2,4,6-tri-tert-butylphenol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 May 2015 to 02 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3650

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

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

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

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

Test guideline 5

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3050

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,4,6-tri-tert-butylphenol

EC Number:

211-989-5

EC Name:

2,4,6-tri-tert-butylphenol

Cas Number:

732-26-3

Molecular formula:

C18H30O

IUPAC Name:

2,4,6-tri-tert-butylphenol

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Appearance: Slightly yellow powder with lumps
- Storage conditions of test material: Room temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:WI(Han), outbred, SPF-Quality
- Age at study initiation: Approximately 11-12 weeks
- Weight at study initiation: Males (mean): 316 g; Females (mean): 217 g
- Fasting period before study: No
- Housing: Pre-mating, animals were housed in groups of 5 animals/sex/cage in plastic cages (height 18 cm); during mating, females were caged together with males on a one-to-one-basis in plastic cages (height 18 cm). Post-mating, males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in plastic cages (height 18 cm). Sterilised sawdust as bedding material and paper as cage-enrichment/nesting material were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet: Free access to pelleted rodent diet
- Water: Free access to tap-water
- Acclimation period: At least 5 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24 °C
- Humidity: 40 to 70 % (relative)
- Air changes: at least 10 room air changes/hour
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES
- From: Experimental starting date: 30 May 2015 (allocation dose range finding study). Dosing for the main study began on the 08 July 2015.
- To: 06 August 2015 (males); 18, 19, 20, 21, 24 and 25 August 2015, and 02 September 2015 (females and/or pups)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Formulations were heated up to a maximum temperature between 56.7 to 61.8 °C, for a maximum of 36 minutes to obtain visual homogeneity.
Formulations were released for dosing when they obtained a temperature below 40 °C. No adjustment was made for specific gravity/density of the test material and formulation. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test material.
The solutions were stored at room temperature.

VEHICLE
- Specific gravity: 0.92
- Amount of vehicle (if gavage): Dose volume was 5 mL/kg. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

CHEMICAL ANALYSIS OF DOSE PREPARATIONS
Analyses were conducted on a single occasion during the treatment phase (day 2 of dosing), according to a validated method. Samples of formulations were analysed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in the vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90 to 110 % of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10 %. Formulations were considered stable if the relative difference before and after storage was maximally 10 %.

ANALYTICAL CONDITIONS
Quantitative analysis was based on the analytical method validated for the test material.
- Instrument: Acquity UPLC system (Waters, Milford, MA, USA)
- Detector: Acquity UPLC TUV detector (Waters)
- Column: Acquity UPLC BEH Shield RP-18, 100 mm x 2.1 mm i.d.,dp = 1.7 μm (Waters)
- Column temperature: 40 ± 1°C
- Injection volume: 10 μL
- Mobile phase: 80/20 (v/v) acetonitrile/water
- Flow: 0.6 mL/min
- UV detection: 220 nm

SAMPLES
Accuracy, homogeneity and stability were determined for formulations prepared for use during treatment.
Duplicate accurately weighed samples of approximately 500 mg were taken from the formulations using a pipette and were transferred into volumetric flasks of 10 mL. For determination of accuracy, samples were taken at middle position (50 % height) or at top, middle and bottom position (90, 50 and 10 % height). The samples taken at 90, 50 and 10 % height were also used for the determination of the homogeneity of the formulations. For determination of stability, additional samples were taken at 50 % height and stored at room temperature under normal laboratory light for 6 hours.
The volumetric flasks were filled up to the mark with acetonitrile. The samples were extracted by shaking for 30 seconds. The solutions were further diluted to obtain an end solution of 50/50 (v/v) acetonitrile/water and concentrations within the calibration range.

PREPARATION OF SOLUTIONS
- Stock and spiking solutions
Stock and spiking solutions of the test material were prepared in acetonitrile at concentrations of 1000 to 4000 mg/L.

- Calibration solutions
Calibration solutions in the concentration range of 0.1 to 20 mg/L were prepared from two stock solutions. The end solution of the calibration solutions was 50/50 (v/v) acetonitrile/water.

-Procedural recovery samples
Approximately 500 mg blank vehicle was spiked with the test material at a target concentration of 0.4 or 10 mg/g. The accuracy samples were treated similarly as the test samples.

SAMPLE INJECTIONS
Calibration solutions were injected in duplicate. Test samples and procedural recovery samples were analysed by single injection.

FORMULAE
Response (R): Peak area test substance [units]

Calibration curve: R = a CN + b
where:
CN = nominal concentration [mg/L]
a = slope [units x L/mg]
b = intercept [units]

Analysed concentration (CA): Ca = [(R – b) / a] x [(V x d) / w] [mg/g]
where:
w = weight sample [mg]
V = volume volumetric flask [mL]
d = dilution factor

Recovery: (CA / CN) x 100 [%]
where:
CN = nominal concentration [mg/g]

Accuracy: (CA / CT) x 100 [%]
where:
CT = target concentration [mg/g]

Relative difference (relative diff.): [(Ct – C0) / C0] x 100 [%]
where:
Ct = mean concentration of stored samples [mg/g]
C0 = mean concentration of non-stored samples [mg/g]

RESULTS
CALIBRATION CURVES
A calibration curve was constructed using six concentrations. For each concentration, two responses were used. Linear regression analysis was performed using the least squares method with a 1/concentration² weighting factor. The coefficient of correlation (r) was > 0.99.

SAMPLES
- Procedural recovery samples
The mean recoveries of the procedural recovery samples fell within the criterion of 90 – 110 %. It demonstrated that the analytical method was adequate for the determination of the test material in the test samples.

- Test samples
Accuracy of preparation
In the control group formulations, no test material was detected. The concentrations analysed in the formulations of the 10 and 30 mg/kg dose groups were in agreement with target concentrations (i.e. mean accuracies between 90 and 110 %).
For the formulation of the 3 mg/kg dose group prepared for use, the mean accuracy was slightly below the target concentration (i.e. 89 % of target). Since the deviation was small, it was considered to have no effect on the integrity of the study and results were accepted.
Homogeneity
The formulations of the 3 and 30 mg/kg dose groups were homogeneous (i.e. coefficient of variation ≤ 10 %).
Stability
Analysis of the 3 and 30 mg/kg dose group formulations after storage yielded a relative difference of ≤ 10 %. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 6 hours.

Duration of treatment / exposure:

- Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
- Females were exposed for 41 to 56 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).

Frequency of treatment:

Once daily, 7 days per week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 3, 10 and 30 mg/kg bw/day (dose Groups 1, 2, 3 and 4 respectively).
Basis:
actual ingested

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for dose levels: Based on the results of a 10-day dose range finding study in which 3 females per dose were treated with the test material at 50, 100 and 250 mg/kg body weight, the dose levels were selected to be 3, 10 and 30 mg/kg.
- Rationale for animal assignment: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20 % of the sex mean.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were examined for mortality/viability at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals, at least immediately after dosing. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) were scored.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION: Yes
- Time schedule: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected animals. Blood samples were drawn from the retro-orbital sinus and collected into tubes prepared with K3-EDTA for haematological parameters (0.5 mL) and with citrate for clotting tests (0.45 mL).
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available.
- How many animals: 5 animals per sex per dose group were evaluated
- The following parameters were examined: White blood cells (WBC; differential leucocyte count: neutrophils, lymphocytes, monocytes, eosinophils and basophils), red blood cells, reticulocytes, red blood cell distribution width (RDW), haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) and platelets. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were also determined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected animals. Blood samples were drawn from the retro-orbital sinus and collected into tubes treated with Li-heparin for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids.
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available.
- How many animals: 5 animals per sex per dose group were evaluated
- The following parameters were examined: Alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium and inorganic phosphate (Inorg. Phos). All parameters were determined in plasma, except for bile acids which were determined in serum.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (all before blood sampling). These tests were performed after observation for clinical signs (including arena observation, if applicable).
- Dose groups that were examined: 5 animals per sex per dose were examined
- Battery of functions tested: Hearing ability, pupillary reflex and static righting reflex. Fore- and hind-limb grip strength were recorded as the mean of three measurements (Series M4-10, Mark-10 Corporation). Locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerised monitoring system, Kinder Scientific LLC); total movements and ambulation were reported (ambulation represents movements characterised by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulation but also smaller or finer movements like grooming, weaving or movements of the head).

Sacrifice and pathology:

SACRIFICE AND NECROPSY
All males and the selected 5 females/group were deprived of food overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food.
All animals surviving to the end of the observation period were deeply anaesthetised using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

Necropsy was conducted on the following days:
- Females which delivered: Lactation days 5 or 7
- Female which failed to deliver (no. 42): Post-coitum Day 25 (female with evidence of mating)
- Males: Following completion of the mating period (a minimum of 28 days of dose administration)

GROSS PATHOLOGY
The numbers of former implantation sites and corpora lutea were recorded for all paired females. Samples of the following tissues and organs were collected and fixed in 10 % buffered formalin (neutral phosphate buffered 4 % formaldehyde solution) for 5 selected animals/sex/group: Adrenal glands, (aorta), brain [cerebellum, midbrain, cortex], caecum, cervix, clitoral gland, colon, coagulation gland, duodenum, epididymides**, eyes (with optic nerve (if detectable) and Harderian gland)**, female mammary gland area, femur including joint, heart, ileum, jejunum, kidneys, (lacrimal gland, exorbital), (larynx), liver, lung infused with formalin, lymph nodes [mandibular, mesenteric], (nasopharynx), (oesophagus), ovaries, (pancreas), Peyer's patches [jejunum, ileum] if detectable, pituitary gland, preputial gland, prostate gland, rectum, (salivary glands [mandibular, sublingual]), sciatic nerve, seminal vesicles, skeletal muscle, (skin), spinal cord [cervical, midthoracic, lumbar], spleen, sternum with bone marrow, stomach, testes**, thymus, thyroid including parathyroid if detectable, (tongue), trachea, urinary bladder, uterus, vagina and all gross lesions.

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

Samples of the following tissues and organs were taken from all remaining animals, including female no. 42 which failed to deliver*: Cervix, clitoral gland, coagulation gland, epididymides**, female mammary gland area, ovaries, preputial gland, prostate gland, seminal vesicles, testes**, uterus, vagina and all gross lesions.

*In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites (using ammonium sulphide solution 20 % and Milli-Ro water).

**Fixed in modified Davidson's solution (using Formaldehyde 37 to 40 %, ethanol, acetic acid (glacial) and Milli-Ro water) and transferred to formalin after fixation for at least 24 hours.

ORGAN WEIGHTS
Terminal body weights were recorded from all males and the selected 5 females/sex/group. The following organ weights were recorded from the 5 selected animals per sex per group on the scheduled day of necropsy: Adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus (including cervix), prostate†, seminal vesicles including coagulating glands† and thyroid including parathyroid.

†Weighed when fixed for at least 24 hours.
The organ weights of the epididymides and testes were taken for all remaining males.

HISTOTECHNOLOGY
All organ and tissue samples, as defined below, were processed, embedded and cut at a thickness of 2 to 4 micrometres. These slides were stained with haematoxylin and eosin. The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin.

HISTOPATHOLOGY
The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
- The additional slides of the testes of the selected 5 males of Groups 1 and 4 and male no. 2 suspected to be infertile to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- Liver of all selected males and females of Groups 2 and 3, spleen of all selected females of Groups 2 and 3 and cecum of all selected males of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4.
- The reproductive organs of control female no. 42 that failed to deliver healthy pups. Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina (male no. 2 was already selected for assessment of reproductive organs).

Other examinations:

Reproductive and developmental toxicity was also investigated within this study. General reproduction data were obtained along with the following indices: Mating index, fertility index, conception index, gestation index, duration of gestation, percentage live males at first litter check, percentage live females at first litter check, percentage of postnatal loss and viability index.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see below

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see below

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

see below

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see below

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see below

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

see below

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see below

Histopathological findings: neoplastic:

no effects observed

Details on results:

MORTALITY
No mortality occurred during the study period.

CLINICAL SIGNS
No clinical signs were noted during daily detailed observations and weekly arena observations that were considered toxicologically relevant.
Salivation seen after dosing among males at 3, 10 and 30 mg/kg and occasionally for a single female at 3 mg/kg was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to taste of the test material.
One female at 30 mg/kg incidentally showed piloerection, which was considered unrelated to treatment, since this was not shown by other animals and was only noted on two days during treatment. The occurrence of alopecia was within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, this was considered a sign of no toxicological relevance.

BODY WEIGHT
At 10 and 30 mg/kg, some females showed weight loss during lactation which was notably different to the weight gain observed for control females. At 10 mg/kg, notable weight loss was recorded of 4 or 9 % compared to Day 1 lactation for three females (nos. 63, 64 and 70). At 30 mg/kg, notable weight loss was recorded for three females (nos. 75, 77 and 80) of 5, 7 or 9 %, respectively, compared to Day 1 lactation.
Mean body weight gain of females at 30 mg/kg appeared slightly lower than controls over the post coitum period, which achieved a level of statistical significance on Days 4 and 7. Mean bodyweight of these females on Day 0 of post coitum was statistically significantly higher than controls, as well as on Day 1 of the mating period. Also, a statistically significant higher body weight gain was recorded for males at 10 and 30 mg/kg during most of the treatment period. None of these changes and other slight statistically significant changes in body weight (gain) were considered toxicologically meaningful, since these remained well within the range considered normal for rats of this age and strain, and showed no clear and consistent trend over time and dose.

FOOD CONSUMPTION
Females at 10 and 30 mg/kg showed an apparent lower food consumption before and after correction for body weight over Days 1 to 4 of lactation (not statistically significant; and with a relatively high standard deviation at 30 mg/kg).
Food consumption before or after correction for body weight was considered unaffected by treatment for females at 3 mg/kg, and for males at all dose levels.

HAEMATOLOGY
The following (statistically significant) changes in haematology parameters distinguished treated females from control females:
- Lower relative neutrophil counts at 30 mg/kg.
- Higher relative lymphocyte counts at 30 mg/kg.
- Higher red blood cell count at 30 mg/kg.
- Lower reticulocyte count at 10 and 30 mg/kg (not statistically significant at 10 mg/kg).
- Lower mean corpuscular volume (MCV) at 10 and 30 mg/kg.
- Lower mean corpuscular haemoglobin (MCH) at 10 and 30 mg/kg.
The changes seen at 10 mg/kg were not considered to be adverse in nature.
The lower prothrombin time (PT) seen at 30 mg/kg was not considered toxicologically relevant since the opposite effect (i.e. an increase) would be expected in case of target organ toxicity.
Haematological parameters of males were considered unaffected by treatment.

CLINICAL BIOCHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Higher total protein in females at 30 mg/kg.
- Higher albumin in females at 30 mg/kg.
- Lower total bilirubin in males and females at 10 and 30 mg/kg.
- Lower urea in females at 30 mg/kg (not statistically significant).
- Higher glucose in females at 30 mg/kg.
- Higher cholesterol in females at 10 and 30 mg/kg.
- Higher potassium in males and females at 30 mg/kg.
- Higher calcium in females at 30 mg/kg.
For one control female (no. 49) and one female at 3 mg/kg (no. 54) a high bile acid level was recorded. No cause for these high values could be established. Given their incidental occurrence and similar presence in the control group, these were considered unrelated to treatment.
Other statistically significant changes in clinical biochemistry parameters were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

FUNCTIONAL OBSERVATIONS
Hearing ability, pupillary reflex and static righting reflex were normal in all selected animals. Grip strength and motor activity was similar between control and high dose animals.
All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period

MACROSCOPIC EXAMINATION
Enlargement of the liver was noted for 3 males (with accentuated lobular pattern for one of these males) and 1 female at 30 mg/kg. The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

ORGAN WEIGHTS
Test material-related higher liver weights (absolute and relative to body weights) were noted in the 30 mg/kg males and 10 and 30 mg/kg females. Relative liver weights were increased 39 and 63 % in males and females at 30 mg/kg, respectively, and 21 % in females at 10 mg/kg.
Any other statistically significant changes in organ weight at the end of the treatment period were not considered to be treatment-related as they occurred in the absence of a dose-related trend and had no histological correlate.

MICROSCOPIC EXAMINATIONS
The following test material-related microscopic findings were noted:
- Liver: hepatocellular hypertrophy was present in males and females treated at 10 and 30 mg/kg up to moderate degree. Hepatocellular necrosis was present in a single male and a single female treated at 30 mg/kg at minimal degree. The higher liver weights in females at 10 and 30 mg/kg along with the combined occurrence of hepatocellular hypertrophy with necrosis at 30 mg/kg were considered to be adverse in nature.
- Cecum: mucosal hypertrophy was present in males treated at 10 and 30 mg/kg up to slight degree. As this occurred in absence of any other indicators of toxicity in this organ, this histopathological lesion was therefore not considered adverse in nature.
- Spleen: decreased haematopoiesis was present in females treated at 10 and 30 mg/kg. This occurred along with lower reticulocyte counts. However, red blood cell counts showed an increase rather than a decrease at this dose level. Also, since there were no other indicators of toxicity in the spleen, these changes were not considered to represent an adverse effect on red blood cell turn over.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test material-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
There was one control treated couple (male 2 and female 42) without offspring. No abnormalities were seen in the reproductive organs, which could account for their lack of offspring.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test material and spermatogenic staging profiles were normal for all males examined.

OTHER
No toxicologically relevant changes in reproductive parameters were noted.
With regard to developmental effects, at 30 mg/kg, the mean number of living pups at first litter check appeared slightly lower than controls. Also, an increased postnatal loss and lower viability index was noted at 10 and 30 mg/kg. At 10 mg/kg, a total of 3 dams showed postnatal loss. At 30 mg/kg, a total of 5 dams showed postnatal loss.
Mean pup body weights for both sexes combined on Day 4 of lactation were approximately 16 and 20 % lower than the control mean at 10 and 30 mg/kg, respectively. Although there were no other developmental changes noted for these pups (macroscopy and clinical signs), the magnitude of changes in pup body weight was considered to represent an adverse effect on pup development. No apparent relationship could be found between maternal food intake and mean pup body weight gain on an individual animal basis.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on higher liver weights at 10 and 30 mg/kg, with hepatocellular hypertrophy and necrosis at 30 mg/kg.

2

Dose descriptor:

NOAEL

Remarks:

(reproduction)

Effect level:

>= 30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The reproduction NOAEL was cited as being at least 30 mg/kg.

3

Dose descriptor:

NOAEL

Remarks:

(developmental)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on lower pup body weight and increased postnatal loss at 10 and 30 mg/kg, and lower mean number of living pups at first litter check at 30 mg/kg.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Mean Body Weights (g)

Time of Measurement		Dose Group (mg/kg)
		0	3	10	30
	MALES
	Pre-mating Period
Day 1, Week 1	Mean N	317 10	315 10	317 10	314 10
Day 8, Week 2	Mean N	340 10	341 10	347 10	345 10
	Mating Period
Day 1, Week 1	Mean N	359 10	357 10	363 10	362 10
Day 8, Week 2	Mean N	359 10	368 10	375 10	369 10
Day 15, Week 3	Mean N	370 10	382 10	391* 10	384 10
	FEMALES
	Pre-mating Period
Day 1, Week 1	Mean N	215 10	216 10	215 10	220 10
Day 8, Week 2	Mean N	226 10	230 10	228 10	232 10
	Mating Period
Day 1, Week 1	Mean N	227 10	233 10	235* 10	235* 10
Day 8, Week 2	Mean N	254 1	262 1	263 1	-
Day 15, Week 3	Mean N	251 1	288 1	-	-
	Post Coitum
Day 0	Mean N	231 9	234 9	238 10	240* 10
Day 4	Mean N	245 9	248 9	253* 10	250 10
Day 7	Mean N	254 9	259 9	261 10	259 10
Day 11	Mean N	268 9	275 10	275 10	272 10
Day 14	Mean N	278 9	288 10	287 10	284 10
Day 17	Mean N	304 9	316 10	316 10	309 10
Day 20	Mean N	345 9	358 10	353 10	347 10
	Lactation
Day 1	Mean N	267 9	275 10	276 10	277 10
Day 4	Mean N	270 9	280 10	271 10	273 10

*/** Dunnett-test based on pooled variance significant at 5 % (*) or 1 % (**) level

Table 2: Summary of Macroscopic Findings

		Group 1 Control	Group 2 3 mg/kg	Group 3 10 mg/kg	Group 4 30 mg/kg
Males	END OF TREATMENT
	Animals examined	10	10	10	10
	Animals without findings	9	9	8	7
	Animals affected	1	1	2	3
	Liver Accentuated lobular pattern Enlarged	0 0	0 0	0 0	1 3
	Preputial glands Reduced in size	1	0	0	0
	Thyroid gland Reduced in size	0	0	1	0
	Adrenal glands Enlarged	0	0	1	0
	Spleen Enlarged	0	0	1	0
	Thymus Focus/foci	0	1	0	0
Females	END OF TREATMENT
	Animals examined	10	10	10	10
	Animals without findings	7	9	9	8
	Animals affected	3	1	1	2
	Stomach Irregular surface	1	1	1	1
	Duodenum Thickened	0	0	0	1
	Jejunum Thickened	0	0	0	1
	Liver Enlarged	0	0	0	1
	Uterus Contains fluid	1	0	0	0
	Clitoral glands Enlarged Discolouration	1 1	0 0	0 0	0 0
	Mesenteric lymph n Discolouration	0	0	0	1

Table 3: Selected Organ/Body Weight Ratio and Organ Weight

END OF TREATMENT	Dose Group (mg/kg)
	0	3	10	30
	Males
Body Weight (g) N	351 10	363 10	371* 10	367 10
Liver weight (g) N	8.07 5	8.68 5	9.24 5	11.38** 5
Liver/bw ratio (%) N	2.25 5	2.40 5	2.52 5	3.12** 5
	Females
Body Weight (g) N	235 5	249 5	246 5	246 5
Liver weight (g) N	7.09 5	7.98 5	8.95** 5	12.08** 5
Liver/bw ratio (%) N	3.01 5	3.20 5	3.64** 5	4.91** 5

*/** Dunnett-test based on pooled variance significant at 5 % (*) or 1 % (**) level

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the NOAEL was determined to be 3 mg/kg (based on higher liver weights at 10 and 30 mg/kg, with hepatocellular hypertrophy and necrosis at 30 mg/kg).

Executive summary:

The repeated dose toxicity of the test material was investigated in a combined repeated dose toxicity study with the reproduction / developmental toxicity screening test conducted in accordance with the standardised guidelines OECD 422 and US EPA OPPTS 870.3650 under GLP conditions.

Based on the results of a 10-day dose range finding study, the dose levels for this study were selected to be 3, 10 and 30 mg/kg.

The test material, formulated in corn oil, was administered daily by oral gavage to SPF-bred Wistar Han rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41 to 56 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), clinical pathology (end of treatment), macroscopy at termination, organ weights and histopathology on a selection of tissues. Reproduction/developmental parameters were also evaluated.

Formulations were analysed once during the study to assess accuracy, homogeneity and stability. Analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

Histopathological examination showed hepatocellular hypertrophy up to moderate degree in both sexes at 10 and 30 mg/kg. This was supported at necropsy by enlargement and/or accentuated lobular pattern of the liver for some animals at 30 mg/kg. At 30 mg/kg, hepatocellular necrosis was present in a single male and a single female at minimal degree. Additionally, higher liver weights (absolute and/or relative to body weights) were recorded at 10 and 30 mg/kg; relative liver weights were increased 39 and 63 % in males and females at 30 mg/kg, respectively, and 21 % in females at 10 mg/kg. The higher liver weights in females at 10 and 30 mg/kg along with the combined occurrence of hepatocellular hypertrophy with necrosis at 30 mg/kg were considered to be adverse in nature.

Mucosal hypertrophy of the cecum in males treated at 10 and 30 mg/kg was noted up to a slight degree only and occurred in absence of any other indicators of toxicity in this organ. This histopathological lesion was therefore not considered adverse in nature.

Decreased splenic haematopoiesis was observed in females treated at 10 and 30 mg/kg, which occurred along with lower reticulocyte counts. However, red blood cell counts showed an increase rather than a decrease at this dose level. Also, since there were no other indicators of toxicity in the spleen, these changes were not considered to represent an adverse effect on red blood cell turn over.

At 30 mg/kg, other changes in blood of females that were considered to be related to treatment consisted of lower relative neutrophil counts, higher relative lymphocyte counts, lower mean corpuscular volume and mean corpuscular haemoglobin, higher total protein, albumin, calcium, cholesterol, potassium and glucose and lower urea and total bilirubin. Higher potassium and lower total bilirubin were also noted for males at 30 mg/kg.

At 10 mg/kg, changes in blood consisted of lower mean corpuscular volume and mean corpuscular haemoglobin in females, lower total bilirubin in males and females, and higher cholesterol in females. In the absence of any concurrent morphological lesions, and given the slight nature of these changes (i.e. within or just outside the range considered normal for rats of this age and strain), these were not considered adverse in nature.

Females at 10 and 30 mg/kg showed lower food consumption during the lactation period, as well as notable weight loss during lactation ranging from 4 to 9 % of Day 1 lactation values. The lower maternal food intake and body weight gain and mean pup body weight gain appeared unrelated on an individual animal basis. Also, these changes were not accompanied by supportive clinical signs or inadequate maternal care. As such, these changes in food intake and body weight gain during lactation were considered not adverse in nature.

No toxicologically relevant clinical signs or changes in functional observation parameters were noted.

No toxicologically relevant changes in reproductive parameters were noted.

With regard to developmental effects, at 30 mg/kg, the mean number of living pups at first litter check appeared slightly lower than controls. Also, an increased postnatal loss and lower viability index was noted at 10 and 30 mg/kg. At 10 mg/kg, a total of 3 dams showed postnatal loss. At 30 mg/kg, a total of 5 dams showed postnatal loss. The significance of these findings is not clear at this stage and are proposed to be further investigated

Mean pup body weights for both sexes combined on Day 4 of lactation were approximately 16 and 20 % lower than the control mean at 10 and 30 mg/kg, respectively. Although there were no other developmental changes noted for these pups (macroscopy and clinical signs), the magnitude of changes in pup body weight was considered to represent an adverse effect on pup development. No apparent relationship could be found between maternal food intake and mean pup body weight gain on an individual animal basis.

Under the conditions of the study, the repeated dose NOAEL was determined to be 3 mg/kg (based on higher liver weights at 10 and 30 mg/kg, with hepatocellular hypertrophy and necrosis at 30 mg/kg).