered parameters of toxicological relevance in females of group 2, such as changes in biochemistry, clinical signs or mortality, the finding of fatty change in this dose group were considered to be not adverse. Hyaline droplet nephrophathy recorded in males is deemed to be related to treatment and is considered to represent an adverse change for the male rat only. Since the hyaline droplets in the male rat relate to accumulation of alpha 2-micoglobulin, and little or none of this protein is present in man, a nephropathy in man that involves the same mechanism is unlikely to occur with the test item and has limited relevance for other species including man.

Because of hyaline droplet nephrophathy noted in males down to the low dose level, no NOEL (No Observed Effect Level) for general toxicity was established in males. NOAEL (No Observed Adverse Effect Level) for general toxicity in females was established at 50 mg/kg body weight/day.

The NOEL (No Observed Effect Level) and NOAEL (No Observed Adverse Effect Level) for reproduction/developmental toxicity was considered to be 50 mg/kg/day, based on a higher post-implantation and post natal loss in the mid and high dose group.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl 3,5,5-trimethylhexanoate

EC Number:

275-073-7

EC Name:

2-ethylhexyl 3,5,5-trimethylhexanoate

Cas Number:

70969-70-9

Molecular formula:

C17H34O2

IUPAC Name:

2-ethylhexyl 3,5,5-trimethylhexanoate

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

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

Examinations

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.

Results and discussion

Results of examinations

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

Effect levels

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

Target system / organ toxicity

1

Critical effects observed:

not specified

2

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

Applicant's summary and conclusion

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.