4-ethoxy-2,3-difluoro-4'-propyl-1,1'-biphenyl

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:

2016-03-02 to 2016-04-24

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:

2016-03-02 to 2016-04-24

Reliability:

1 (reliable without restriction)

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)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The test guideline OECD 422 is designed for use with the rat and the Wistar WI (Han) rat was chosen since it is widely accepted by authorities as an appropriate experimental model, with documented susceptibility to a wide range of toxic substances.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10-12 weeks
- Weight at study initiation: males: 301 (268 – 344) females. 205 (186 – 228)
- Fasting period before study: no
- Housing: conventional conditions, with means to hide, pre-mating: 3 animals/sex/cage, mating: 1 male and 1 female per cage, after mating: 1 animal/cage and females were provided nesting material, not mated animals: 3 animals/sex/cage
- Diet: ad libitum, Ssniff V1534 (batch: 1324730), but for approx. 18 h before blood collection food was withheld
- Water: ad libitum, tap water, bottles freshly filled twice a week
- Acclimation period: at least 9 days

DETAILS OF FOOD AND WATER QUALITY:
The diet is checked periodically by an independent and approved laboratory, according to the specifications of the manufacturer. Analysis includes both qualitative and quantitative evaluation for heavy metals, aflatoxins, pesticides, and antibiotics. The drinking water is periodically analyzed according to the German regulations for human drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.9-24.5
- Humidity (%): 42.4-60.6
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Stability and homogeneity data of the test item in 0.5 % aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium) revealed that the preparation was homogeneous and stable for at least 9 days at room temperature. Therefore, the formulations were prepared weekly. The preparations of the test item formulations were stored under normal room conditions and constant magnetic stirring. Exposure to light was kept to a minimum. Before the first application the formulations were stirred overnight.

VEHICLE
- Justification for use and choice of vehicle: A suitable vehicle for the test substance and the application route with sufficient historical control data was used.
- Concentration in vehicle: 20, 60, 120/200 mg/mL
- Amount of vehicle: 5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: max. 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After unsuccessful pairing no replacement of first male by another male with proven fertility was done.
- Further matings after two unsuccessful attempts: not applicable
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Determination of test item concentration was performed for 2 preparations per group: each time at the beginning of a preparation period and at the end of a preparation period. Analysis were performed using a HPLC method with UV detection. An external standard was used. The limit of detection (LOD) and the limit of quantification (LOQ) of this method was analyzed as 0.022 mg/L and 0.072 mg/L, respectively. The preparations were stored under normal room conditions. Exposure to light was kept to a minimum. At all days of sampling, the concentrations of the test item in the dose formulations were within the predefined acceptance limits (±15 % of the nominal concentration). No test item was detected in the control formulations. In conclusion, the results indicate the accurate use of the test item and 0.5 % aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium) as vehicle during this study.

Duration of treatment / exposure:

Males of all groups and group 4 (1000/600 mg/kg bw/d) females received the test item for 41 days, whereas groups 1-3 females had a treatment period of up to 47 days.

Frequency of treatment:

once daily, 7 times a week

Details on study schedule:

F1 generation was not used for mating.

Dose / conc.:

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

Remarks:

group 1
applicable for males and females

Dose / conc.:

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

Remarks:

group 2
applicable for males and females

Dose / conc.:

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

Remarks:

group 3
applicable for males and females

Dose / conc.:

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

Remarks:

group 4
applicable only for females
reduced from 1000 mg/kg bw/d due to premature sacrifice after moribund condition of 4 females and strongly reduced body weight gain in the surviving females
as different subsets were used reduction for surviving females was done between study day 2 and 9

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

group 4
applicable for males during complete exposure period and few days for females

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The test item was tested for acute toxicity in Wistar (Crl:WI (Han)) rats after single dose oral administration of 2000 mg/kg bw. No mortality or signs of toxicity were seen in the rats (3 males, 3 females). Therefore, the LD50 was considered to be higher than 2000 mg/kg bw after single oral administration. In a 14-day dose range finding study the test item was clinically well tolerated in Wistar (Crl:WI (Han)) rats at 300 and 1000 mg/kg bw. Therefore, for the current study, 1000 mg/kg bw was selected as the high dose with the aim of inducing mild to moderate systemic toxic effects, 300 and 100 mg/kg bw were selected as the mid and low dose, respectively, to enable a dose-related response and to define a NOAEL.
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemistry: approx. 18 h
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Dose range finding studies: A 14-day dose range finding study using the test item at 300 and 1000 mg/kg bw in Wistar (Crl:WI (Han)) rats was conducted. The test item was clinically well tolerated in the DRF.

Positive control:

No positive control was used.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least daily
- Cage side observations included: mortality, behavior and appearance of each animal

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first dose (day -1) and thereafter weekly
- Detailed clinical observations checked in table No.1 were included. For the detailed clinical observations the animals were taken out of the home cage, palpated and then transferred in a separate type IV Makrolon cage.

BODY WEIGHT: Yes
- Time schedule for examinations: The males and females were weighed before treatment and thereafter once a week. Additional body weight measurements could be done, if considered necessary (e.g. animals with clinical signs). From the date of mating the females (group 1-3) were weighed daily, within 24 hours of parturition (day 0 post-partum), and on day 4 post-partum.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was recorded by weighing the food per cage which had not been consumed. Food consumption of group 4 males and females was recorded weekly until the end of treatment. Food consumption was recorded once a week before mating of all animals. No food consumption was measured for the males (group 1-3) further on. Food consumption measurements of the females (group 1-3) were started again after the positive vaginal smears, then on days 7, 14, 21 post coitum and again on day 4 post-partum.

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

OPHTHALMOSCOPIC EXAMINATION: Yes, as part of detailed clinical observations

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 13 for all groups, day 42 for group 4 animals and male group 1 animals
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, approx. 18 h
- How many animals: half of each sex per group
- Parameters checked in table No. 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 13 for all groups, day 42 for group 4 animals and male group 1 animals
- Animals fasted: Yes, approx. 18 h
- How many animals: half of each sex per group
- Parameters checked in table No. 3 were examined.

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: Yes, using half of the animals of each sex per group
- Time schedule for collection of urine: day 13 for all groups, day 42 for group 4 animals and male group 1 animals
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, approx. 18 h
- Parameters checked in table No. 4 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 5 and on day 4 post-partum
- Dose groups that were examined: week 5: numerical first 5 males of all groups and females of group 4; day 4 postpartum: 5 pregnant females per group 1-3
- Battery of functions tested: sensory activity, grip strength and motor activity. Parameters of the functional observational battery (FOB) are listed in table No. 5. The parameters were examined in the cage, during removal from the cage, in the observation arena and during handling.

IMMUNOLOGY: No

Oestrous cyclicity (parental animals):

During mating daily vaginal smears were taken from the females, their cycles were monitored and the presence of sperm was examined.
No further parameters were examined.

Sperm parameters (parental animals):

Parameters examined in P male parental generations: testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no, all pups were killed on day 4 postpartum

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
- Males (all groups) and non-mated group 4 females: All surviving animals in week 6
- Maternal animals (groups 1-3): All surviving animals on postpartum day 4

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues were prepared for microscopic examination as indicated in table No. 7. For all animals, except those that were prematurely killed in the course of the study, organ weights were determined as listed in table No. 6.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Table No. 8 gives the minimum extent of investigated parameters performed for each pup.

HISTOPATHOLOGY / ORGAN WEIGTHS: Not performed.

Statistics:

Statistics were performed for the data of the males and of the pregnant females of the fertility part (starting on GD 0) that either deliver pups or were diagnosed “pregnant” during necropsy (resorptions). From females that were in the end diagnosed as non-pregnant, the parameters recorded after the pre-mating period were not used for statistical evaluation.
To compare the treatment groups with the control group, the following statistical procedures were applied separately for each sex and each measuring point. To take the number of dose groups into account all the test procedures used maintain a multiple significance level of α = 0.05.
Absolute body weight, body weight gain (differences to baseline values on day 0 or differences to baseline values on GD 0 for pregnant females), body temperature, food consumption, organ weights (relative and absolute), clinical pathology parameters (hematology, clinical chemistry serum parameters, specific gravity and urine weight), and motor activity (total time period of 60 min), dose groups were compared with those of the control, using the multiple two-sided Dunnett-Test.
For all numerical parameters of the FOB, dose groups and the control groups were compared, using the non-parametric 2-sided Kruskal-Wallis test followed by the Wilcoxon-test rank sum test.
For all parameters (for urinalysis only specific gravity and urine weight) mean values, standard deviation (except for numerical FOB parameters), and number of animals (N) are calculated.
For each numerical parameter of the FOB also min, max, median, and the frequencies of scores were calculated.
Some findings (posture, convulsions, vocalization, abnormalities, and stereotypy) were listed as incidences per treatment group in a table.
For the reproductive toxicology parameters, the statistical evaluations as shown in table No. 9 were done.

Reproductive indices:

- Conceiving days: Incidence / group of days 1-5 and days 6-14
- Pregnancy days: Incidence / group of days 21, 22, or 23
- Females showing evidence of copulation: Incidence of females/group
- Females achieving pregnancy: Incidence of pregnant females/group
- Pre-Implantation loss: Incidence per group: Females with 0, 1, 2, 3 or more
- Pre-natal loss = post-implantation loss: Incidence per group: Females with 0, 1, 2, 3 or more

Offspring viability indices:

- Post-natal loss: Incidence per group: Females with 0, 1, 2, 3 or more
- Abnormal pups: Incidence of abnormal pups (external gross abnormalities, gross pathological alterations, behavioral abnormalities and runts)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The following symptoms were observed during the whole study (day -1 to 42) for group 1-4 males and group 4 females or until successful mating (day -1 up to maximally day 20) for group 1-3 females:
Single 300, 1000 and 1000/600 mg/kg bw/d males and females showed hair loss for 9-36 days. These findings were without a dose relationship and also observed in 1 male and 2 females of the control group. Therefore, these findings are considered incidental. One of the control females that showed hair loss also showed a skin wound for 5 days. This skin wound was already present before the first treatment with vehicle and therefore considered incidental.
One 300 mg/kg bw/d male showed salivation for more than 5 minutes on 3 days. Since this symptom occurred sporadically and was not accompanied by other symptoms, it is considered incidental.
The 4 1000 mg/kg bw/d females (animal Nos. 85, 86, 90 and 93) that were sacrificed in moribund conditions on days 9 and 10 showed the following clinical signs before removal: piloerection, hypothermia (by touch) and high-legged gait. These animal showed body weight loss of up to 17.5 % . In addition, animals Nos. 85 and 86 showed sunken flanks and animal No. 86 also reddish discharge from the nostrils.
Two of the surviving 1000/600 mg/kg bw/d females (animal Nos. 91 and 96) showed piloerection and body weight loss of up to 8.0 % between day 1 and 14.

The following symptoms were observed in pregnant females (gestation day 0 to necropsy):
Single 100 and 300 mg/kg bw/d pregnant females showed hair loss and/or skin wounds for 2-6 days. These findings were without a dose relationship and also observed in pregnant females of the control group. Therefore, these findings are considered incidental.

No effects on abnormalities, convulsions, discharge, excretions, eye, gait, mucous membrane, posture, respiration, response to handling, secretions, stereotypies and vocalisation could be observed in all dose groups at all time points. The only symptoms observed were hair loss (fur) and wounds (skin). However, both symptoms were observed in control (0 mg/kg bw/d) and dose group animals with a similar incidence and no dose relationship was seen. Therefore, these findings are considered incidental.

Mortality:

mortality observed, treatment-related

Description (incidence):

Four 1000 mg/kg bw/d females were sacrificed in moribund condition between day 9 and 10. Therefore, the dose was reduced for the surviving group 4 females from 1000 mg/kg bw/d to 600 mg/kg bw/d and mating for group 4 animals was omitted. Since the study was performed using different subsets, the surviving females were between study day 2 and 9, when the dose was reduced to 600 mg/kg bw/d.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight was significantly increased in 100 mg/kg bw/d males by approximately 9 % (compared to control) on day 42. In 1000 mg/kg bw/d males mean body weight was slightly decreased by approximately 7-8 % (compared to control) on day 14 and 42. These findings were without a dose relationship, but the decrease on day 14 was accompanied by decreased food consumption during week 1 (no food consumption was measured for group 1-3 males after the premating period). Therefore, a treatment relationship of these effects is considered questionable. In 1000/600 mg/kg bw/d females mean body weight was significantly decreased on days 7 and 14 by approximately 8-9 % (compared to control). Since the dose was reduced to 600 mg/kg bw/d in the surviving females between study day 2 and 9 the mean body weight slightly increased between day 21 and 35, but on day 42 the mean body weight dropped again, reaching a value that was only slightly above the pre-dose value of day 1. In the prematurely sacrificed females the individual body weight loss (individual value compared to pre-dose individual value on day 1) ranged between 13.3 and 17.5 %. In the period between day 7 and 14 the individual body weight loss in the surviving females ranged between 1.2 and 8.0 %. In 300 mg/kg bw/d females the mean body weight was significantly decreased during the premating period on days 7 and 14 by approximately 6-7 % (compared to control) and the individual body weight loss (individual value compared to pre-dose individual value on day 1) ranged between 0.7 and 8.3 %. During the whole pregnancy the mean body weight was decreased by approximately 2-8 % (compared to control), mostly statistically significant. However, no body weight loss was observed. During lactation the mean body weight was significantly decreased in 300 mg/kg bw/d females on days 0 and 4 p.p. by approximately 6-7 % (compared to control). All other changes in mean body weight are not considered treatment-related since they are they are mostly not statistically significant, do not differ from effects in control animals and/or do not show any dose-dependency.

In 1000 mg/kg bw/d males mean body weight gain was significantly decreased compared to control from day 1-42. 300 and 1000/600 mg/kg bw/d females did not show body weight gain during the premating period (day 1-14). After day 14 body weight gain was seen in the 1000/600 mg/kg bw/d females. However, the values were still relatively low; even if no direct comparison to pregnant control females was possible. During pregnancy mean body weight gain was significantly decreased in 300 mg/kg bw/d females compared to control. During lactation (day 0-4 p.p.) mean body weight gain was decreased in 100 and 300 mg/kg bw/d females compared to control; however, without reaching statistical significance. All other changes in mean body weight gain are not considered treatment-related since they are they are mostly not statistically significant, are isolated variations and/or do not show any dose-dependency.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During the whole premating period food consumption was significantly decreased in 1000 mg/kg bw/d males (compared to control). After the first 14 days food consumption increased in 1000 mg/kg/d males (compared to the premating period), but during the last week (day 35-42) food consumption dropped again to a value comparable to that of the premating period. During the whole premating period food consumption was significantly decreased in 300 mg/kg bw/d females (compared to control). During pregnancy food consumption was significantly decreased in 300 mg/kg bw/d females except for the first week of pregnancy. During the whole premating period food consumption was significantly decreased in 1000/600 mg/kg bw/d females (compared to control). All other changes in food consumption are not considered treatment-related since they are they are not statistically significant, do not differ from effects in control animals and/or do not show any dose-dependency.

Food efficiency:

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No effects on the eye were observed during detailed clinical observations.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Alterations in hematology were slight in degree, sporadic, without dose-dependency and in the range of internal reference intervals. They are not treatment-related.
On day 13 RBC was decreased in males of 100 mg/kg bw/d group and PLT was increased for 1000/600 mg/kg bw/d females. On day 42 no alterations were detected. Alterations of the surviving 1000/600 mg/kg bw/d females on day 42 could not directly be compared to control females at this time point. They were compared to values of control group values from day 13.
Alterations of coagulation parameters were not seen.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Regarding clinical chemistry, the decrease of ASAT in males was slight in degree, within known reference interval and without pathological relevance. The slight increase of triglycerides and bile acid in high dose females was also slight in degree and within known reference interval and not considered treatment-related.
On day 13 ASAT was decreased in 300 and 1000/600 mg/kg bw/d males. In 1000/600 mg/kg bw/d females TRIG and BA were increased. On day 42 ASAT was decreased in 1000/600 mg/kg bw/d males. Alterations of the surviving 1000/600 mg/kg/d females on day 42 could not directly be compared to control females at this time point. They were compared to values of control group values from day 13.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Urinalysis revealed slight changes in individual animals without any dose-dependency. The findings are not suggestive for a pathological disorder and not related to the treatment.
On day 13 BLO was increased in 1 of 6 males and OX-U was increased in 1 of 6 females of the 300 mg/kg bw/d group. In the 1000/600 mg/kg bw/d group KET and ESQ-U were increased in 1 of 6 male rats, mean values of UWG was decreased and SG increased. On day 42 KET was increased in males of the 1000/600 mg/kg bw/d group.
Alterations of the surviving 1000/600 mg/kg/d females on day 42 could not directly be compared to control females at this time point. They were compared to values of control group values from day 13.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

In the functional observational battery (FOB) males on day 29, surviving 1000/600 mg/kg bw/d females (group 4) on day 30 and group 1-3 females on day 4 p.p. showed no treatment-related changes in autonomous, neuromuscular, sensomotoric and central nervous system.

Autonomous Nervous System
No significant effects on lacrimation, salivation, pupil response, piloerection, defecation (number of fecal boluses, feces consistency), urination (number of urine pools, urine stain size), and palpebral closure were seen in all males on day 29, in the surviving 1000/600 mg/kg bw/d females (group 4) on day 30 and in group 1-3 females on day 4 p.p..
Due to different reproductive/hormonal status FOB measurements of 1000/600 mg/kg bw/d (group 4) unmated females on day 30 cannot directly be compared to control females (group 1; 0 mg/kg bw/d) on day 4 p.p.. However, the FOB results of the surviving 1000/600 mg/kg bw/d females on day 30 were comparable to control females (group 1; 0 mg/kg bw/d) on day 4 p.p. and in line with in-house experience of FOB measurements in this rat strain.

Neuromuscular System
No significant effects on gait, forelimb grip strength, hindlimb grip strength, hindlimb foot splay, righting reflex, mobility, or muscle tone were observed in all males on day 29, in the surviving 1000/600 mg/kg bw/d females (group 4) on day 30 and in group 1-3 females on day 4 p.p..

Sensomotoric System
No significant effects on approach response, click response, touch and tail pinch response could be observed in all males on day 29, in the surviving 1000/600 mg/kg bw/d females (group 4) on day 30 and in group 1-3 females on day 4 p.p..

Central Nervous System
No significant effects on ease of removal and handling, arousal, fur appearance, raising, raising behavior, or catalepsies could be observed in all males on day 29, in the surviving 1000/600 mg/kg bw/d females (group 4) on day 30 and in group 1-3 females on day 4 p.p..

Body Temperature
No significant changes of body temperature in treated animals versus control were observed on day 29 in males, in the surviving 1000/600 mg/kg bw/d females (group 4) on day 30 and in group 1-3 females on day 4 p.p.

Motor Activity
Total counts were dose-dependently increased on day 29 in males, however, statistical significance was reached in 1000 mg/kg bw/d males only. Corresponding effects were observed on the derived motor activity parameters: counts in 5-minute segments, total distance, on-time, rearing time and rearing number.
Motor activity of the surviving 1000/600 mg/kg bw/d (group 4) unmated females on day 30 cannot directly be compared to control females (group 1; 0 mg/kg bw/d) on day 4 p.p.. However, considering the different reproductive/hormonal status and taking in- house experience with motor activity measurements with this rat strain into account, the motor activity results of the surviving 1000/600 mg/kg bw/d females were considered to be within the normal range of control females.

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):

1000/600 mg/kg bw/d: Histopathology examination revealed treatment-related findings in the liver of male and female rats. In males and females, an increased incidence of minimal multifocal hepatocellular necroses was found compared to controls. In females only, the incidence of minimal multifocal mononuclear infiltrates was increased compared to control females. In males, this lesion was seen more often in controls than in treated animals, so the toxicologic relevance is questionable. Moreover, all females (including those that were prematurely killed) showed a reduced hepatocellular glycogen content most likely due to body weight loss.

300 mg/kg bw/d and 100 mg/kg bw/d: No treatment related organ lesions were found in rats dosed with 300 or 100 mg/kg bw/d.

Decedents: prematurely killed females dosed with 1000 mg/kg bw/d: Histopathology revealed no treatment-related lesions that might further explain the bad health condition of the animals leading to premature killing on study day 9 and 10. In the bone marrow, a mild decreased cellularity was noted in all four females. In addition, in the thymus, a minimal lymphoid depletion of was seen in cortical regions in three females. Both findings were considered secondary to the moribund state of health and not directly related to the test item.

Other findings
All other findings are considered to be spontaneous in nature e.g. marked reduced lactation noted in one group 3 female or a consequence of pregnancy e.g. increased EMH (extramedullary hematopoiesis) in the spleen of female control and treated animals (group 3).

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No effects were observed for testis weight and epididymis weight.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

All control, 100 and 300 mg/kg/d females paired, showed evidence of conception, and achieved pregnancy. No treatment-related effects on male or female fertility were observed.

Conceiving days 1-5 were evident in all control and 300 mg/kg bw/d females (n=12 per group), and in 11 (of 12) 100 mg/kg bw/d females. One 100 mg/kg bw/d female conceived later (day 6).

Pregnancy duration varied between 21 and 23 days, with most females delivering after 22 pregnancy days. Control females had either 22 (10/12) or 23 (2/12) days of pregnancy, 100 mg/kg bw/d females had 21 days (1/11) or 22 days (10/11) of pregnancy, and 300 mg/kg bw/d females had either 21 days (1/12), 22 days (7/12), or 23 days (4/12) of pregnancy. However, these slight variations are not considered treatment-related due to a missing dose-dependency of effects and the in-house experience for background findings with this rat strain into account.

Taking the high number of pre-implantation losses in the control, the total number of pre-natal losses per group, the missing dose-dependency of effects and the in-house experience for background findings with this rat strain into account, no treatment-relationship could be established.

Live young on day 0 and 4 p.p. were observed in 12/12 control females. In the 100 mg/kg bw/d group, live young were observed in 11/12 females on day 0 and 4 p.p. and in the 300 mg/kg bw/d females live young were observed in 12/12 females on day 0 p.p. and in 11/12 females on day 4 p.p..

Pre-implantation loss (corpora lutea minus implantations) was observed in all groups and most pronounced in control females (33 in controls, 9 in 100 mg/kg bw/d females and 7 in 300 mg/kg bw/d females). No treatment-relationship could be established.

Pre-natal loss (implantations minus live births) was observed in all groups including control: In the control group 2 females showed 1 resorption and another two 3 resorptions and 1 female had 2 resorptions and 1 stillborn pup. In the 100 mg/kg bw/d group 2 females had 2 resorptions, another 2 females had 3 resorptions and 1 female had 10 resorptions. In the 300 mg/kg bw/d group 2 females had 1 resorption, 1 female had 2 resorptions, 1 female 3 resorptions, 1 female 5 resorptions and 1 female showed 2 resorptions and delivered 6 dead pups. The latter animal also showed 3 post-natal losses which are related to a spontaneous defect of the mammary gland at histopathology. Taking the high number of pre-implantation losses in the control, the total number of pre-natal losses per group, the missing dose-dependency of effects and the in-house experience for background findings with this rat strain into account, no treatment- relationship could be established.

Post-natal loss (live births minus alive at post-natal day 4) was observed in one control female and two 300 mg/kg bw/d females. In the control group one female with one post-natal loss was seen. In the 300 mg/kg bw/d group one females with one post-natal loss and another female with three post- natal losses were seen. The female with the three post-natal losses showed a spontaneous defect of the mammary gland at histopathology. Therefore, the three post-natal losses of this animal are considered as a secondary effect of a spontaneous lesion. No treatment- relationship could be established.

The number of corpora lutea was slightly reduced in 300 mg/kg bw/d females. The number of implants was slightly increased in 100 mg/kg bw/d females. However, in the control and 300 mg/kg bw/d females the number of implants was comparable. The number of resorptions was slightly increased in 100 and 300 mg/kg bw/d females, but without dose relationship. The number of live pups on day 0 p.p. was slightly increased in 100 mg/kg bw/d females. However, in the control and 300 mg/kg bw/d females the number of live pups on day 0 p.p. was comparable. The number of live pups on day 4 p.p. was slightly reduced in 300 mg/kg bw/d females. However, this was mainly related to the pups that died due to the spontaneous defect of the mammary gland in the corresponding dam.

Overall, the variations in the above mentioned parameters are very low, mainly without a dose relationship and not mutually corroborative or related to secondary spontaneous effects. Therefore, they were considered incidental.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One abnormal live pup was observed in one 100 mg/kg bw/d female, this pup showed a hematoma at the lower jaw. A hematoma is not a malformation, but a common spontaneous observation arising due to handling by the dam. In addition, no dose-dependency was seen. Therefore, this finding was considered incidental. Another abnormal live pup was observed in one 300 mg/kg bw/d female, this pup showed decreased body temperature (by touch) with bluish skin. The dam of this pup showed a spontaneous defect of the mammary gland at histopathology and the symptoms observed in the pup are related to the dam`s inability to provide (enough) milk. Therefore, these findings were considered not treatment-related. All other females had no abnormal pups and no runts were observed in any group.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

On day 0 p.p. and between day 0 and 4 p.p., no dead pups occurred at 100 mg/kg bw/d and the number of dead pups in the 300 mg/kg bw/d group were slightly higher compared to control. However, except for 1 dead pup one day 4 p.p., this slight increase was related to the dead pups of one maternal animal that showed a spontaneous mammary gland lesion. Therefore, this increase is not considered treatment-related.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean pup weight of male and female pups at birth and on day 4 p.p. did not show any statistically significant differences between treatment groups and control. A trend towards a slightly decreased pup weights (approximately 10 % in both genders) was observed on day 4 p.p. in the 100 and 300 mg/kg bw/d groups. However, on day 0 p.p., no dose-relationship of weight changes could be established. Therefore, these changes were considered incidental.

Litter weight at birth and on day 4 p.p. did not show any statistically significant differences between treatment groups and control. A trend towards a slightly decreased pup weights (approximately 10 % in both genders) was observed on days 0 and 4 p.p. in the 300 mg/kg bw/d group. However, on day 0 p.p., litter weight was increased to nearly the same extend in 100 mg/kg bw/d males and no dose-relationship of weight changes could be established. In addition, the number of pups in one litter of the 300 mg/kg bw/d group was reduced due to secondary spontaneous effects in the mammary gland of one dam. Therefore, these changes were not considered treatment-related.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Description (incidence and severity):

Sex ratio
Sex ratios on day 0 and 4 p.p. in the control was slightly shifted towards females with a ratio of 0.88 and 0.89, respectively. In the 100 mg/kg bw/d group the sex rations were slightly shifted towards males with ratios of 1.20 and in the 300 mg/kg bw/d group, the ratio was 1.00. Therefore, it is concluded that no treatment-related change can be observed. Taking the missing dose-dependency of effects and the in-house experience for background findings with this rat strain into account, no treatment-relationship could be established.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects were observed up to the highest dose tested in mated animals (300 mg/kg bw/d).

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

The no adverse effect level (NOAEL) of the test item regarding systemic toxicity and reproduction parameters is considered to be 300 mg/kg bw/d.

Executive summary:

A combined repeated dose oral toxicity with the reproduction/developmental toxicity screening test according to OECD 422 was conducted using Crl:WI (Han) rats to evaluate the test item properties after repeated exposure. The test item was administered orally by gavage, once daily, 7 times a week to 3 groups of male rats at doses of 100, 300 or 1000 mg/kg bw/d and female rats at doses of 100, 300 or 1000/600 mg/kg bw/d. Males of all groups and group 4 (1000/600 mg/kg bw/d) females received the test item for 41 days, whereas groups 1-3 females had a treatment period of up to 47 days. A similarly constituted control group received the vehicle, 0.25 % aqueous hydroxypropyl methylcellulose (Methocel®K4M Premium) and served to generate contemporary control data. All dose groups consisted of 12 male and 12 female rats each.

Due to premature sacrifice of 4 females of group 4 and strongly reduced body weight gain in the surviving group 4 females after dose reduction from 1000 to 600 mg/kg bw/d, mating of group 4 animals was omitted and group 4 animals were gang-housed (3 animals/sex).

In animals of groups 1-3 treatment was commenced 14 days before mating, and continued during the mating period (females only until positive sperm finding) and thereafter until day 41 for males or day 4 post-partum (p.p.) for pregnant females. Unmated group 4 animals were treated for 41 days.

 

During the course of the study following parameters were assessed:

Observations/Measurements	Time schedule
Adults (males and females)
Appearance and behavior	Daily
Mortality	Daily
Detailed Clinical Observations	Day -1, thereafter weekly
Functional observational battery Males Females group 4 Females group 1-3	Day 29 Day 30 Day 4 p.p.
Motor activity Males Females group 4 Females group 1-3	Day 29 Day 30 Day 4 p.p.
Body weight Males Females group 4 Females group 1-3	Once a week Once a week Once a week, from mating daily, day 0 and 4 p.p
Food consumption Males Females group 4 Females group 1-3	Once a week Once a week Once a week before mating, after positive vaginal smear on days 7, 14, 21
Hematology and Coagulation All animals Males and females group 4	day 13 day 42
Clinical chemistry All animals Males and females group 4	day 13 day 42
Urinalysis All animals Males and females group 4	day 13 day 42
Reproductive parameters (pregnant females)
Conceiving day for each dam	daily vaginal smears during mating period
Delivery day for each dam	once between day 20 – 23
Female showing evidence of copulation	between day 1 – 7 during mating period
Female achieving pregnancy	between day 1 – 7 during mating period
Live young born per dam	on day 0 p.p.
Live young per dam	on day 4 p.p.
Pup weight (live) per dam	on day 0 p.p.
Pup weight (live) per dam	on day 4 p.p.
External gross abnormalities	between day 0-4 p.p.
Behavioral abnormalities	between day 0-4 p.p.
Runts	on day 0 p.p.
Sex ratio at birth	on day 0 p.p.
Sex ratio	on day 4 p.p.

 

At the end of the treatment period, the adult animals were subjected to a detailed necropsy and examination for gross pathological alterations. Organ weights were recorded and histopathological examinations performed on selected organs.

The offspring were weighed, sexed, and observed for mortality, clinical signs, appearance and behavior. All surviving pups were subjected to external macroscopy, evisceration with internal sexing of animals and examination for gross pathology changes.

Formulation analysis revealed that the anticipated test item concentrations were met in the formulations of all treatment groups at the beginning and at the end of usage. No test item was detected in the control formulations.

Four 1000 mg/kg bw/d females were sacrificed in moribund condition between day 9 and 10. Therefore, the dose was reduced for the surviving group 4 females from 1000 mg/kg bw/d to 600 mg/kg bw/d and mating for group 4 animals was omitted. Since the study was performed using different subsets, the surviving females were between study day 2 and 9, when the dose was reduced to 600 mg/kg bw/d.

The 1000 mg/kg bw/d females that were sacrificed in moribund conditions on days 9 and 10 showed the following clinical signs before removal: piloerection, hypothermia (by touch) and high-legged gait. Two of the surviving 1000/600 mg/kg bw/d females showed piloerection between day 9 and 14.

The detailed clinical observations performed before the first dose and thereafter weekly revealed no treatment-related findings.

In the functional observational battery (FOB) males on day 29, surviving 1000/600 mg/kg bw/d females (group 4) on day 30 and group 1-3 females on day 4 p.p. showed no treatment-related changes in autonomous, neuromuscular, sensomotoric and central nervous system. The motor activity investigations revealed a dose-dependent decrease of total counts on day 29 in males, however, statistical significance was reached in 1000 mg/kg bw/d males only. Corresponding effects were observed on the derived motor activity parameters: counts in 5-minute segments, total distance, on-time, rearing time and rearing number. The increases of motor activity parameters were slight in degree and no further central nervous system effects were observed in the FOB. Therefore, the changes in motor activity parameters were not considered adverse.

Mean body weight was significantly increased in 100 mg/kg bw/d males by approximately 9 % (compared to control) on day 42. In 1000 mg/kg bw/d males mean body weight was slightly decreased by approximately 7-8 % (compared to control) on day 14 and 42. These findings were without a dose relationship, but the decrease on day 14 was accompanied by decreased food consumption during week 1 (no food consumption was measured for group 1-3 males after the premating period). Therefore, a treatment relationship of these effects is considered questionable. In 1000/600 mg/kg bw/d females mean body weight was significantly decreased on days 7 and 14 by approximately 8-9 % (compared to control). Since the dose was reduced to 600 mg/kg bw/d in the surviving females between study day 2 and 9 the mean body weight slightly increased between day 21 and 35, but on day 42 the mean body weight dropped again, reaching a value that was only slightly above the pre-dose value of day 1. In the prematurely sacrificed females the individual body weight loss (individual value compared to pre-dose individual value on day 1) ranged between 13.3 and 17.5 %. In the period between day 7 and 14 the individual body weight loss in the surviving females ranged between 1.2 and 8.0 %. In 300 mg/kg bw/d females the mean body weight was significantly decreased during the premating period on days 7 and 14 by approximately 6-7 % (compared to control) and the individual body weight loss (individual value compared to pre-dose individual value on day 1) ranged between 0.7 and 8.3 %. During the whole pregnancy the mean body weight was decreased by approximately 2-8 % (compared to control), mostly statistically significant. However, no body weight loss was observed. During lactation the mean body weight was significantly decreased in 300 mg/kg bw/d females on days 0 and 4 p.p. by approximately 6-7 % (compared to control).

Mean body weight gain was significantly decreased compared to control from day 1-42 in 1000 mg/kg bw/d males. 300 and 1000/600 mg/kg bw/d females did not show body weight gain during the premating period (day 1-14). After day 14 body weight gain was seen in the 1000/600 mg/kg bw/d females. However, the values were still relatively low; even if no direct comparison to pregnant control females was possible. During pregnancy mean body weight gain was significantly decreased in 300 mg/kg bw/d females compared to control. During lactation (day 0-4 p.p.) mean body weight gain was decreased (not statistically significant) in 100 and 300 mg/kg bw/d females compared to control.

Food consumption was significantly decreased in 1000 mg/kg bw/d males (compared to control) during the whole premating period. After the first 14 days food consumption increased in 1000 mg/kg bw/d males (compared to the premating period), but during the last week (day 35-42) food consumption dropped again to a value comparable to that of the premating period. During the whole premating period food consumption was significantly decreased in 300 mg/kg bw/d females (compared to control). During pregnancy food consumption was significantly decreased in 300 mg/kg bw/d females except for the first week of pregnancy. During the whole premating period food consumption was significantly decreased in 1000/600 mg/kg bw/d females (compared to control).

Hematology, coagulation, clinical chemistry and urinalysis evaluation on day 13 (100, 300 and 1000/600 mg/kg bw/d) and 42 (1000/600 mg/kg bw/d) did not reveal any treatment-related effects.

At gross pathology, all animals of both sexes (early and scheduled sacrifice) did not show any treatment-related findings.

Terminal body weight was decreased in 300 mg/kg bw/d females, 1000 mg/kg bw/d males and in the surviving 1000/600 mg/kg bw/d females.

Determination of organ weights revealed a decrease of absolute and relative liver weights in 1000/600 mg/kg bw/d females, most likely as a consequence of the catabolic metabolism status. Histopathology revealed a corresponding reduced hepatocellular glycogen content in all 1000/600 mg/kg bw/d females (including those that were prematurely killed). In 100, 300 and 1000 mg/kg bw/d males increases of absolute and relative liver weights was noted. Histopathology of males and females dosed with 1000 mg/kg bw/d and 1000/600 mg/kg bw/d, respectively revealed an increased incidence of minimal multifocal hepatocellular necroses compared to controls. In females only, the incidence of minimal multifocal mononuclear infiltrates was increased compared to control females. In males, this lesion was seen more often in controls than in treated animals. Therefore, the toxicological relevance is questionable. The four 1000 mg/kg bw/d females that were prematurely sacrificed showed a mild decreased cellularity in the bone marrow. In three of these animals a minimal lymphoid depletion in the cortex of the thymus was seen. Both findings were considered secondary to the moribund condition of the animals.

All control, 100 and 300 mg/kg bw/d females paired, showed evidence of conception, and achieved pregnancy. No treatment-related effects on male or female fertility were observed.

Conceiving days 1-5 were evident in all control and 300 mg/kg bw/d females (n=12 per group), and in 11 (of 12) 100 mg/kg bw/d females. One 100 mg/kg bw/d female conceived later (day 6).

Pregnancy duration varied between 21 and 23 days, with most females delivering after 22 pregnancy days and no treatment-related effects were observed.

Live young on day 0 and 4 p.p. were observed in 12/12 control females. In the 100 mg/kg bw/d group, live young were observed in 11/12 females on day 0 and 4 p.p. and in the 300 mg/kg bw/d females live young were observed in 12/12 females on day 0 p.p. and in 11/12 females on day 4 p.p..

No treatment-related effects were observed on the number of abnormal pups (external gross abnormalities, gross pathological alterations, behavioral abnormalities and runts), pre- implantation loss (corpora lutea minus implantations), pre-natal loss (implantations minus live births), post-natal loss (live births minus alive at post-natal day 4), numbers of the corpora lutea, implants, resorptions, and live pups (on day 0 and 4 p.p.), dead pups (on day 0 and 4 p.p.), sex ratio (on day 0 and 4 p.p.), weight of male and female pups at birth and on day 4 p.p. and litter weight at birth and on day 4 p.p..

Daily oral treatment by gavage of 100 and 300 mg/kg bw/d of the test item to rats according to the study design was tolerated whereas 1000/600 mg/kg bw/d was clinically not tolerated and lead to early sacrifice of 4 female animals.

All doses (100, 300 and 1000 mg/kg bw/d) were clinically tolerated over a treatment period of 41 days in males. Slight effects were seen on motor activity, mean body weight, mean body weight gain and food consumption. In females 100 and 300 mg/kg bw/d were clinically tolerated over a treatment period of approximately up to 7 weeks. At 300 mg/kg bw/d females showed slight effects on mean body weight, mean body weight gain and food consumption and at 100 mg/kg bw/d females only showed a slight effect on mean body weight gain. In the surviving females of group 4 (1000/600 mg/kg bw/d) the mean body weight gain after dose reduction from 1000 to 600 mg/kg bw/d was strongly reduced and the mean body weight at the beginning and end of treatment was nearly the same.

According to the effects of in-life body weights, decreases terminal body weight were seen in 300 mg/kg bw/d females, 1000 mg/kg bw/d males and in the surviving 1000/600 mg/kg bw/d females at necropsy. In addition, decreases of absolute and relative liver weights were observed in 1000/600 mg/kg bw/d females, most likely as a consequence of the catabolic metabolism status. At histopathology, the following treatment-related and non-adverse alterations were noted in the liver of 1000 mg/kg bw/d males and surviving 1000/600 mg/kg bw/d females: multifocal hepatocellular necroses (both sexes), multifocal mononuclear infiltrates and reduced hepatocellular glycogen content (both females only). The 1000 mg/kg bw/d females that were prematurely sacrificed showed a decreased cellularity in the bone marrow and lymphoid depletion in the cortex of the thymus. Both findings were considered secondary to the moribund condition of the animals.

Regarding reproduction parameters no treatment-related effects were noted at the clinically tolerated doses of 100 and 300 mg/kg bw/d.

In conclusion, the no adverse effect level (NOAEL) regarding systemic toxicity and reproduction parameters is considered to be 300 mg/kg bw/d.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-ethoxy-2,3-difluoro-4'-propyl-1,1'-biphenyl

EC Number:

638-734-4

Cas Number:

157248-24-3

Molecular formula:

C₁₇H₁₈F₂O

IUPAC Name:

4-ethoxy-2,3-difluoro-4'-propyl-1,1'-biphenyl

Test material form:

solid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The test guideline OECD 422 is designed for use with the rat and the Wistar WI (Han) rat was chosen since it is widely accepted by authorities as an appropriate experimental model, with documented susceptibility to a wide range of toxic substances.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10-12 weeks
- Weight at study initiation: males: 301 (268 – 344) females. 205 (186 – 228)
- Fasting period before study: no
- Housing: conventional conditions, with means to hide, pre-mating: 3 animals/sex/cage, mating: 1 male and 1 female per cage, after mating: 1 animal/cage and females were provided nesting material, not mated animals: 3 animals/sex/cage
- Diet: ad libitum, Ssniff V1534 (batch: 1324730), but for approx. 18 h before blood collection food was withheld
- Water: ad libitum, tap water, bottles freshly filled twice a week
- Acclimation period: at least 9 days

DETAILS OF FOOD AND WATER QUALITY:
The diet is checked periodically by an independent and approved laboratory, according to the specifications of the manufacturer. Analysis includes both qualitative and quantitative evaluation for heavy metals, aflatoxins, pesticides, and antibiotics. The drinking water is periodically analyzed according to the German regulations for human drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.9-24.5
- Humidity (%): 42.4-60.6
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Remarks:

0.5 % aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Stability and homogeneity data of the test item in 0.5 % aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium) revealed that the preparation was homogeneous and stable for at least 9 days at room temperature. Therefore, the formulations were prepared weekly. The preparations of the test item formulations were stored under normal room conditions and constant magnetic stirring. Exposure to light was kept to a minimum. Before the first application the formulations were stirred overnight.

VEHICLE
- Justification for use and choice of vehicle: A suitable vehicle for the test substance and the application route with sufficient historical control data was used.
- Concentration in vehicle: 20, 60, 120/200 mg/mL
- Amount of vehicle: 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Determination of test item concentration was performed for 2 preparations per group: each time at the beginning of a preparation period and at the end of a preparation period. Analysis were performed using a HPLC method with UV detection. An external standard was used. The limit of detection (LOD) and the limit of quantification (LOQ) of this method was analyzed as 0.022 mg/L and 0.072 mg/L, respectively. The preparations were stored under normal room conditions. Exposure to light was kept to a minimum. At all days of sampling, the concentrations of the test item in the dose formulations were within the predefined acceptance limits (±15 % of the nominal concentration). No test item was detected in the control formulations. In conclusion, the results indicate the accurate use of the test item and 0.5 % aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium) as vehicle during this study.

Duration of treatment / exposure:

Males of all groups and group 4 (1000/600 mg/kg bw/d) females received the test item for 41 days, whereas groups 1-3 females had a treatment period of up to 47 days.

Frequency of treatment:

once daily, 7 times a week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

group 1
applicable for males and females

Doses / concentrations 2

Dose / conc.:

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

Remarks:

group 2
applicable for males and females

Doses / concentrations 3

Dose / conc.:

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

Remarks:

group 3
applicable for males and females

Doses / concentrations 4

Dose / conc.:

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

Remarks:

group 4
applicable only for females
reduced from 1000 mg/kg bw/d due to premature sacrifice after moribund condition of 4 females and strongly reduced body weight gain in the surviving females
as different subsets were used reduction for surviving females was done between study day 2 and 9

Doses / concentrations 5

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

group 4
applicable for males during complete exposure period and few days for females

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The test item was tested for acute toxicity in Wistar (Crl:WI (Han)) rats after single dose oral administration of 2000 mg/kg bw. No mortality or signs of toxicity were seen in the rats (3 males, 3 females). Therefore, the LD50 was considered to be higher than 2000 mg/kg bw after single oral administration. In a 14-day dose range finding study the test item was clinically well tolerated in Wistar (Crl:WI (Han)) rats at 300 and 1000 mg/kg bw. Therefore, for the current study, 1000 mg/kg bw was selected as the high dose with the aim of inducing mild to moderate systemic toxic effects, 300 and 100 mg/kg bw were selected as the mid and low dose, respectively, to enable a dose-related response and to define a NOAEL.
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemistry: approx. 18 h
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Dose range finding studies: A 14-day dose range finding study using the test item at 300 and 1000 mg/kg bw in Wistar (Crl:WI (Han)) rats was conducted. The test item was clinically well tolerated in the DRF.

Positive control:

No positive control was used.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least daily
- Cage side observations included: mortality, behavior and appearance of each animal

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first dose (day -1) and thereafter weekly
- Detailed clinical observations checked in table No.1 were included. For the detailed clinical observations the animals were taken out of the home cage, palpated and then transferred in a separate type IV Makrolon cage.

BODY WEIGHT: Yes
- Time schedule for examinations: The males and females were weighed before treatment and thereafter once a week. Additional body weight measurements could be done, if considered necessary (e.g. animals with clinical signs). From the date of mating the females (group 1-3) were weighed daily, within 24 hours of parturition (day 0 post-partum), and on day 4 post-partum.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was recorded by weighing the food per cage which had not been consumed. Food consumption of group 4 males and females was recorded weekly until the end of treatment. Food consumption was recorded once a week before mating of all animals. No food consumption was measured for the males (group 1-3) further on. Food consumption measurements of the females (group 1-3) were started again after the positive vaginal smears, then on days 7, 14, 21 post coitum and again on day 4 post-partum.

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

OPHTHALMOSCOPIC EXAMINATION: Yes, as part of detailed clinical observations

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 13 for all groups, day 42 for group 4 animals and male group 1 animals
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, approx. 18 h
- How many animals: half of each sex per group
- Parameters checked in table No. 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 13 for all groups, day 42 for group 4 animals and male group 1 animals
- Animals fasted: Yes, approx. 18 h
- How many animals: half of each sex per group
- Parameters checked in table No. 3 were examined.

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: Yes, using half of the animals of each sex per group
- Time schedule for collection of urine: day 13 for all groups, day 42 for group 4 animals and male group 1 animals
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, approx. 18 h
- Parameters checked in table No. 4 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 5 and on day 4 post-partum
- Dose groups that were examined: week 5: numerical first 5 males of all groups and females of group 4; day 4 postpartum: 5 pregnant females per group 1-3
- Battery of functions tested: sensory activity, grip strength and motor activity. Parameters of the functional observational battery (FOB) are listed in table No. 5. The parameters were examined in the cage, during removal from the cage, in the observation arena and during handling.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals were examined for gross pathological alterations. For all animals, except those that were prematurely killed in the course of the study, organ weights were determined as listed in table No. 6.

HISTOPATHOLOGY: Yes, see table No. 7. All tissues from decedents (prematurely killed females, group 4) were processed and examined microscopically to the same extent as listed for control animals.

Other examinations:

Furthermore, reproductive toxicity evaluation was performed as part of the study. For details on this please refer to the respective IUCLID section.

Statistics:

Statistics were performed for the data of the males and of the pregnant females of the fertility part (starting on gestation day 0) that either deliver pups or were diagnosed “pregnant” during necropsy (resorptions). From females that were in the end diagnosed as non-pregnant, the parameters recorded after the pre-mating period were not used for statistical evaluation.
Body temperature, body weight (gain), food consumption, organ weights, hematology, clinical chemistry, motor activity, functional observational battery:
To compare the treatment groups with the control group, the following statistical procedures were applied separately for each sex and each measuring point. To take the number of dose groups into account all the test procedures used maintain a multiple significance level of α= 0.05.
Absolute body weight, body weight gain (differences to baseline values on day 0 or differences to baseline values on gestations day 0 for pregnant females), body temperature, food consumption, organ weights (relative and absolute), clinical pathology parameters (hematology, clinical chemistry serum parameters, specific gravity and urine weight), and motor activity (total time period of 60 minutes), dose groups were compared with those of the control, using the multiple two-sided Dunnett-Test.
For all numerical parameters of the FOB, dose groups and the control groups were compared, using the non-parametric 2-sided Kruskal-Wallis test followed by the Wilcoxon-test rank sum test.
For all parameters (for urinalysis only specific gravity and urine weight) mean values, standard deviation (except for numerical FOB parameters), and number of animals (N) are calculated.
For each numerical parameter of the FOB also minimum, maximum, median, and the frequencies of scores were calculated.
Some findings (posture, convulsions, vocalization, abnormalities, and stereotypy) were listed as incidences per treatment group in a table.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The following symptoms were observed during the whole study (day -1 to 42) for group 1-4 males and group 4 females or until successful mating (day -1 up to maximally day 20) for group 1-3 females:
Single 300, 1000 and 1000/600 mg/kg bw/d males and females showed hair loss for 9-36 days. These findings were without a dose relationship and also observed in 1 male and 2 females of the control group. Therefore, these findings are considered incidental. One of the control females that showed hair loss also showed a skin wound for 5 days. This skin wound was already present before the first treatment with vehicle and therefore considered incidental.
One 300 mg/kg bw/d male showed salivation for more than 5 minutes on 3 days. Since this symptom occurred sporadically and was not accompanied by other symptoms, it is considered incidental.
The 4 1000 mg/kg bw/d females (animal Nos. 85, 86, 90 and 93) that were sacrificed in moribund conditions on days 9 and 10 showed the following clinical signs before removal: piloerection, hypothermia (by touch) and high-legged gait. These animal showed body weight loss of up to 17.5 % . In addition, animals Nos. 85 and 86 showed sunken flanks and animal No. 86 also reddish discharge from the nostrils.
Two of the surviving 1000/600 mg/kg bw/d females (animal Nos. 91 and 96) showed piloerection and body weight loss of up to 8.0 % between day 1 and 14.

The following symptoms were observed in pregnant females (gestation day 0 to necropsy):
Single 100 and 300 mg/kg bw/d pregnant females showed hair loss and/or skin wounds for 2-6 days. These findings were without a dose relationship and also observed in pregnant females of the control group. Therefore, these findings are considered incidental.

No effects on abnormalities, convulsions, discharge, excretions, eye, gait, mucous membrane, posture, respiration, response to handling, secretions, stereotypies and vocalisation could be observed in all dose groups at all time points. The only symptoms observed were hair loss (fur) and wounds (skin). However, both symptoms were observed in control (0 mg/kg bw/d) and dose group animals with a similar incidence and no dose relationship was seen. Therefore, these findings are considered incidental.

Mortality:

mortality observed, treatment-related

Description (incidence):

Four 1000 mg/kg bw/d females were sacrificed in moribund condition between day 9 and 10. Therefore, the dose was reduced for the surviving group 4 females from 1000 mg/kg bw/d to 600 mg/kg bw/d and mating for group 4 animals was omitted. Since the study was performed using different subsets, the surviving females were between study day 2 and 9, when the dose was reduced to 600 mg/kg bw/d.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight was significantly increased in 100 mg/kg bw/d males by approximately 9 % (compared to control) on day 42. In 1000 mg/kg bw/d males mean body weight was slightly decreased by approximately 7-8 % (compared to control) on day 14 and 42. These findings were without a dose relationship, but the decrease on day 14 was accompanied by decreased food consumption during week 1 (no food consumption was measured for group 1-3 males after the premating period). Therefore, a treatment relationship of these effects is considered questionable. In 1000/600 mg/kg bw/d females mean body weight was significantly decreased on days 7 and 14 by approximately 8-9 % (compared to control). Since the dose was reduced to 600 mg/kg bw/d in the surviving females between study day 2 and 9 the mean body weight slightly increased between day 21 and 35, but on day 42 the mean body weight dropped again, reaching a value that was only slightly above the pre-dose value of day 1. In the prematurely sacrificed females the individual body weight loss (individual value compared to pre-dose individual value on day 1) ranged between 13.3 and 17.5 %. In the period between day 7 and 14 the individual body weight loss in the surviving females ranged between 1.2 and 8.0 %. In 300 mg/kg bw/d females the mean body weight was significantly decreased during the premating period on days 7 and 14 by approximately 6-7 % (compared to control) and the individual body weight loss (individual value compared to pre-dose individual value on day 1) ranged between 0.7 and 8.3 %. During the whole pregnancy the mean body weight was decreased by approximately 2-8 % (compared to control), mostly statistically significant. However, no body weight loss was observed. During lactation the mean body weight was significantly decreased in 300 mg/kg bw/d females on days 0 and 4 p.p. by approximately 6-7 % (compared to control). All other changes in mean body weight are not considered treatment-related since they are they are mostly not statistically significant, do not differ from effects in control animals and/or do not show any dose-dependency.

In 1000 mg/kg bw/d males mean body weight gain was significantly decreased compared to control from day 1-42. 300 and 1000/600 mg/kg bw/d females did not show body weight gain during the premating period (day 1-14). After day 14 body weight gain was seen in the 1000/600 mg/kg bw/d females. However, the values were still relatively low; even if no direct comparison to pregnant control females was possible. During pregnancy mean body weight gain was significantly decreased in 300 mg/kg bw/d females compared to control. During lactation (day 0-4 p.p.) mean body weight gain was decreased in 100 and 300 mg/kg bw/d females compared to control; however, without reaching statistical significance. All other changes in mean body weight gain are not considered treatment-related since they are they are mostly not statistically significant, are isolated variations and/or do not show any dose-dependency.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During the whole premating period food consumption was significantly decreased in 1000 mg/kg bw/d males (compared to control). After the first 14 days food consumption increased in 1000 mg/kg/d males (compared to the premating period), but during the last week (day 35-42) food consumption dropped again to a value comparable to that of the premating period. During the whole premating period food consumption was significantly decreased in 300 mg/kg bw/d females (compared to control). During pregnancy food consumption was significantly decreased in 300 mg/kg bw/d females except for the first week of pregnancy. During the whole premating period food consumption was significantly decreased in 1000/600 mg/kg bw/d females (compared to control). All other changes in food consumption are not considered treatment-related since they are they are not statistically significant, do not differ from effects in control animals and/or do not show any dose-dependency.

Food efficiency:

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No effects on the eye were observed during detailed clinical observations.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Alterations in hematology were slight in degree, sporadic, without dose-dependency and in the range of internal reference intervals. They are not treatment-related.
On day 13 RBC was decreased in males of 100 mg/kg bw/d group and PLT was increased for 1000/600 mg/kg bw/d females. On day 42 no alterations were detected. Alterations of the surviving 1000/600 mg/kg bw/d females on day 42 could not directly be compared to control females at this time point. They were compared to values of control group values from day 13.
Alterations of coagulation parameters were not seen.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Regarding clinical chemistry, the decrease of ASAT in males was slight in degree, within known reference interval and without pathological relevance. The slight increase of triglycerides and bile acid in high dose females was also slight in degree and within known reference interval and not considered treatment-related.
On day 13 ASAT was decreased in 300 and 1000/600 mg/kg bw/d males. In 1000/600 mg/kg bw/d females TRIG and BA were increased. On day 42 ASAT was decreased in 1000/600 mg/kg bw/d males. Alterations of the surviving 1000/600 mg/kg/d females on day 42 could not directly be compared to control females at this time point. They were compared to values of control group values from day 13.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Urinalysis revealed slight changes in individual animals without any dose-dependency. The findings are not suggestive for a pathological disorder and not related to the treatment.
On day 13 BLO was increased in 1 of 6 males and OX-U was increased in 1 of 6 females of the 300 mg/kg bw/d group. In the 1000/600 mg/kg bw/d group KET and ESQ-U were increased in 1 of 6 male rats, mean values of UWG was decreased and SG increased. On day 42 KET was increased in males of the 1000/600 mg/kg bw/d group.
Alterations of the surviving 1000/600 mg/kg/d females on day 42 could not directly be compared to control females at this time point. They were compared to values of control group values from day 13.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

In the functional observational battery (FOB) males on day 29, surviving 1000/600 mg/kg bw/d females (group 4) on day 30 and group 1-3 females on day 4 p.p. showed no treatment-related changes in autonomous, neuromuscular, sensomotoric and central nervous system.

Autonomous Nervous System
No significant effects on lacrimation, salivation, pupil response, piloerection, defecation (number of fecal boluses, feces consistency), urination (number of urine pools, urine stain size), and palpebral closure were seen in all males on day 29, in the surviving 1000/600 mg/kg bw/d females (group 4) on day 30 and in group 1-3 females on day 4 p.p..
Due to different reproductive/hormonal status FOB measurements of 1000/600 mg/kg bw/d (group 4) unmated females on day 30 cannot directly be compared to control females (group 1; 0 mg/kg bw/d) on day 4 p.p.. However, the FOB results of the surviving 1000/600 mg/kg bw/d females on day 30 were comparable to control females (group 1; 0 mg/kg bw/d) on day 4 p.p. and in line with in-house experience of FOB measurements in this rat strain.

Neuromuscular System
No significant effects on gait, forelimb grip strength, hindlimb grip strength, hindlimb foot splay, righting reflex, mobility, or muscle tone were observed in all males on day 29, in the surviving 1000/600 mg/kg bw/d females (group 4) on day 30 and in group 1-3 females on day 4 p.p..

Sensomotoric System
No significant effects on approach response, click response, touch and tail pinch response could be observed in all males on day 29, in the surviving 1000/600 mg/kg bw/d females (group 4) on day 30 and in group 1-3 females on day 4 p.p..

Central Nervous System
No significant effects on ease of removal and handling, arousal, fur appearance, raising, raising behavior, or catalepsies could be observed in all males on day 29, in the surviving 1000/600 mg/kg bw/d females (group 4) on day 30 and in group 1-3 females on day 4 p.p..

Body Temperature
No significant changes of body temperature in treated animals versus control were observed on day 29 in males, in the surviving 1000/600 mg/kg bw/d females (group 4) on day 30 and in group 1-3 females on day 4 p.p.

Motor Activity
Total counts were dose-dependently increased on day 29 in males, however, statistical significance was reached in 1000 mg/kg bw/d males only. Corresponding effects were observed on the derived motor activity parameters: counts in 5-minute segments, total distance, on-time, rearing time and rearing number.
Motor activity of the surviving 1000/600 mg/kg bw/d (group 4) unmated females on day 30 cannot directly be compared to control females (group 1; 0 mg/kg bw/d) on day 4 p.p.. However, considering the different reproductive/hormonal status and taking in- house experience with motor activity measurements with this rat strain into account, the motor activity results of the surviving 1000/600 mg/kg bw/d females were considered to be within the normal range of control females.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/d, males: A slight treatment-related decrease of terminal body weights was noted. Absolute liver weights showed a tendency to increased mean values and relative liver weights were increased significantly. Although no histomorphological correlate was seen, these weight changes were considered treatment-related. All other organ weight changes were considered to be spontaneous and incidental in nature.
300 mg/kg bw/d, males: No relevant terminal body changes were seen that could be related to treatment. Increased absolute liver and relative liver weights were seen. Although no histomorphological correlate was found, these changes were considered treatment-related. All other organ weight changes were considered to be spontaneous and incidental in nature.
100 mg/kg bw/d, males: No relevant terminal body and changes were seen that could be related to treatment. Increased absolute and relative liver weights were seen. Although no histomorphological correlate was found, these changes were considered treatment-related. All other organ weight changes were considered to be spontaneous and incidental in nature.

1000/600 mg/kg bw/d, females: In surviving females (unmated), a treatment-related decrease of terminal body weights was noted. Absolute and relative liver weights were reduced, most likely as a consequence of the reduced terminal body weights.
300 mg/kg bw/d, females: A treatment-related decrease of terminal body weights was noted. Relative liver weights were slightly increased. As absolute liver weights were unchanged and no histomorphological correlate was noted, these relative weight changes were considered rather related to the terminal body weight loss. All other organ weight changes were considered to be spontaneous and incidental in nature.
100 mg/kg bw/d, females: No treatment-related body and organ weight changes were noted.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At gross pathology, no treatment-related findings were seen.
Skin lesions noted in single treated females (group 2) were considered to be related to mating and not to treatment with the test material.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

1000/600 mg/kg bw/d: Histopathology examination revealed treatment-related findings in the liver of male and female rats. In males and females, an increased incidence of minimal multifocal hepatocellular necroses was found compared to controls. In females only, the incidence of minimal multifocal mononuclear infiltrates was increased compared to control females. In males, this lesion was seen more often in controls than in treated animals, so the toxicologic relevance is questionable. Moreover, all females (including those that were prematurely killed) showed a reduced hepatocellular glycogen content most likely due to body weight loss.

300 mg/kg bw/d and 100 mg/kg bw/d: No treatment related organ lesions were found in rats dosed with 300 or 100 mg/kg bw/d.

Decedents: prematurely killed females dosed with 1000 mg/kg bw/d: Histopathology revealed no treatment-related lesions that might further explain the bad health condition of the animals leading to premature killing on study day 9 and 10. In the bone marrow, a mild decreased cellularity was noted in all four females. In addition, in the thymus, a minimal lymphoid depletion of was seen in cortical regions in three females. Both findings were considered secondary to the moribund state of health and not directly related to the test item.

Other findings
All other findings are considered to be spontaneous in nature e.g. marked reduced lactation noted in one group 3 female or a consequence of pregnancy e.g. increased EMH (extramedullary hematopoiesis) in the spleen of female control and treated animals (group 3).

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Description (incidence and severity):

Reproductive toxicity evaluation was performed as part of the study. For details on this please refer to the respective IUCLID section.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

The no adverse effect level (NOAEL) of the test item regarding systemic toxicity is considered to be 300 mg/kg bw/d.

Executive summary:

A combined repeated dose oral toxicity with the reproduction/developmental toxicity screening test according to OECD TG 422 was conducted using Crl:WI (Han) rats to evaluate the test item properties after repeated exposure. The test item was administered orally by gavage, once daily, 7 times a week to 3 groups of male rats at doses of 100, 300 or 1000 mg/kg bw/d and female rats at doses of 100, 300 or 1000/600 mg/kg bw/d. Males of all groups and group 4 (1000/600 mg/kg bw/d) females received the test item for 41 days, whereas groups 1-3 females had a treatment period of up to 47 days. A similarly constituted control group received the vehicle, 0.25 % aqueous hydroxypropyl methylcellulose (Methocel^®K4M Premium) and served to generate contemporary control data. All dose groups consisted of 12 male and 12 female rats each.

Due to premature sacrifice of 4 females of group 4 and strongly reduced body weight gain in the surviving group 4 females after dose reduction from 1000 to 600 mg/kg bw/d, mating of group 4 animals was omitted and group 4 animals were gang-housed (3 animals/sex).

In animals of groups 1-3 treatment was commenced 14 days before mating, and continued during the mating period (females only until positive sperm finding) and thereafter until day 41 for males or day 4 post-partum (p.p.) for pregnant females. Unmated group 4 animals were treated for 41 days.

During the course of the study following parameters were assessed:

Observations/Measurements	Time schedule
Adults (males and females)
Appearance and behavior	Daily
Mortality	Daily
Detailed Clinical Observations	Day -1, thereafter weekly
Functional observational battery Males Females group 4 Females group 1-3	Day 29 Day 30 Day 4 p.p.
Motor activity Males Females group 4 Females group 1-3	Day 29 Day 30 Day 4 p.p.
Body weight Males Females group 4 Females group 1-3	Once a week Once a week Once a week, from mating daily, day 0 and 4 p.p
Food consumption Males Females group 4 Females group 1-3	Once a week Once a week Once a week before mating, after positive vaginal smear on days 7, 14, 21
Hematology and Coagulation All animals Males and females group 4	day 13 day 42
Clinical chemistry All animals Males and females group 4	day 13 day 42
Urinalysis All animals Males and females group 4	day 13 day 42

At the end of the treatment period, the adult animals were subjected to a detailed necropsy and examination for gross pathological alterations. Organ weights were recorded and histopathological examinations performed on selected organs.

Formulation analysis revealed that the anticipated test item concentrations were met in the formulations of all treatment groups at the beginning and at the end of usage. No test item was detected in the control formulations.

Four 1000 mg/kg bw/d females were sacrificed in moribund condition between day 9 and 10. Therefore, the dose was reduced for the surviving group 4 females from 1000 mg/kg bw/d to 600 mg/kg bw/d and mating for group 4 animals was omitted. Since the study was performed using different subsets, the surviving females were between study day 2 and 9, when the dose was reduced to 600 mg/kg bw/d.

The 1000 mg/kg bw/d females that were sacrificed in moribund conditions on days 9 and 10 showed the following clinical signs before removal: piloerection, hypothermia (by touch) and high-legged gait. Two of the surviving 1000/600 mg/kg bw/d females showed piloerection between day 9 and 14.

The detailed clinical observations performed before the first dose and thereafter weekly revealed no treatment-related findings.

In the functional observational battery (FOB) males on day 29, surviving 1000/600 mg/kg bw/d females (group 4) on day 30 and group 1-3 females on day 4 p.p. showed no treatment-related changes in autonomous, neuromuscular, sensomotoric and central nervous system. The motor activity investigations revealed a dose-dependent decrease of total counts on day 29 in males, however, statistical significance was reached in 1000 mg/kg bw/d males only. Corresponding effects were observed on the derived motor activity parameters: counts in 5-minute segments, total distance, on-time, rearing time and rearing number. The increases of motor activity parameters were slight in degree and no further central nervous system effects were observed in the FOB. Therefore, the changes in motor activity parameters were not considered adverse.

Mean body weight was significantly increased in 100 mg/kg bw/d males by approximately 9 % (compared to control) on day 42. In 1000 mg/kg bw/d males mean body weight was slightly decreased by approximately 7-8 % (compared to control) on day 14 and 42. These findings were without a dose relationship, but the decrease on day 14 was accompanied by decreased food consumption during week 1 (no food consumption was measured for group 1-3 males after the premating period). Therefore, a treatment relationship of these effects is considered questionable. In 1000/600 mg/kg bw/d females mean body weight was significantly decreased on days 7 and 14 by approximately 8-9 % (compared to control).Since the dose was reduced to 600 mg/kg bw/d in the surviving females between study day 2 and 9 the mean body weight slightly increased between day 21 and 35, but on day 42 the mean body weight dropped again, reaching a value that was only slightly above the pre-dose value of day 1. In the prematurely sacrificed females the individual body weight loss (individual value compared to pre-dose individual value on day 1) ranged between 13.3 and 17.5 %. In the period between day 7 and 14 the individual body weight loss in the surviving females ranged between 1.2 and 8.0 %. In 300 mg/kg bw/d females the mean body weight was significantly decreased during the premating period on days 7 and 14 by approximately 6-7 % (compared to control) and the individual body weight loss (individual value compared to pre-dose individual value on day 1) ranged between 0.7 and 8.3 %. During the whole pregnancy the mean body weight was decreased by approximately 2-8 % (compared to control), mostly statistically significant. However, no body weight loss was observed. During lactation the mean body weight was significantly decreased in 300 mg/kg bw/d females on days 0 and 4 p.p. by approximately 6-7 % (compared to control).

Mean body weight gain was significantly decreased compared to control from day 1-42 in 1000 mg/kg bw/d males. 300 and 1000/600 mg/kg bw/d females did not show body weight gain during the premating period (day 1-14). After day 14 body weight gain was seen in the 1000/600 mg/kg bw/d females. However, the values were still relatively low; even if no direct comparison to pregnant control females was possible. During pregnancy mean body weight gain was significantly decreased in 300 mg/kg bw/d females compared to control. During lactation (day 0-4 p.p.) mean body weight gain was decreased (not statistically significant) in 100 and 300 mg/kg bw/d females compared to control.

Food consumption was significantly decreased in 1000 mg/kg bw/d males (compared to control) during the whole premating period. After the first 14 days food consumption increased in 1000 mg/kg bw/d males (compared to the premating period), but during the last week (day 35-42) food consumption dropped again to a value comparable to that of the premating period. During the whole premating period food consumption was significantly decreased in 300 mg/kg bw/d females (compared to control). During pregnancy food consumption was significantly decreased in 300 mg/kg bw/d females except for the first week of pregnancy. During the whole premating period food consumption was significantly decreased in 1000/600 mg/kg bw/d females (compared to control).

Hematology, coagulation, clinical chemistry and urinalysis evaluation on day 13 (100, 300 and 1000/600 mg/kg bw/d) and 42 (1000/600 mg/kg bw/d) did not reveal any treatment-related effects.

At gross pathology, all animals of both sexes (early and scheduled sacrifice) did not show any treatment-related findings.

Terminal body weight was decreased in 300 mg/kg bw/d females, 1000 mg/kg bw/d males and in the surviving 1000/600 mg/kg bw/d females.

Determination of organ weights revealed a decrease of absolute and relative liver weights in 1000/600 mg/kg bw/d females, most likely as a consequence of the catabolic metabolism status. Histopathology revealed a corresponding reduced hepatocellular glycogen content in all 1000/600 mg/kg bw/d females (including those that were prematurely killed). In 100, 300 and 1000 mg/kg bw/d males increases of absolute and relative liver weights was noted. Histopathology of males and females dosed with 1000 mg/kg bw/d and 1000/600 mg/kg bw/d, respectively revealed an increased incidence of minimal multifocal hepatocellular necroses compared to controls. In females only, the incidence of minimal multifocal mononuclear infiltrates was increased compared to control females. In males, this lesion was seen more often in controls than in treated animals. Therefore, the toxicological relevance is questionable. The four 1000 mg/kg bw/d females that were prematurely sacrificed showed a mild decreased cellularity in the bone marrow. In three of these animals a minimal lymphoid depletion in the cortex of the thymus was seen. Both findings were considered secondary to the moribund condition of the animals.

Daily oral treatment by gavage of 100 and 300 mg/kg bw/d of the test item to rats according to the study design was tolerated whereas 1000/600 mg/kg bw/d was clinically not tolerated and lead to early sacrifice of 4 female animals.

All doses (100, 300 and 1000 mg/kg bw/d) were clinically tolerated over a treatment period of 41 days in males. Slight effects were seen on motor activity, mean body weight, mean body weight gain and food consumption. In females 100 and 300 mg/kg bw/d were clinically tolerated over a treatment period of approximately up to 7 weeks. At 300 mg/kg bw/d females showed slight effects on mean body weight, mean body weight gain and food consumption and at 100 mg/kg bw/d females only showed a slight effect on mean body weight gain. In the surviving females of group 4 (1000/600 mg/kg bw/d) the mean body weight gain after dose reduction from 1000 to 600 mg/kg bw/d was strongly reduced and the mean body weight at the beginning and end of treatment was nearly the same.

According to the effects of in-life body weights, decreases terminal body weight were seen in 300 mg/kg bw/d females, 1000 mg/kg bw/d males and in the surviving 1000/600 mg/kg bw/d females at necropsy. In addition, decreases of absolute and relative liver weights were observed in 1000/600 mg/kg bw/d females, most likely as a consequence of the catabolic metabolism status. At histopathology, the following treatment-related and non-adverse alterations were noted in the liver of 1000 mg/kg bw/d males and surviving 1000/600 mg/kg bw/d females: multifocal hepatocellular necroses (both sexes), multifocal mononuclear infiltrates and reduced hepatocellular glycogen content (both females only). The 1000 mg/kg bw/d females that were prematurely sacrificed showed a decreased cellularity in the bone marrow and lymphoid depletion in the cortex of the thymus. Both findings were considered secondary to the moribund condition of the animals.

In conclusion, the no adverse effect level (NOAEL) regarding systemic toxicity is considered to be 300 mg/kg bw/d.