1-(dimethylamino)propan-2-ol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Under the conditions of a combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD TG 422, GLP), the oral administration of the test item by gavage to Wistar rats did not cause adverse effects on fertility and reproduction up to the highest tested dose. Thus, the NOAEL for reproductive performance and fertility was set to 240 mg/kg bw/d for male and female Wistar rats.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, D-55116 Mainz

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch number of test material:
A029-2018.
- Expiration date of the lot/batch:
16 Oct 2019.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Ambient (room temperature).
- Stability under storage conditions:
The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor.
- Stability under test conditions:
The stability of the test substance in deionized water over a period of 7 days wasproven. As the test substance preparations were stored no longer than this time period, the stability was guaranteed.
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium:
Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that the test substance was distributed homogeneously in deionized water.

FORM AS APPLIED IN THE TEST: Test substance preparations in deionized water.

Species:

rat

Strain:

Wistar

Remarks:

Wistar Rat; Crl:WI(Han)

Details on species / strain selection:

The rat is the preferred animal species for reproduction studies according to the various test guidelines and the Wistar strain was selected. This Wistar rat strain (Crl:WI(Han)) was selected since extensive historical control data were available on these Wistar rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany.
- Females nulliparous and non-pregnant: yes.
- Age at study initiation: About 11 - 12 weeks (male animals), about 10 weeks (female animals).
- Weight at study initiation: 385.7 (male), 229.6 (female).
- Fasting period before study: no.
- Housing: During pre-treatment: Polysulfonate cages Typ 2000P (H-Temp), floor area about 2065 cm2 (610 x 435 x 215 mm); supplied by TECHNIPLAST, Hohenpeißenberg, Germany. During pre-mating, mating, gestation, lactation, males after mating and females after weaning: Polycarbonate cages type III. For motor activity (MA) measurements the animals were housed individually in polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany, with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2) and small amounts of bedding material. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. Wooden gnawing blocks (Lignocel Block Large) were supplied by J. Rettenmaier & Söhne GmbH + Co. KG, Rosenberg, Germany, and used for environmental enrichment.
- Diet: ad libitum. The food used was ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA (new name Granovit AG), Kaiseraugst, Switzerland.
- Water: ad libitum.
- Acclimation period: 28 days.

DETAILS OF FOOD AND WATER QUALITY:
- On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants, the diet was found to be suitable. Fed. Reg. Vol. 44, No. 91 of 09 May 1979, p. 27354 (EPA), served as a guideline for maximum tolerable chemical contaminants. The number of microorganisms did not exceed 1*10^5/g food.
- On the basis of the analytical findings the drinking water was found to be suitable. German “Trinkwasserverordnung” (Drinking Water Regulation) served as a guideline for maximum tolerable contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C.
- Humidity (%): 45-65%.
- Air changes (per hr): 15.
- Photoperiod (hrs dark / hrs light): 12 hours (12 hours light from 06.00-18.00 h, 12 hours dark from 18.00-06.00 h).

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
- Test substance preparations in deionized water.

EXPOSURE
- After the acclimatization period, the test substance was administered orally via gavage to the F0 generation parental animals, daily at the same time in the morning (exception: no administration to animals being in labor). The treatment lasted up to one day prior to sacrifice. The animals of the control group were treated in the same way with the vehicle only (deionized water). The calculation of the administered volume was generally based on the most recent individual body weights.

VEHICLE
- Concentration in vehicle: 0, 0.40, 1.20, 3.75/2.40 g/100 mL.
- Amount of vehicle (if gavage): 10 mL/kg bw/d.

Details on mating procedure:

- M/F ratio per cage: 1:1.
- Length of cohabitation: overnight (from about 16.00 h until 06.30 - 09.00 h of the following morning) for a maximum of 2 weeks.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy (gestation day (GD) 0)
- After successful mating each pregnant female was caged (how): Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that the test material was distributed homogenously in deionized water. The mean values and single values of the test substance in deionized water were found to be in a range of 90%-110% of the nominal concentrations. The mean values of samples 11-13 was found to be 136.7% of the nominal concentrations and therefore did not meet the specification of the test facility.

Duration of treatment / exposure:

Males: 31 days, females: 59 days.
The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

Frequency of treatment:

Daily at the same time in the morning (exception: no administration to animals being in labor).

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Remarks:

High dose from study day 10 onwards.

Dose / conc.:

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

Remarks:

Initial high dose up to day 9.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A test study was performed beforehand to select proper dose levels for the present OECD 422 study. The test substance was administered daily by gavage to groups of 4 male and 4 female Wistar rats at dose levels of 0 (test group 0), 100 (test group 1), 250 (test group 2) and 750 mg/kg bw/d (test group 3) over a period of 2 weeks. After the second administration, the following findings occurred in test group 3 (750 mg/kg bw/d): All male animals showed piloerection and semi-closed eyelids, three male animals showed twitching, and one male animal showed tremors. In females, semi-closed eyelids occurred in all animals, hypothermia and poor general condition was observed in 2 female animals. Body weight loss was determined for all male and female animals between study days 0 and 2. All animals of test group 3 (750 mg/kg bw/d) had to be sacrificed in a moribund condition on study day 2. At necropsy, all male and female animals showed foci in the glandular stomach and thickening of the duodenum wall. One male and one female also showed foci in the forestomach and in the jejunum. Peyer’s patches were enlarged in all male and one female animal. The male and female animals of test groups 0, 1 and 2 (control, 100 and 250 mg/kg bw/d, respectively) were sacrificed as scheduled after 2 weeks of treatment. In test group 2 (250 mg/kg bw/d), one male animal showed enlarged Peyer’s patches at necropsy. Based on these results, the following dose levels were selected for the present study: 40 mg/kg bw/d as low-dose level, 120 mg/kg bw/d as mid-dose level and 375 mg/kg bw/d as high-dose level.
- During the course of the premating period, reduced food consumption and body weight loss occurred in female animals of test group 3 (375 mg/kg bw/d). In addition, one female animal of test group 3 was found dead on study day 5 and respiration sounds were observed in 2 male and 2 female animals of the same test group. Therefore, the dose level was reduced to 240 mg/kg bw/d from study day 10 onwards.
- Fasting period before blood sampling for clinical biochemistry: yes. Fasting period: 16 to 20 hours fasting before blood sampling for clinical biochemistry.

Positive control:

not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes (for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity; parturition and lactation behavior of the dams).
- Time schedule: at least once daily.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: prior to the first administration and thereafter at weekly intervals
- Examined parameters: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size.

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period (to randomize the animals), study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning) with the following exceptions for the females:
-- During the premating phase, body weight was determined once a week, i.e. on study days 0, 7 and 13.
-- During the mating period, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
-- Females with litter were weighed on the day of parturition (PND 0), PNDs 4, 7, 10 and 13.
-- Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as were the males.
-- Females without litter and after weaning (PND 13) were weighed once a week.
- The body weight change of the animals was calculated from these results.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
- Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
-- Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
-- Food consumption of the females with evidence of sperm was determined for GD 7, 14 and 20.
-- Food consumption of the females which gave birth to a litter was determined for PND 4, 7, 10 and 13.
- Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

HAEMATOLOGY: Yes.
- Time schedule for collection of blood: at termination (males); at PND 14 (females).
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Yes (about 16-20 hours).
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group.
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes.
- Time schedule for collection of blood: at termination (males); at PND 14 (females).
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Yes (about 16-20 hours).
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group.
- Parameters checked in table 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes; functional observational battery and motor activity assessment (see "OTHER")

OTHER:

FUNCTIONAL OBSERVATIONAL BATTERY (FOB):
- A functional observational battery (FOB) was performed in all animals at the end of the administration period starting at about 10:00 h. At least one hour before the start of the FOB the rats were transferred to single-animal polycarbonate cages type III (floor area of about 800 cm2)) supplied by TECNIPLAST (Hohenpeissenberg, Germany) with wire covers from Ehret (Emmendingen, Germany) and small amounts of bedding material. Drinking water was provided ad libitum, but no food was offered during the measurements. The FOB started with passive observations without disturbing the rats, followed by removal from the home cage, open field observations in a standard arena and sensory motor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
- Examined parameters:
-- Home cage observations (10-30 seconds): posture, tremors, convulsions, abnormal movements, gait, other findings.
-- Open field observations (at least for 2 minutes): behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/ pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/ stereotypes, gait, activity/ arousal level, feces excreted within 2 minutes (appearance/ consistency), urine excreted within 2 minutes (amount/ color), rearing within 2 minutes, other findings.
-- Sensory motor tests/ reflexes: reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory startle response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings.

MOTOR ACTIVITY ASSESSMENT:
- Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed.

THYROID HORMONES: only adult males.
- Time schedule for collection of blood: all males at termination.
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Yes (about 16-20 hours).
- How many animals: all surviving males at termination.
- Parameters checked in table 3 were examined.

Oestrous cyclicity (parental animals):

ESTROUS CYCLE:
- For all females of the pool estrous cycle normality was evaluated before the beginning of the administration period.
- In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear.
- Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all female F0 rats.

Sperm parameters (parental animals):

Parameters examined in parental males:
testis weight, epididymides weight, prostate weight, weight of seminal vesicles with coagulating glands; histopathological examination of epididymides, prostate gland, seminal vesicles, testes; spermatogenic staging profiles.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes.
- If yes, maximum of 4 pubs/sex/litter as nearly as possible; excess pups were killed and discarded.

PUB NUMBER AND STATUS AT DELIVERY:
- All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

PUB VIABILITY/MORTALITY:
- In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. Dead pups were evaluated by the methods, which are described in detail in “Pup Necropsy observations”.
- The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PNDs 1-4, 5-7 and 8-13 were determined. Pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups per litter was calculated on the day of birth (PND 0), and on lactation days 4, 7 and 13. Furthermore, viability and survival indices were calculated.

SEX RATIO:
- On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy.
- The sex ratio was calculated at PND 0 and PND 13.

PUB CLINICAL OBSERVATIONS:
- The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

PUB BODY WEIGHT DATA:
- The pups were weighed on the day after birth (PND 1) as well as on PNDs 4, 7 and 13. Pups' body weight change was calculated from these results.
- The individual weights were always determined at about the same time of the day (in the morning) and on PND 4 immediately before standardization of the litters.
- “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.

NIPPLE/AREOLA ANLAGEN:
- All surviving male pups were examined for the presence of nipple/areola anlagen on PND 13 of the lactation phase. The number of nipple/areola anlagen was counted.

ANOGENITAL DISTANCE:
- Anogenital distance (AGD; defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements was done blind to treatment in a randomized order, using a measuring ocular, on all live male, female and uncertain pups on day 1 after birth.

PUB NECROPSY OBSERVATIONS:
- On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated, and the organs were assessed macroscopically.
- On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for further processing.
- All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically.
- All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

THYROID HORMONES IN PUBS:
- Blood samples were taken from one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia.
- Blood samples from the PND 13 pups were assessed for serum levels for thyroid hormones (T4 and TSH).

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals of the main groups on study day 31.
- Maternal animals: All surviving animals of the main groups on study day 59.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 4 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 (surplus pups after litter standardization) and 13 (remaining pups after litter standardization) days of age.
- These animals were subjected to postmortem examinations (macroscopic and microscopic (if required) as follows: the pups were examined externally and eviscerated, and the organs were assessed macroscopically).

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

HISTOPATHOLOGY
- Thyroid glands/parathyroid glands of one male and one female pup per litter at 13 days of age were fixed in neutral buffered 4% formaldehyde solution for possible further processing.

Statistics:

See table 5

Reproductive indices:

MALE REPRODUCTION DATA:
- The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.
- For the males, mating and fertility indices were calculated for F1 litters (for formulas see "Any other information on materials and methods").

FEMALE REPRODUCTION AND DELIVERY DATA:
- The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females.
- For the females, mating, fertility and gestation indices were calculated for F1 litters (for formulas see "Any other information on materials and methods").

Offspring viability indices:

- viability index, survival index (for formulas see "Any other information on materials and methods").

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Summary clinical observations for males and females (except gestation and lactation periods):
- During the course of the premating period, respiration sounds were observed in male animal Nos. 32 and 33 of test group 3 (375 mg/kg bw/d) starting on study day 9 until study day 17 and 18, respectively. The same was true for female animal Nos. 132 and 138 of the same test group between study days 9 to 13. In addition, male animal No. 31 of test group 3 showed respiration sounds between study days 28 to 31. The findings were assessed to be related to treatment.
- No treatment-related, adverse findings were observed in male and female animals of test groups 1 and 2 (40 and 120 mg/kg bw/d) during the premating, mating and postmating (males only) phases in any test group.
- During pre- and postmating, salivation shortly after treatment was observed in 8 male animals of test group 3 (375 and 240 mg/kg bw/d). From the temporary, short appearance immediately after dosing (or shortly before) it was concluded that this type of finding was induced by a bad taste of the test substance or local affection of the upper digestive tract.
- On premating day 13, female animal No. 122 of test group 2 (120 mg/kg bw/d) showed a mass at the right anogenital region (palpable through skin, size less than 1.5 cm). The finding was assessed to be incidental and not related to treatment.

Summary clinical observations for females during gestation:
- Female animal No. 138 of test group 3 (375 and 240 mg/kg bw/d) showed respiration sounds between gestation days (GD) 0 to 5. From GD 6 onwards, the finding did not occur again. The finding was assessed to be related to treatment.
- Between GD 0 to 5, female animal No. 122 of test group 2 (120 mg/kg bw/d) still showed a mass at the right anogenital region (palpable to rough skin, size less than 1.5 cm). From GD 6 onwards, the finding did not occur again. The finding was assessed to be incidental and
not related to treatment.
- All other females of test groups 0 to 3 (control, 40, 120 as well as 375 and 240 mg/kg bw/d, respectively) did not show any treatment-related or spontaneous findings.

Clinical observations for females during lactation:
- Neither treatment-related nor spontaneous findings were observed in any female animal between postnatal days (PND) 0 to 13.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Female animal No. 140 of test group 3 (375 mg/kg bw/d) was found dead on premating day 5 in the present study. The cause for the premature death of this individual could not be determined after pathological examinations and no other animal died ahead of schedule. Thus, after completion of all related examinations, its occurrence was assessed to be spontaneous in nature and not related to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- No significant changes in body weight parameters, i.e. mean body weights and body weight change values, were observed for any test group (except the significantly lower body weight change value in female animals of test group 3 between premating days 7 to 13).
- However, although a slight mean body weight loss between premating days 0 to 7 could be observed in female animals of any test group including the control, the female animals of test group 3 lost about 10 g within this week. These animals gained weight during the following interval (premating days 7 to 13) during which the dose level was reduced from 375 to 240 mg/kg bw/d. The body weight loss in females of test group 3 (375 mg/kg bw/d) during the first week of application was assessed to be treatment-related and adverse.
- In the following study phases, i.e. the gestation and lactation periods, mean body weight of female animals in test group 3 (375 and 240 mg/kg bw/d) was always below the controls as well as the low- and mid-dose groups. However, the values for mean body weights and body weight change did not differ significantly.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- During the premating period, food consumption was significantly decreased in female animals of test group 3 (375 and 240 mg/kg bw/d). During the gestation and lactation periods, no significant changes in food consumption occurred.
- In male animals of test groups 1 to 3 (40, 120 as well as 375 and 240 mg/kg bw/d, respectively) as well as in female animals of test groups 1 and 2, no changes in food consumption were observed at any time point.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No treatment-related, adverse findings were observed.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- No treatment-related changes among hematological parameters were observed.
- At the end of the administration period, in males of test group 3 (375 and 240 mg/kg bw/d) prothrombin time (Hepatoquick’s test, HQT) was significantly shortened. However, the values were within the historical control range (males, HQT 35.0-40.5 sec). In dams of test group 2 (120 mg/kg bw/d), relative eosinophil counts were significantly increased, but the change was not dose-dependent. Therefore, the mentioned alterations were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

- No treatment-related, adverse changes among clinical chemistry parameters were observed.
- At the end of the administration period, in males of test groups 2 and 3 (120 as well as 375 and 240 mg/kg bw/d, respectively) cholesterol levels were significantly increased, and in dams of test group 3, inorganic phosphate levels were significantly higher compared to controls. Both changes were the only relevant clinical-pathology alterations among these individuals. Therefore, the changes were regarded as treatment-related, but non-adverse (ECETOC Technical Report No. 85, 2002).
- In males of test group 3 (375 and 240 mg/kg bw/d), inorganic phosphate levels were significantly increased, and in females of the same test group, chloride levels were significantly decreased. However, the values were within historical control ranges (males, inorganic phosphate 1.43-2.06 mmol/L; females, chloride 89.6-97.0 mmol/L). Therefore, these alterations were regarded as incidental and not treatment-related.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

- All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and
without any relation to treatment.
- The stages of spermatogenesis in the testes of males of the high dose test group were comparable to those of the controls. In high dose females the different stages of functional bodies in the ovaries were present and comparable to the control animals.
- Premature decedent: Female animal No. 140 of test group 3 (375 and 240 mg/kg bw/d) which died prematurely showed stress-related findings in adrenal cortex (diffuse cortical hyperplasia), glandular stomach (erosion/ulcer), axillary lymph nodes (apoptotic necrosis of lymphoid cells), spleen (PALS decreased), and thymus (starry sky cells increased). Furthermore, there was myeloid hyperplasia in the bone marrow. The cause of death of this animal could not be determined.
- Fertility: Female animal No. 134, which was not pregnant, showed inactive interstitial glands in the ovary and minimal squamous metaplasia in the uterus. Because the changes occurred only in an individual female animal, they were considered to be incidental and not related to treatment. The other female animal (No. 139), which was not pregnant, as well as the male mating partners of these two females (Nos. 34, 39) did not show relevant histopathological findings.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

DETAILED CLINICAL OBSERVATIONS:
- During detailed clinical observations (DCO), in 3 male and in 2 female animals of test group 3 (375 and 240 mg/kg bw/d) respiration sounds were observed. The findings were assessed to be related to treatment.
- Female animal No. 122 of test group 2 (120 mg/kg bw/d) showed a mass at the right anogenital region (palpable through skin, size less than 1.5 cm). The finding was assessed to be incidental and not related to treatment.

FUNCTIONAL OBSERVATIONAL BATTERY:
- Deviations from "zero values" were obtained in quantitative parameters in male and female animals. Without a dose-response relationship or occurred in single animals only, these observations were considered as incidental.
- The following examinations were performed during FOB and are assessed individually:
-- Home cage observations: No test substance-related effects were observed.
-- Open field observations: No test substance-related effects were observed.
-- Sensorimotor tests/reflexes: No test substance-related effects were observed.
-- Quantitative parameters: No test substance-related effects were observed.

MOTOR ACTIVITY MEASUREMENT:
- Regarding the overall motor activity and single intervals, no test substance-related deviations were noted for male and female animals of any test group.

THYROID HORMONES:
- In parental males of test groups 1, 2 and 3 (40, 120 as well as 375 and 240 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

ESTROUS CYCLE:
- Estrous cycle data revealed regular cycles in most of the female animals of all test groups including the control. The mean estrous cycle duration in test groups 0 to 3 was at or between 3.7 to 3.9 days. A treatment-related change regarding the estrous cycle parameters did not occur in any test group.
- Prolonged estrous was observed in a single female animal of test group 3 (375 and 240 mg/kg bw/d), and prolonged diestrous could be observed in 1 animal of test group 3, in 2 animals of test groups 0 and 2 as well as in 3 animals of test group 1. A dose-response relationship did not occur.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The stages of spermatogenesis in the testes of males of the high dose test group were comparable to those of the controls.

Reproductive performance:

no effects observed

Description (incidence and severity):

POSTIMPLANTATION LOSS:
- The postimplantation loss was 6.7% in control group 0, 5.9% in test group 1 (40 mg/kg bw/d), 6.1% in test group 2 (120 mg/kg bw/d) and 6.9% in test group 3 (375 and 240 mg/kg bw/d).
- These values were within the normal range of biological variation inherent in the strain of rats used for this study.

MALE REPRODUCTION DATA:
- No direct, treatment-related changes with regard to the male mating index was observed in any test group.
- The male mating indices calculated after the mating period to produce F1 litter were 100% in test groups 0 to 2.
- The male mating index for test group 3 (375 and 240 mg/kg bw/d) was 88.9% because female animal No. 140 was found dead on premating day 5 and, consequently, mating was not carried out for male animal No. 40.

MALE FERTILITY INDEX:
- Fertility was proven in nearly all of the F0 parental males of test groups 0 to 2 (control; 40 and 120 mg/kg bw/d) within the scheduled mating interval to produce F1 litter. Male animal Nos. 34 and 39 of test group 3 (375 and 240 mg/kg bw/d), which were mated with female Nos. 134 and 139, did not generate F1 pups. No implants were found at necropsy in female
animal No. 139.
- Thus, the male fertility index was 100.0% in test groups 0 and 2, 77.8% in test group 1.
- These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

FEMALE MATING INDEX:
- The female mating index calculated after the mating period for F1 litter was 100% in test groups 0 to 2. For test group 3 the female mating index was 88.9% because animal No. 140 was found dead on premating day 5.
- The mean duration until sperm was detected (GD 0) was 2.8 days for test group 0 (control), 3.1 days for test group 1 (40 mg/kg bw/d), 2.5 days for test group 2 (120 mg/kg bw/d) and test group 3 (375 and 240 mg/kg bw/d).
- These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

FEMALE FERTILITY NDEX:
- All sperm positive rats of test groups 0 to 2 (control; 40 and 120 mg/kg bw/d) delivered pups. Thus, the female fertility index was 100% in test groups 0 and 2. In test group 3 (375 and 240 mg/kg bw/d), all but one (No. 134) female
animals were sperm positive. As female animal No. 139 was sperm positive but had no implants and did not generate pups, the female fertility index was 77.8%.
- These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
- The mean duration of gestation was 22.2 days in test group 0 (control), and 22.1 days in test groups 1 (40 mg/kg bw/d), 2 (120 mg/kg bw/d) and 3 (375 and 240 mg/kg bw/d).

GESTATION INDEX:
- The gestation index was 100% in all test groups.

LIVE BIRTH INDICES:
- The rate live birth indices were 100% in all groups.

PUB NUMBER AND STATUS AT DELIVERY:
- The mean number of delivered F1 pups per dam was 12.3 in control group 0, 11.3 in test group 1 (40 mg/kg bw/d), 12.6 in test group 2 (120 mg/kg bw/d) and 11.4 in test group 3 (375 and 240 mg/kg bw/d).

- The test substance was administered daily by gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 40 mg/kg bw/d (test group 1), 120 mg/kg bw/d (test group 2) and 375 mg/kg bw/d (test group 3). Deionized water served as vehicle, control animals were dosed daily with the vehicle only.
- The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.
- Regarding clinical examinations, obvious signs of general systemic toxicity were observed in female animals of test group 3 (375 mg/kg bw/d) during the premating period when treated at 375 mg/kg bw/d. Food consumption was significantly reduced and body weight loss occurred during the first week of treatment. Respiration sounds were observed in 2 male and 2 female animals of the same test group. Male animal No. 31 of test group 3 showed respiration sounds between study days 28 to 31. These changes were assessed to be related to treatment and adverse. Furthermore, one female animal of test group 3 was found dead on study day 5. Because no explanation for the premature death of this individual could be given after pathological examinations and no other animal died ahead of schedule, its occurrence was assessed to be spontaneous in nature and not related to treatment. However, having the results of the range-finding study in mind together with the occurrence of the mentioned findings and the premature death of one animal during the first days of application, a reduction of the dose level seemed to be necessary. Thus, it was reduced to 240 mg/kg bw/d from study day 10 onwards.
- Fertility indices for male and female animals were not impaired by test-substance administration even at a dose level 240 mg/kg bw/d (formerly 375 mg/kg bw/d). In addition, live birth indices of pups in all test groups were not influenced. The viability index as indicator for pup mortality was not altered.
- Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose level of the compound of 375 and 240 mg/kg bw/d.
- Regarding pathology, no treatment-related findings were identified. All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at this dose.

Dose descriptor:

LOAEL

Remarks:

general systemic toxicity

Effect level:

240 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

food consumption and compound intake

Dose descriptor:

NOAEL

Remarks:

reproductive performance and fertility

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect observed up the highest tested dose.

Critical effects observed:

no

Food consumption and compound intake (if feeding study):

not examined

Remarks on result:

other: not applicable for OECD TG 422

Clinical signs:

no effects observed

Description (incidence and severity):

PUB CLINICAL OBSERVATIONS:
- In each test group including control group some pups died during the lactation period.
- All other F1 pups in test groups 0 to 3 did not show clinical signs up to scheduled sacrifice on PND 4 and PND 13.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

PUB VIABILITY INDEX/MORTALITY:
- The rate of liveborn pups in all test groups was not affected by the test substance, as indicated by live birth indices of 100% in test groups 0, 2 and 3 (control, 120 as well as 375 and 240 mg/kg bw/d). The live birth index in test group 1 (40 mg/kg bw/d) was 89.3% because of two pups of female animal No. 117 died on the day of birth.
- The viability index indicating pup mortality between PND 0 and 4 was 100% in test groups 0 (control) and 1 (40 mg/kg bw/d), 98.3% in test group 2 (120 mg/kg bw/d) and 95.3% in test group 3 (375 and 240 mg/kg bw/d).
- The survival index indicating pup mortality between PND 4 and 13 was 100% in all test groups including test group 3 (375 and 240 mg/kg bw/d).
- The values for test groups 0, 1, 2 and 3 reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

PUB BODY WEIGHT DATA:
- Mean pup body weights and pup body weight change values were similar in all test groups.
- Eleven male runts and seven female runts were found in one litter of test group 2 (120 mg/kg bw/d; female animal No. 125). Two female runts were found in different litters of test groups 0 (control; pup Nos. 109-14 and 110-11) and in one litter of test group 3 (375 and 240 mg/kg bw/d; pup Nos. 133-05 and 133-06).
- A relation to dosing was not observed, test substance-related effects did not occur.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

PUB SEX RATIO:
- In test groups 0, 1, 2 and 3 (control, 40, 120 as well as 375 and 240 mg/kg bw/d, respectively), the sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups. Slight differences were regarded to be spontaneous in nature.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

ANOGENITAL DISTANCE:
- In test groups 1 - 3 (40, 120 as well as 375 and and 240 mg/kg bw/d, respectively), the apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

NIPPLE/AREOLA ANLAGEN
- In test groups 1 - 3 (40, 120 as well as 375 and and 240 mg/kg bw/d, respectively), the apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

PUB NECROPSY OBSERVATION:
- One female pup (No. 119-07) of test group 1 (40 mg/kg bw/d) showed a dilated renal pelvis in the right kidney and one female pup (No. 121-10) of test group 2 (120 mg/kg bw/d) showed a dilated renal pelvis in the left kidney. These findings were assessed to be incidental and not related to the test substance.
- One male (No. 133-07) and one female pup (No. 135-12) of test group 3 (375 and 240 mg/kg bw/d) were cannibalized and could not be found in the respective cage on PND 1.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

THYROID HORMONES:
- In male and female pubs of all dosing groups, no treatment-related alterations of T4 and TSH levels were observed.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to the highest tested dose.

Critical effects observed:

no

Remarks on result:

other: not applicable for OECD TG 422

Reproductive effects observed:

no

Table 6: Male fertility indices for F0 males

 

	Test group 0 (0 mg/kg bw/d)	Test group 1 (40 mg/kg bw/d)	Test group 2 (120 mg/kg bw/d)	Test group 3 (375 and 240 mg/kgbw/d)
Male fertility index [%]	100.0	100.0	100.0	77.8

* p≤0.05; ** p≤0.01

 

Table 7:Female fertility index

 

	Test group 0 (0 mg/kg bw/d)	Test group 1 (40 mg/kg bw/d)	Test group 2 (120 mg/kg bw/d)	Test group 3 (375 and 240 mg/kgbw/d)
Female fertility index [%]	100.0	100.0	100.0	77.8

* p≤0.05; ** p≤0.01

 

Table 8: Sex ratio of live F1 pups

 

PND 0	Test group 0 (0 mg/kg bw/d)	Test group 1 (40 mg/kg bw/d)	Test group 2 (120mg/kg bw/d)	Test group 3 (375 and 240 mg/kgbw/d)
Live males [%]	52.8	51.3	52.2	55.8
Live females [%]	47.2	48.7	47.8	44.2
PND 13	Test group 0 (0 mg/kg bw/d)	Test group 1 (40 mg/kg bw/d)	Test group 2 (120mg/kg bw/d)	Test group 3 (375 and 240 mg/kgbw/d)
Live males [%]	48.8	50.0	51.2	57.1
Live females [%]	51.2	50.0	48.8	42.7

 

Table 9:Relative organ weights

 

	Female animals
Test group (mg/kg bw/d)	1 (40)	2 (120)	3 (375/240)
Spleen	97%	84%*	90%

* p≤0.05; ** p≤0.01

 

Table 10:Summary - Clinical Observation, males, pre-mating

 

			Test group 0 (0 mg/kg bw/d)	Test group 1 (40 mg/kg bw/d)	Test group 2 (120 mg/kg bw/d)	Test group 3 (375 and 240 mg/kgbw/d)
Day 0àday 31	Animals examined	N	10	10	10	10
	Animals with signs	N	0	0	0	8
	Headsalivation	N	0	0	0	8
	Dead Sacrificed scheduled	N	10	10	10	10
	Normal NAD	N	10	10	10	10
	Reapiration sounds	N	0	0	0	3

 

Table 11:Summary - Clinical Observation, females, pre-mating

 

			Test group 0 (0 mg/kg bw/d)	Test group 1 (40 mg/kg bw/d)	Test group 2 (120 mg/kg bw/d)	Test group 3 (375 and 240 mg/kgbw/d)
Day 0àday 13	Animals examined	N	10	10	10	10
	Animals with signs	N	0	0	0	2
	Dead Found dead	N	0	0	0	1
	Normal NAD	N	10	10	10	10
	Mass Palpable through skin	N	0	0	1	0
	Reapiration sounds	N	0	0	0	2

 

 

Table 12:Summary – Food consumption, females, in-life

 

		Test Group0/F 0 mg/kgbw/d	Test Group1/F 40 mg/kgbw/d	Test Group2/F 120mg/kgbw/d	Test Group3/F 375/240 mg/kgbw/d
d 0 -> 7	Mean [g]	16.1 n	15.4	16.0	13.3**
	S.d.	1.2	1.3	0.4	1.4
	N	5	5	5	5
	Deviation Vs Control [%]		-4.7	-0.9	-17.6
d 7 -> 13	Mean [g]	15.2 n	14.7	14.2	12.8*
	S.d.	1.6	1.4	0.2	1.0
	N	5	5	5	5
	Deviation Vs Control [%]		-3.2	-6.9	-16.1

Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

d= day; n=DUNNETT

 

Table 13:Summary – Body Weights - BW / Body Weights [g], females, in-life

 

		Test Group0/F 0 mg/kgbw/d	Test Group1/F 40 mg/kgbw/d	Test Group2/F 120mg/kgbw/d	Test Group3/F 375/240 mg/kgbw/d
d 0	Mean [g]	231.6n	227.5	230.2	119.2
	S.d.	13.0	15.8	14.5	10.1
	N	10	10	10	10
	Deviation Vs Control [%]		-1.8	-0.6	-1.0
d 7	Mean [g]	230.9n	225.7	227.7	219.3
	S.d.	12.1	10.5	11.6	6.1
	N	10	10	10	9
	Deviation Vs Control [%]		-2.3	-1.4	-5.0
d 13	Mean [g]	234.1n	228.5	231.7	225.0
	S.d.	14.5	9.9	10.5	7.1
	N	10	10	10	9
	Deviation Vs Control [%]		-2.4	-1.0	-3.9

Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

n=DUNNETT

 

Table 14: Summary Mating Report

 

		Test Group 0/F 0 mg/kgbw/d		Test Group 1/F 40 mg/kgbw/d	Test Group 2/ F 120 mg/kgbw/d		Test Group 3/F 240 mg/kgbw/d
No. of females mated	N	10		10	10	9
- Inseminated	N	10	f-	10	10	8
Female mating index	%	100.0		100.0	100.0	88.9
-- Pregnant	N	10	f-	10	10	7
Female fertility index	%	100.0		100.0	100.0	87.5
No. of males mated	N	10		10	10	9
- With inseminated females	N	10	f-	10	10	8
Male mating index	%	100.0		100.0	100.0	88.9
- With pregnant females	N	10	f-	10	10	7
Male fertility index	%	100.0		100.0	100.0	77.8
Females with defined Day 0 pc	N	10		10	10	8
Mating days until Day 0 pc	Mean	2.8	x+	3.1	2.5	2.5
	S.d.	1.4		1.5	1.1	1.7
	N	10		10	10	8
Days 0 To 4	N	9		9	10	7
	%	90.0		90.0	100.0	87.5
Days 5 To 9	N	1		1	0	1
	%	10.0		10.0	0.0	12.5
Days 10 To 14	N	0		0	0	0
	%	0.0		0.0	0.0	0.0

Statistic Profile = Fisher's exact test (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics f=FISHER-EXACT; x=WILCOX

 

 

Table 15: Summary Pregnancy Status Report – Reproduction

 

		Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 40 mg/kgbw/d	Test Group 2/ F 120 mg/kgbw/d		Test Group 3/F 240 mg/kgbw/d
No. of females at start	N	10	10	10	9
No. of females at mated	N	10	10	10	9
Without evidence og mating	N	0	0	0	1
- Pregnant	N	0	0	0	0
- Not pregnant	N	0	0	0	1
Females with defined Day 0 pc	N	10	10	10	8
Pregnant	N	10	10	10	7
- sacrificed scheduled	N	10	10	10	7
Not pregnant	N	0	0	0	2
- sacrificed scheduled	N	0	0	0	2
Pregnant, not delivering	N	0	0	0	0
Delivering	N	10	10	10	7
-- With liveborn pubs	N	10	10	10	7
	%	100.0	100.0	100.0	100.0
-- With all pubs stillborn	N	0	0	0	0
	%	0.0	0.0	0.0	0.0

 

 

Table 16: Summary Delivery Report

 

		Test Group 0/F 0 mg/kgbw/d		Test Group 1/F 40 mg/kgbw/d	Test Group 2/ F 120 mg/kgbw/d		Test Group 3/F 240 mg/kgbw/d
No. of females at start	N	10		10	10	9
No. of females at mated	N	10	f-	10	10	9
	%	100.0		100.0	100.0	100.0
Pregnant	N	10	f-	10	10	7
	%	100.0		100.0	100.0	77.8
Dead	N	10		10	10	9
Without Delivery	N	0		0	0	2
- Pregnant	N	0		0	0	0
- Not pregnant	N	0		0	0	2
-- Delivering	N	10	f-	10	10	7
	%	100.0		100.0	100.0	100.0
-- With liveborn pubs	N	10	f-	10	10	7
Gestation Index	%	100.0		100.0	100.0	100.0
Gestation days	Mean	22.2	n	22.1	22.1	22.1
	S.d.	0.4		0.3	0.6	0.4
	%	10		10	10	7
-- With stillborn pubs	N	0		1	0	0
	%	0.0	f+	10.0	0.0	0.0
-- With all pubs stillborn	N	0		0	0	0
	%	0.0	f+	0.0	0.0	0.0

Statistic Profile = Fisher's exact test (one-sided-), Dunnett test (two-sided), Fisher's exact test (one-sided+), *p<=0.05, ** p <=0.01, X = Group excluded from statistics

F=FISHER-EXACT; n=DUNNETT

 

 

Table 17: Summary Litter Report

 

		Test Group 0/F 0 mg/kgbw/d		Test Group 1/F 40 mg/kgbw/d	Test Group 2/ F 120 mg/kgbw/d		Test Group 3/F 240 mg/kgbw/d
Total Number of Pregnant Females	N	10		10	10	7
Total number of litters	N	10		10	10	7
With liveborn pubs	N	10	f-	10	10	7
	%	100.0		100.0	100.0	100-0
With stillborn pubs	N	0	f+	1	0	0
	%	0.0		10.0	10.0	0.0
With all pubs stillborn	N	0	f+	0	0	0
	%	0.0		0.0	0.0	0.0
Implantation Sites	N	131		122	134	86
	Mean	13.1	x-	12.2	13.4	12.3
	S.d.	1.9		1.8	1.8	1.5
	N	10		10	10	7
Pubs delivered	N	122		115	126	80
	Mean	12.2	x-	11.5	12.6	11.4
	S.d.	2.2		2.1	2.3	2.3
	N	10		10	10	7
Postimplantation Loss	Mean%	6.7	x+	5.9	6.1	6.9
	S.d.	11.0		7.4	9.4	15.5
	N	10		10	10	7
Pubs liveborn	N	122		113	126	80
	%%	100.0		98.3	100.0	100.0
	Mean	12.2	x-	11.3	12.6	11.4
	S.d.	2.2		2.2	2.3	2.3
	N	10		10	10	7

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-),Fisher's exact test (one-sided+), *p<=0.05, ** p <=0.01, X = Group excluded from statistics

F=FISHER-EXACT; n=DUNNETT

 

Table 18: Summary Litter Report – Pup status

 

		Test Group 0/F 0 mg/kgbw/d		Test Group 1/F 40 mg/kgbw/d	Test Group 2/ F 120 mg/kgbw/d		Test Group 3/F 240 mg/kgbw/d
Pubs stillborn	N	0		2	0	0
	%	0.0		1.7	0.0	0.0
	Mean	0.0	x+	0.2	0.0	0.0
	S.d.	0.0		0.6	0.0	0.0
	N	10		10	10	7
Perinatal Loss	Mean%	0.0	x+	1.8	0.0	0.0
	S.d.	0.0		5.7	0.0	0.0
	N	10		10	10	7

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-),Fisher's exact test (one-sided+), *p<=0.05, ** p <=0.01, X = Group excluded from statistics

F=FISHER-EXACT; n=DUNNETT

 

 

Table 19: Summary Litter Report - Dead Pups

 

		Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 40 mg/kgbw/d	Test Group 2/ F 120 mg/kgbw/d		Test Group 3/F 240 mg/kgbw/d
Litters with liveborn pups	N	10	10	10	7
Pubs delivered	N	122	115	126	8
found dead [pup] / Dead	N	0	0	2	1
	%	0.0	0.0	1.6	1.2
Stillborn / Dead	N	0	2	0	0
	%	0.0	1.7	0.0	0.0
Alive / Alive	N	122	113	126	80
	%	100.0	98.3	100.0	100.0
cannibalizing [pup] / Dead	N	1	0	0	2
	%	0.8	0.0	0.0	2.5
sacrificed scheduled [pup] / Dead	N	79	80	80	54
	%	64.8	69.6	63.5	67.5
Culled / Dead	N	42	33	44	23
	%	34.4	28.7	34.9	28.8
Litters not surviving Day 13	N	0	0	0	0
	%	0.0	0.0	0.0	0.0

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-),Fisher's exact test (one-sided+), *p<=0.05, ** p <=0.01, X = Group excluded from statistics

 

 

Table 20: Summary Litter Report - Pups Died

 

		Test Group 0/F 0 mg/kgbw/d		Test Group 1/F 40 mg/kgbw/d	Test Group 2/ F 120 mg/kgbw/d	Test Group 3/F 240 mg/kgbw/d
Litters with liveborn pups	N	10		10	10	7
Pubs delivered	N	122		115	126	80
Day 0 To 0	N	0		0	0	0
	%	0		0	0	0
Day 1 To 4	N	0		0	2	3
	%	0		0	1.6	3.8
Day 5 To 7	N	0		0	0	0
	%	0		0	0	0
Day 8 To 13	N	0		0	0	0
	%	0		0	0	0
Pups surviving days 0 To 4	N	123		113	124	77
Viability Index	Mean%	100.0	x-	100	98.3	95.3
	S.d.	0.0		0.0	3.8	6.1
	N	10		10	10	7
Pups surviving days 4 To 13	N	81		80	80	54
Viability Index	Mean%	100.0	NA	100.0	100.0	100.0
	S.d.	0.0		0.0	0.0	0.0
	N	10		10	10	7

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-),Fisher's exact test (one-sided+), *p<=0.05, ** p <=0.01, x =WILCOX; NA = No Test Applicable

 

 

Table 21: Summary Pup Clinical Observation during Lactation

 

Males			Test Group 0/M 0 mg/kgbw/d	Test Group 1/M 40 mg/kgbw/d	Test Group 2/ M 120 mg/kgbw/d		Test Group 3/M 240 mg/kgbw/d
Day 0 -->13	Animals examined	N	65	60	66	42
	normal NAD	N	65	59	66	42
Females			Test Group 0/ F 0 mg/kg bw/d	Test Group 1/ F 40 mg/kg bw/d	Test Group 2/ F 120 mg/kg bw/d	Test Group 3/ F 240 mg/kg bw/d
Day 0 -->13	Animals examined	N	57	55	60	38
	Animals with signs	N	1	1	1	2
	normal NAD	N	57	54	60	38
	reproduction not assessed (died)	N	1	1	1	2

 

 

Table 22: Summary Pup Report Body Weights - BW / Body Weights [g]

 

		Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 40 mg/kgbw/d	Test Group 2/ F 120 mg/kgbw/d	Test Group 3/F 240 mg/kgbw/d
day 1 Runt	Males	0	0	11	2
	Females	2	0	7	0
day 1 Males	Mean	7.0 n	6.9	6.8	6.8
	S.d.	0.6	0.4	1.0	0.7
	N	10	10	10	7
	Deviation Vs Control [%]		-0.7	-3.0	-2.5
day 1 Females	Mean	6.5 n	6.5	6.3	6.7
	S.d.	0.7	0.4	1.0	0.7
	N	10	10	10	6
	Deviation Vs Control [%]		0.0	-3.0	2.5
day 1 Males + Females	Mean	6.8 n	6.7	6.5	6.7
	S.d.	0.7	0.4	1.0	0.7
	N	10	10	10	7
	Deviation Vs Control [%]		-0.4	-3.3	-1.0
day 4 Males	Mean	10.7 n	10.6	10.3	10.4
	S.d.	1.3	0.8	1.7	1.1
	N	10	10	10	7
	Deviation Vs Control [%]		-0.8	-3.9	-3.1
day 4 Females	Mean	10.1 n	10.2	9.7	10.4
	S.d.	1.4	0.9	1.7	1.2
	N	10	10	10	6
	Deviation Vs Control [%]		1.0	-3.8	2.2
day 4 Males + Females	Mean	10.4 n	10.4	10.0	10.3
	S.d.	1.3	0.8	1.7	1.1
	N	10	10	10	7
	Deviation Vs Control [%]		-0.1	-4.2	-1.5
	Mean	17.4 n	17.1	16.7	16.5
day 7 Males	S.d.	1.5	1.3	2.8	1.8
	N	10	10	10	7
	Deviation Vs Control [%]		-1.3	-3.7	-4.9
	Mean	16.2 n	16.4	16.0	16.4
day 7 Females	S.d.	1.6	1.4	2.4	1.7
	N	10	10	10	6
	Deviation Vs Control [%]		1.8	-1.0	1.3
	Mean	16.8 n	16.8	16.4	16.3
day 7 Males + Females	S.d.	1.5	1.3	2.6	1.7
	N	10	10	10	7
	Deviation Vs Control [%]		0.2	-2.3	-2.6
	Mean	32.3 n	31.9	31.1	31.2
day 13 Males	S.d.	1.9	1.8	3.6	2.1
	N	10	10	10	7
	Deviation Vs Control [%]		-1.0	-3.5	-3.2
	Mean	30.7 n	31.0	30.2	30.8
day 13 Females	S.d.	2.4	1.9	2.8	2.0
	N	10	10	10	6
	Deviation Vs Control [%]		1.0	-1.4	0.5
	Mean	31.5 n	31.5	30.7	31.0
day 13 Males + Females	S.d.	2.1	1.8	3.2	2.0
	N	10	10	10	7
	Deviation Vs Control [%]		0.0	-2.4	-1.6

Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

n=DUNNETT

 

 

Table 23: Summary Pup Report AG Distance + AG Index of males

 

		Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 40 mg/kgbw/d	Test Group 2/ F 120 mg/kgbw/d	Test Group 3/F 240 mg/kgbw/d
AG Distance	Mean	2.97 n	2.99	2.91	2.91
[mm]	S.d.	0.29	0.27	0.16	0.15
day 1 Males	N	10	10	10	7
AG Index Cubic Root	Mean	1.56 n	1.57	1.54	1.54
[---]	S.d.	0.14	0.16	0.06	0.07
day 1 Males	N	10	10	10	7
AG Distance	Mean	1.47 n	1.45	1.44	1.51
[mm]	S.d.	0.08	0.04	0.08	0.14
day 1 Females	N	10	10	10	6
AG Index Cubic Root	Mean	0.79 n	0.78	0.78	0.80
[---]	S.d.	0.04	0.02	0.02	0.07
day 1 Females	N	10	10	10	6

Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

n=DUNNETT

 

 

Table 24: Summary Pup Report Nipple development

 

		Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 40 mg/kgbw/d	Test Group 2/ F 120 mg/kgbw/d	Test Group 3/F 240 mg/kgbw/d
Nipple development	Passed
[lncidence]	-N	25	21	31	16
day 13 Males	-%	64	52	76	53
	Failed
	-N	14	19	10	14
	-%	36	48	24	47
Nipple development [%]	Mean	62.50 x+	51.17	75.00	52.38
[%]	S.d.	37.73	39.85	23.57	38.70
day 13 Males	N	10	10	10	7
Nipple Number	Mean	1.30 x+	1.08	1.46	1.19
[%]	S.d.	1.09	1.11	0.70	0.90
day 13 Males	N	10	10	10	7

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

x=WILCOX

 

 

Table 25: Summary - Pup Necropsy Observation

 

Males			Test Group 0/M 0 mg/kgbw/d	Test Group 1/M 40 mg/kgbw/d	Test Group 2/ M 120 mg/kgbw/d		Test Group 3/M 240 mg/kgbw/d
Day 0 -->13 With unscheduled data	Animals examined	N	65	60	66	42
	Animals with signs	N	0	0	0	1
		%	0.0	0.0	0.0	2.4
	General Not assessed	N	0	0	0	1
		%	0.0	0.0	0.0	2.4
	normal NAD	N	65	60	66	41
		%	100.0	100.0	100.0	97.6
Females			Test Group 0/ F 0 mg/kg bw/d	Test Group 1/ F 40 mg/kg bw/d	Test Group 2/ F 120 mg/kg bw/d	Test Group 3/ F 240 mg/kg bw/d
Day 0 -->13 With unscheduled data	Animals examined	N	57	55	60	38
	Animals with signs	N	0	1	1	1
		%	0.0	1.8	1.7	2.6
	Renal pelvis dilated	N	0	1	1	0
		%	0.0	1.8	1.7	0.0
	General Not assessed	N	0	0	0	1
		%	0.0	0.0	0.0	2.6
	normal NAD	N	57	55	60	37
		%	100.0	100.0	100.0	97.4

 

 

Conclusions:

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of the test substance to male and female Wistar rats revealed signs of systemic toxicity at a dose level of 375 mg/kg bw/d taking clinical findings in male and female animals into account.
Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 120 mg/kg bw/d for male for female Wistar rats.
The NOAEL for reproductive performance and fertility was also set to 240 mg/kg bw/d for male and female Wistar rats.
The NOAEL for developmental toxicity was 240 mg/kg bw/d.

Executive summary:

In a GLP-compliant OECD TG 422-study, the test substancewas administered daily by gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 40 mg/kg bw/d (test group 1), 120 mg/kg bw/d (test group 2) and 375 mg/kg bw/d (test group 3). Deionized waterserved as vehicle, control animals were dosed daily with the vehicle only.

The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

 During the course of the premating period, reduced food consumption and body weight loss occurred in female animals of test group 3 (375 mg/kg bw/d). In addition, one female animal of test group 3 was found dead on study day 5 and respiration sounds were observed in 2 male and 2 female animals of the same test group starting on study day 9. Therefore, the dose level was reduced to 240 mg/kg bw/d from study day 10 onwards.

 

Observations

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances.

F0 animals were mated for a maximum of two weeks after the beginning of treatment to produce a litter (F1 generation pups). As soon as sperm was detected in the vaginal smear, mating was discontinued. F0 animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the postimplantation loss in all F0 females.

A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals.

Food consumption of the F0 parents was determined regularly once weekly before and after the mating period, as well as in dams during gestation days 7, 14 and 20 and lactation days 4, 7, 10 and 13.

In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, and on postnatal days (PND) 0, 4, 7, 10 and 13.

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PNDs 1, 4, 7 and on PND 13 and their viability was recorded. On day 1 after birth the anogenital distance (AGD) was determined on all live male, female and uncertain pups. On PND 4, the individual litters were standardized in such a way that, whenever possible, each litter contains 4 male and 4 female pups (as a rule, the first 4 surviving pups/sex in each litter were taken for further rearing). On PND 13, all male F1 pups were examined for retention of nipples/areolae. The number of nipples/areolae anlagen were counted.

At necropsy on PNDs 4 and 13, all pups were sacrificed with CO₂under isoflurane anesthesia and examined macroscopically for external and visceral findings. Blood samples were taken from all surplus pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally, blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and transferred to the Laboratory Pathology for possible further processing.

Towards the end of the administration period, a functional observational battery was performed, and motor activity was measured in 5 parental animals per sex and test group. Clinico-chemical and hematological examinations were performed in 5 parental animals per sex and group.

All F0 parental animals were sacrificed by decapitation under isoflurane anesthesia and were assessed by gross pathology. Weights of selected organs were recorded, and a histopathological examination was performed.

 

Results

Analyses:

The various analyses confirmed

• the stability of the test-substance preparations for a period of at least 7 days at room temperature,
• the homogeneous distribution of the test substance in the vehicle,
• the correctness of the prepared concentrations.

Effects:

The following test substance-related, relevant findings were noted:

 

Test group 3: 375 mg/kg bw/d (from premating day 0 to 9) and 240 mg/kg bw/d (from premating day 10 onwards)

 F0 PARENTAL ANIMALS

Clinical Examinations

• Starting on premating day 9, respiration sounds were observed in 2 male animals (finding was observable until study days 17 and 18). The same was true for 2 female animals between study days 9 to 13. In addition, another male animal showed respiration sounds between study days 28 to 31.
• Significantly reduced food consumption was observed in female animals during the first week of application, i.e. -18%, as well as between premating days 7-13, i.e. -16%.
• Body weight loss by -10 g (maximum value) was observed in female animals during the first week of application. Between premating days 7-13, the animals gained weight again but were still below their starting weight at premating day 0.

Reproductive Performance, Clinical Pathology and Pathology

• No treatment-related, adverse effects were observed¹⁾.

F1 PUPS

Clinical Examinations/ Gross Findings

•  No treatment-related, adverse effects were observed.

 Testgroup 2: 120 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical Examination, Reproductive Performance, Clinical Pathology and Pathology

• No treatment-related, adverse effects were observed.

F1 PUPS

Clinical Examinations/ Gross Findings

• No treatment-related, adverse effects were observed.

Test group 1: 40 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical Examination, Reproductive Performance, Clinical Pathology and Pathology

•  No treatment-related, adverse effects were observed.

F1 PUPS

Clinical Examinations/ Gross Findings

• No treatment-related, adverse effects were observed.

Conclusion

Under the conditions of thisCombined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage ofN,N-Dimethylisopropanolamine to male and female Wistar ratsrevealed signs of systemic toxicity at a dose level of 375 mg/kg bw/d taking clinical findings in male and female animals into account.

Thus,the no observed adverse effect level (NOAEL) forgeneral systemic toxicitywas 120 mg/kg bw/d for male for female Wistar rats.

The NOAELforreproductive performanceandfertilitywas also set to 240 mg/kg bw/d formale and female Wistar rats.

TheNOAELfordevelopmental toxicitywas 240 mg/kg bw/d.

 

¹⁾Note: One female animal of test group 3 was found dead on study day 5. Because no explanation for the premature death of this individual could be given after pathological examinations and no other animal died during the administration period, its occurrence was assessed to be spontaneous in nature and not related to treatment

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

240 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a GLP-compliant OECD 422 -study, the test substancewas administered daily by gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 40 mg/kg bw/d (test group 1), 120 mg/kg bw/d (test group 2) and 375 mg/kg bw/d (test group 3). Deionized waterserved as vehicle, control animals were dosed daily with the vehicle only.

The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

During the course of the premating period, reduced food consumption and body weight loss occurred in female animals of test group 3 (375 mg/kg bw/d). In addition, one female animal of test group 3 was found dead on study day 5 and respiration sounds were observed in 2 male and 2 female animals of the same test group starting on study day 9. Therefore, the dose level was reduced to 240 mg/kg bw/d from study day 10 onwards.

 

Observations

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances.

F0 animals were mated for a maximum of two weeks after the beginning of treatment to produce a litter (F1 generation pups). As soon as sperm was detected in the vaginal smear, mating was discontinued. F0 animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the postimplantation loss in all F0 females.

A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals.

Food consumption of the F0 parents was determined regularly once weekly before and after the mating period, as well as in dams during gestation days 7, 14 and 20 and lactation days 4, 7, 10 and 13.

In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, and on postnatal days (PND) 0, 4, 7, 10 and 13.

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PNDs 1, 4, 7 and on PND 13 and their viability was recorded. On day 1 after birth the anogenital distance (AGD) was determined on all live male, female and uncertain pups. On PND 4, the individual litters were standardized in such a way that, whenever possible, each litter contains 4 male and 4 female pups (as a rule, the first 4 surviving pups/sex in each litter were taken for further rearing). On PND 13, all male F1 pups were examined for retention of nipples/areolae. The number of nipples/areolae anlagen were counted.

At necropsy on PNDs 4 and 13, all pups were sacrificed with CO2under isoflurane anesthesia and examined macroscopically for external and visceral findings. Blood samples were taken from all surplus pups or 2 preferably female pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally, blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and transferred to the Laboratory Pathology for possible further processing.

Towards the end of the administration period, a functional observational battery was performed, and motor activity was measured in 5 parental animals per sex and test group. Clinico-chemical and hematological examinations were performed in 5 parental animals per sex and group.

All F0 parental animals were sacrificed by decapitation under isoflurane anesthesia and were assessed by gross pathology. Weights of selected organs were recorded, and a histopathological examination was performed.

 

Results

Analyses

The various analyses confirmed:

·        the stability of the test-substance preparations for a period of at least 7 days at room temperature,

·        the homogeneous distribution of the test substance in the vehicle,

·        the correctness of the prepared concentrations.

 

Effects

The following test substance-related, relevant findings were noted:

 

Test group 3: 375 mg/kg bw/d (from premating day 0 to 9) and 240 mg/kg bw/d (from premating day 10 onwards)

F0 PARENTAL ANIMALS

Clinical Examinations

·        Starting on premating day 9, respiration sounds were observed in 2 male animals. The finding was observable until study days 17 and 18.

·        Significantly reduced food consumption was observed in female animals during the first week of application, i.e. -18%, as well as between premating days 7-13, i.e. -16%.

·        Body weight loss by -10 g was observed in female animals during the first week of application. Between premating days 7-13, the animals gained weight again but were still below their starting weight at premating day 0.

Reproductive Performance, Clinical Pathology and Pathology

·        No treatment-related, adverse effects were observed(One female animal of test group 3 was found dead on study day 5. Because no explanation for the premature death of this individual could be given after pathological examinations and no other animal died ahead of schedule, its occurrence was assessed to be spontaneous in nature and not related to treatment)

F1 PUPS

Clinical Examinations/ Gross Findings

·        No treatment-related, adverse effects were observed.

 

Testgroup 2: 120 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical Examination, Reproductive Performance, Clinical Pathology and Pathology

·        No treatment-related, adverse effects were observed.

F1 PUPS

Clinical Examinations/ Gross Findings

·        No treatment-related, adverse effects were observed.

 

Test group 1: 40 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical Examination, Reproductive Performance, Clinical Pathology and Pathology

·        No treatment-related, adverse effects were observed.

F1 PUPS

Clinical Examinations/ Gross Findings

·        No treatment-related, adverse effects were observed.

 

Conclusion

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of the test substance to male and female Wistar rats revealed signs of systemic toxicity at a dose level of 375 mg/kg bw/d taking clinical findings in male and female animals into account.

Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 120 mg/kg bw/d for male for female Wistar rats.

The NOAEL for reproductive performance and fertility was also set to 240 mg/kg bw/d for male and female Wistar rats.

The NOAEL for developmental toxicity was 240 mg/kg bw/d.

Effects on developmental toxicity

Description of key information

Under the conditions of a combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD TG 422, GLP), the oral administration of the test item by gavage to Wistar rats caused no signs of developmental toxicity up to the highest tested dose. Thus, the NOAEL for developmental toxicity was 240 mg/kg bw/d.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Remarks:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, D-55116 Mainz

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch number of test material:
A029-2018.
- Expiration date of the lot/batch:
16 Oct 2019.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Ambient (room temperature).
- Stability under storage conditions:
The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor.
- Stability under test conditions:
The stability of the test substance in deionized water over a period of 7 days wasproven. As the test substance preparations were stored no longer than this time period, the stability was guaranteed.
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium:
Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that the test substance was distributed homogeneously in deionized water.

FORM AS APPLIED IN THE TEST: Test substance preparations in deionized water.

Species:

rat

Strain:

Wistar

Remarks:

Wistar Rat; Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Charles River Laboratories, Research Models and Services, Sulzfeld, Germany.
- Females nulliparous and non-pregnant: yes.
- Age at study initiation: About 11 - 12 weeks (male animals), about 10 weeks (female animals).
- Weight at study initiation: 385.7 (male), 229.6 (female).
- Fasting period before study:no.
- Housing: During pre-treatment: Polysulfonate cages Typ 2000P (H-Temp), floor area about 2065 cm2 (610 x 435 x 215 mm); supplied by TECHNIPLAST, Hohenpeißenberg, Germany. During pre-mating, mating, gestation, lactation, males after mating and females after weaning: Polycarbonate cages type III. For motor activity (MA) measurements the animals were housed individually in polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany, with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2) and small amounts of bedding material. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. Wooden gnawing blocks (Lignocel Block Large) were supplied by J. Rettenmaier & Söhne GmbH + Co. KG, Rosenberg, Germany, and used for environmental enrichment.
- Diet: ad libitum. The food used was ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA (new name Granovit AG), Kaiseraugst, Switzerland.
- Water: ad libitum.
- Acclimation period: 28 days.

DETAILS OF FOOD AND WATER QUALITY:
- On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants, the diet was found to be suitable. Fed. Reg. Vol. 44, No. 91 of 09 May 1979, p. 27354 (EPA), served as a guideline for maximum tolerable chemical contaminants. The number of microorganisms did not exceed 1*10^5/g food.
- On the basis of the analytical findings the drinking water was found to be suitable. German “Trinkwasserverordnung” (Drinking Water Regulation) served as a guideline for maximum tolerable contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C.
- Humidity (%): 45-65%.
- Air changes (per hr): 15.
- Photoperiod (hrs dark / hrs light): 12 hours (12 hours light from 06.00-18.00 h, 12 hours dark from 18.00-06.00 h).

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
- Test substance preparations in deionized water.

EXPOSURE
- After the acclimatization period, the test substance was administered orally via gavage to the F0 generation parental animals, daily at the same time in the morning (exception: no administration to animals being in labor). The treatment lasted up to one day prior to sacrifice. The animals of the control group were treated in the same way with the vehicle only (deionized water). The calculation of the administered volume was generally based on the most recent individual body weights.

VEHICLE
- Concentration in vehicle: 0, 0.40, 1.20, 3.75/2.40 g/100 mL.
- Amount of vehicle (if gavage): 10 mL/kg bw/d.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that the test material was distributed homogenously in deionized water. The mean values and single values of the test substance in deionized water were found to be in a range of 90%-110% of the nominal concentrations. The mean values of samples 11-13 was found to be 136.7% of the nominal concentrations and therefore did not meet the specification of the test facility.

Details on mating procedure:

- M/F ratio per cage: 1:1.
- Length of cohabitation: overnight (from about 16.00 h until 06.30 - 09.00 h of the following morning) for a maximum of 2 weeks.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy (gestation day (GD) 0)
- After successful mating each pregnant female was caged (how): Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.

Duration of treatment / exposure:

Males: 31 days, females: 59 days.
The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

Frequency of treatment:

Daily at the same time in the morning (exception: no administration to animals being in labor).

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Remarks:

High dose from study day 10 onwards.

Dose / conc.:

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

Remarks:

Initial high dose up to day 9.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A test study was performed beforehand to select proper dose levels for the present OECD 422 study. The test substance was administered daily by gavage to groups of 4 male and 4 female Wistar rats at dose levels of 0 (test group 0), 100 (test group 1), 250 (test group 2) and 750 mg/kg bw/d (test group 3) over a period of 2 weeks. After the second administration, the following findings occurred in test group 3 (750 mg/kg bw/d): All male animals showed piloerection and semi-closed eyelids, three male animals showed twitching, and one male animal showed tremors. In females, semi-closed eyelids occurred in all animals, hypothermia and poor general condition was observed in 2 female animals. Body weight loss was determined for all male and female animals between study days 0 and 2. All animals of test group 3 (750 mg/kg bw/d) had to be sacrificed in a moribund condition on study day 2. At necropsy, all male and female animals showed foci in the glandular stomach and thickening of the duodenum wall. One male and one female also showed foci in the forestomach and in the jejunum. Peyer’s patches were enlarged in all male and one female animal. The male and female animals of test groups 0, 1 and 2 (control, 100 and 250 mg/kg bw/d, respectively) were sacrificed as scheduled after 2 weeks of treatment. In test group 2 (250 mg/kg bw/d), one male animal showed enlarged Peyer’s patches at necropsy. Based on these results, the following dose levels were selected for the present study: 40 mg/kg bw/d as low-dose level, 120 mg/kg bw/d as mid-dose level and 375 mg/kg bw/d as high-dose level.
- During the course of the premating period, reduced food consumption and body weight loss occurred in female animals of test group 3 (375 mg/kg bw/d). In addition, one female animal of test group 3 was found dead on study day 5 and respiration sounds were observed in 2 male and 2 female animals of the same test group. Therefore, the dose level was reduced to 240 mg/kg bw/d from study day 10 onwards.
- Fasting period before blood sampling for clinical biochemistry: yes. Fasting period: 16 to 20 hours fasting before blood sampling for clinical biochemistry.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes (for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity; parturition and lactation behavior of the dams).
- Time schedule: at least once daily.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: prior to the first administration and thereafter at weekly intervals
- Examined parameters: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size.

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period (to randomize the animals), study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning) with the following exceptions for the females:
-- During the premating phase, body weight was determined once a week, i.e. on study days 0, 7 and 13.
-- During the mating period, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
-- Females with litter were weighed on the day of parturition (PND 0), PNDs 4, 7, 10 and 13.
-- Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as were the males.
-- Females without litter and after weaning (PND 13) were weighed once a week.
- The body weight change of the animals was calculated from these results.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
- Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
-- Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
-- Food consumption of the females with evidence of sperm was determined for GD 7, 14 and 20.
-- Food consumption of the females which gave birth to a litter was determined for PND 4, 7, 10 and 13.
- Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

HAEMATOLOGY: Yes.
- Time schedule for collection of blood: at termination (males); at PND 14 (females).
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Yes (about 16-20 hours).
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group.
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes.
- Time schedule for collection of blood: at termination (males); at PND 14 (females).
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Yes (about 16-20 hours).
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group.
- Parameters checked in table 2 were examined.

SACRIFICE
- Male animals: All surviving animals of the main groups on study day 31.
- Maternal animals: All surviving animals of the main groups on study day 59.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 4 were prepared for microscopic examination and weighed, respectively.

NEUROBEHAVIOURAL EXAMINATION: Yes; functional observational battery and motor activity assessment (see "OTHER")

OTHER:

FUNCTIONAL OBSERVATIONAL BATTERY (FOB):
- A functional observational battery (FOB) was performed in all animals at the end of the administration period starting at about 10:00 h. At least one hour before the start of the FOB the rats were transferred to single-animal polycarbonate cages type III (floor area of about 800 cm2)) supplied by TECNIPLAST (Hohenpeissenberg, Germany) with wire covers from Ehret (Emmendingen, Germany) and small amounts of bedding material. Drinking water was provided ad libitum, but no food was offered during the measurements. The FOB started with passive observations without disturbing the rats, followed by removal from the home cage, open field observations in a standard arena and sensory motor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.
- Examined parameters:
-- Home cage observations (10-30 seconds): posture, tremors, convulsions, abnormal movements, gait, other findings.
-- Open field observations (at least for 2 minutes): behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/ pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/ stereotypes, gait, activity/ arousal level, feces excreted within 2 minutes (appearance/ consistency), urine excreted within 2 minutes (amount/ color), rearing within 2 minutes, other findings.
-- Sensory motor tests/ reflexes: reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory startle response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings.

MOTOR ACTIVITY ASSESSMENT:
- Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed.

ESTROUS CYCLE:
- For all females of the pool estrous cycle normality was evaluated before the beginning of the administration period.
- In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear.
- Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all female F0 rats.

MALE REPRODUCTION DATA:
- The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.
- For the males, mating and fertility indices were calculated for F1 litters (for formulas see "Any other information on materials and methods").

FEMALE REPRODUCTION AND DELIVERY DATA:
- The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females.
- For the females, mating, fertility and gestation indices were calculated for F1 litters (for formulas see "Any other information on materials and methods").

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes.
Examinations included:
- Uterus weight: Yes.
- Number of implantations: Yes.

Fetal examinations:

Fetal examinations are not applicable to OECD 422

LITTER/PUB DATA:

PUB NUMBER AND STATUS AT DELIVERY:
- All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

PUB VIABILITY/MORTALITY:
- In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. Dead pups were evaluated by the methods, which are described in detail in “Pup Necropsy observations”.
- The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PNDs 1-4, 5-7 and 8-13 were determined. Pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups per litter was calculated on the day of birth (PND 0), and on lactation days 4, 7 and 13. Furthermore, viability and survival indices were calculated.

SEX RATIO:
- On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy.
- The sex ratio was calculated at PND 0 and PND 13.

PUB CLINICAL OBSERVATIONS:
- The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

PUB BODY WEIGHT DATA:
- The pups were weighed on the day after birth (PND 1) as well as on PNDs 4, 7 and 13. Pups' body weight change was calculated from these results.
- The individual weights were always determined at about the same time of the day (in the morning) and on PND 4 immediately before standardization of the litters.
- “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.

NIPPLE/AREOLA ANLAGEN:
- All surviving male pups were examined for the presence of nipple/areola anlagen on PND 13 of the lactation phase. The number of nipple/areola anlagen was counted.

ANOGENITAL DISTANCE:
- Anogenital distance (AGD; defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements was done blind to treatment in a randomized order, using a measuring ocular, on all live male, female and uncertain pups on day 1 after birth.

PUB NECROPSY OBSERVATIONS:
- On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated, and the organs were assessed macroscopically.
- On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for further processing.
- All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically.
- All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

THYROID HORMONES IN PUBS:
- Blood samples were taken from one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia.
- Blood samples from the PND 13 pups were assessed for serum levels for thyroid hormones (T4 and TSH).

Statistics:

See table 5.

Indices:

- For the males, mating and fertility indices were calculated for F1 litters (for formulas see "Any other information on materials and methods").
- For the females, mating, fertility and gestation indices were calculated for F1 litters (for formulas see "Any other information on materials and methods").
- For the pubs viability index, survival index (for formulas see "Any other information on materials and methods").

Historical control data:

Historical control data to allow comparison with concurrent controls were provided by the test facility for the following parameters: maternal body weights, mating data, delivery data, litter data, sex ratio, pup weights, anogenital distance and anogenital index, presence of areolas/nipples, pup necropsy observations, clinical pathology, organ weight (spleen).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Summary clinical observations for males and females (except gestation and lactation periods):
- During the course of the premating period, respiration sounds were observed in male animal Nos. 32 and 33 of test group 3 (375 mg/kg bw/d) starting on study day 9 until study day 17 and 18, respectively. The same was true for female animal Nos. 132 and 138 of the same test group between study days 9 to 13. In addition, male animal No. 31 of test group 3 showed respiration sounds between study days 28 to 31. The findings were assessed to be related to treatment.
- No treatment-related, adverse findings were observed in male and female animals of test groups 1 and 2 (40 and 120 mg/kg bw/d) during the premating, mating and postmating (males only) phases in any test group.
- During pre- and postmating, salivation shortly after treatment was observed in 8 male animals of test group 3 (375 and 240 mg/kg bw/d). From the temporary, short appearance immediately after dosing (or shortly before) it was concluded that this type of finding was induced by a bad taste of the test substance or local affection of the upper digestive tract.
- On premating day 13, female animal No. 122 of test group 2 (120 mg/kg bw/d) showed a mass at the right anogenital region (palpable through skin, size less than 1.5 cm). The finding was assessed to be incidental and not related to treatment.

Summary clinical observations for females during gestation:
- Female animal No. 138 of test group 3 (375 and 240 mg/kg bw/d) showed respiration sounds between gestation days (GD) 0 to 5. From GD 6 onwards, the finding did not occur again. The finding was assessed to be related to treatment.
- Between GD 0 to 5, female animal No. 122 of test group 2 (120 mg/kg bw/d) still showed a mass at the right anogenital region (palpable to rough skin, size less than 1.5 cm). From GD 6 onwards, the finding did not occur again. The finding was assessed to be incidental and
not related to treatment.
- All other females of test groups 0 to 3 (control, 40, 120 as well as 375 and 240 mg/kg bw/d, respectively) did not show any treatment-related or spontaneous findings.

Clinical observations for females during lactation:
- Neither treatment-related nor spontaneous findings were observed in any female animal between postnatal days (PND) 0 to 13.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Female animal No. 140 of test group 3 (375 mg/kg bw/d) was found dead on premating day 5 in the present study. The cause for the premature death of this individual could not be determined after pathological examinations and no other animal died ahead of schedule. Thus, after completion of all related examinations, its occurrence was assessed to be spontaneous in nature and not related to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- No significant changes in body weight parameters, i.e. mean body weights and body weight change values, were observed for any test group (except the significantly lower body weight change value in female animals of test group 3 between premating days 7 to 13).
- However, although a slight mean body weight loss between premating days 0 to 7 could be observed in female animals of any test group including the control, the female animals of test group 3 lost about 10 g within this week. These animals gained weight during the following interval (premating days 7 to 13) during which the dose level was reduced from 375 to 240 mg/kg bw/d. The body weight loss in females of test group 3 (375 mg/kg bw/d) during the first week of application was assessed to be treatment-related and adverse.
- In the following study phases, i.e. the gestation and lactation periods, mean body weight of female animals in test group 3 (375 and 240 mg/kg bw/d) was always below the controls as well as the low- and mid-dose groups. However, the values for mean body weights and body weight change did not differ significantly.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- During the premating period, food consumption was significantly decreased in female animals of test group 3 (375 and 240 mg/kg bw/d). During the gestation and lactation periods, no significant changes in food consumption occurred.
- In male animals of test groups 1 to 3 (40, 120 as well as 375 and 240 mg/kg bw/d, respectively) as well as in female animals of test groups 1 and 2, no changes in food consumption were observed at any time point.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No treatment-related, adverse findings were observed.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- No treatment-related changes among hematological parameters were observed.
- At the end of the administration period, in males of test group 3 (375 and 240 mg/kg bw/d) prothrombin time (Hepatoquick’s test, HQT) was significantly shortened. However, the values were within the historical control range (males, HQT 35.0-40.5 sec). In dams of test group 2 (120 mg/kg bw/d), relative eosinophil counts were significantly increased, but the change was not dose-dependent. Therefore, the mentioned alterations were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

- No treatment-related, adverse changes among clinical chemistry parameters were observed.
- At the end of the administration period, in males of test groups 2 and 3 (120 as well as 375 and 240 mg/kg bw/d, respectively) cholesterol levels were significantly increased, and in dams of test group 3, inorganic phosphate levels were significantly higher compared to controls. Both changes were the only relevant clinical-pathology alterations among these individuals. Therefore, the changes were regarded as treatment-related, but non-adverse (ECETOC Technical Report No. 85, 2002).
- In males of test group 3 (375 and 240 mg/kg bw/d), inorganic phosphate levels were significantly increased, and in females of the same test group, chloride levels were significantly decreased. However, the values were within historical control ranges (males, inorganic phosphate 1.43-2.06 mmol/L; females, chloride 89.6-97.0 mmol/L). Therefore, these alterations were regarded as incidental and not treatment-related.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- Absolute weights: None of the mean absolute weight parameters showed significant differences when compared to the control group 0.
- Relative organ weights When compared to the control group 0 (set to 100%), the mean relative weight of the spleen of test group 2 (120 mg/kg bw/d) females was significantly decreased.
- In female animals, no other mean relative weight parameters showed significant differences when compared to the control group 0. The same was true for the relative weight parameters in male animals.
- The mean relative spleen weight of female animals in test group 2 (120 mg/kg bw/d), i.e. 0.182%, was within the historical control range (0.173-0.257%). and a dose-response relationship could not be observed. In addition, no histopathological findings related to spleen were noted in the survivors of test group 3. Therefore, the decreased mean relative weight of the spleen in test group 2 females was regarded as incidental.

Gross pathological findings:

no effects observed

Description (incidence and severity):

- All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
- Premature decedent: No findings were noted in female animal No. 140 of test group 3 (375 and 240 mg/kg bw/d) which died prematurely.
- Fertility: Female animal Nos. 134, 139 of test group 3 (375 and 240 mg/kg bw/d), which were not pregnant, as well as the male mating partners (Nos. 34, 39) did not show relevant gross lesions.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

- All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and
without any relation to treatment.
- The stages of spermatogenesis in the testes of males of the high dose test group were comparable to those of the controls. In high dose females the different stages of functional bodies in the ovaries were present and comparable to the control animals.
- Premature decedent: Female animal No. 140 of test group 3 (375 and 240 mg/kg bw/d) which died prematurely showed stress-related findings in adrenal cortex (diffuse cortical hyperplasia), glandular stomach (erosion/ulcer), axillary lymph nodes (apoptotic necrosis of lymphoid cells), spleen (PALS decreased), and thymus (starry sky cells increased). Furthermore, there was myeloid hyperplasia in the bone marrow. The cause of death of this animal could not be determined.
- Fertility: Female animal No. 134, which was not pregnant, showed inactive interstitial glands in the ovary and minimal squamous metaplasia in the uterus. Because the changes occurred only in an individual female animal, they were considered to be incidental and not related to treatment. The other female animal (No. 139), which was not pregnant, as well as the male mating partners of these two females (Nos. 34, 39) did not show relevant histopathological findings.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

DETAILED CLINICAL OBSERVATIONS:
- During detailed clinical observations (DCO), in 3 male and in 2 female animals of test group 3 (375 and 240 mg/kg bw/d) respiration sounds were observed. The findings were assessed to be related to treatment.
- Female animal No. 122 of test group 2 (120 mg/kg bw/d) showed a mass at the right anogenital region (palpable through skin, size less than 1.5 cm). The finding was assessed to be incidental and not related to treatment.

ESTROUS CYCLE:
- Estrous cycle data revealed regular cycles in most of the female animals of all test groups including the control. The mean estrous cycle duration in test groups 0 to 3 was at or between 3.7 to 3.9 days. A treatment-related change regarding the estrous cycle parameters did not occur in any test group.
- Prolonged estrous was observed in a single female animal of test group 3 (375 and 240 mg/kg bw/d), and prolonged diestrous could be observed in 1 animal of test group 3, in 2 animals of test groups 0 and 2 as well as in 3 animals of test group 1. A dose-response relationship did not occur.

MALE REPRODUCTION DATA:
- No direct, treatment-related changes with regard to the male mating index was observed in any test group.
- The male mating indices calculated after the mating period to produce F1 litter were 100% in test groups 0 to 2.
- The male mating index for test group 3 (375 and 240 mg/kg bw/d) was 88.9% because female animal No. 140 was found dead on premating day 5 and, consequently, mating was not carried out for male animal No. 40.

MALE FERTILITY INDEX:
- Fertility was proven in nearly all of the F0 parental males of test groups 0 to 2 (control; 40 and 120 mg/kg bw/d) within the scheduled mating interval to produce F1 litter. Male animal Nos. 34 and 39 of test group 3 (375 and 240 mg/kg bw/d), which were mated with female Nos. 134 and 139, did not generate F1 pups. No implants were found at necropsy in female
animal No. 139.
- Thus, the male fertility index was 100.0% in test groups 0 and 2, 77.8% in test group 1.
- These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

FEMALE MATING INDEX:
- The female mating index calculated after the mating period for F1 litter was 100% in test groups 0 to 2. For test group 3 the female mating index was 88.9% because animal No. 140 was found dead on premating day 5.
- The mean duration until sperm was detected (GD 0) was 2.8 days for test group 0 (control), 3.1 days for test group 1 (40 mg/kg bw/d), 2.5 days for test group 2 (120 mg/kg bw/d) and test group 3 (375 and 240 mg/kg bw/d).
- These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

FUNCTIONAL OBSERVATIONAL BATTERY:
- Deviations from "zero values" were obtained in quantitative parameters in male and female animals. Without a dose-response relationship or occurred in single animals only, these observations were considered as incidental.
- The following examinations were performed during FOB and are assessed individually:
-- Home cage observations: No test substance-related effects were observed.
-- Open field observations: No test substance-related effects were observed.
-- Sensorimotor tests/reflexes: No test substance-related effects were observed.
-- Quantitative parameters: No test substance-related effects were observed.

MOTOR ACTIVITY MEASUREMENT:
- Regarding the overall motor activity and single intervals, no test substance-related deviations were noted for male and female animals of any test group.

THYROID HORMONES:
- In parental males of test groups 1, 2 and 3 (40, 120 as well as 375 and 240 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.

Details on results:

- The test substance was administered daily by gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 40 mg/kg bw/d (test group 1), 120 mg/kg bw/d (test group 2) and 375 mg/kg bw/d (test group 3). Deionized water served as vehicle, control animals were dosed daily with the vehicle only.
- The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.
- Regarding clinical examinations, obvious signs of general systemic toxicity were observed in female animals of test group 3 (375 mg/kg bw/d) during the premating period when treated at 375 mg/kg bw/d. Food consumption was significantly reduced and body weight loss occurred during the first week of treatment. Respiration sounds were observed in 2 male and 2 female animals of the same test group. Male animal No. 31 of test group 3 showed respiration sounds between study days 28 to 31. These changes were assessed to be related to treatment and adverse. Furthermore, one female animal of test group 3 was found dead on study day 5. Because no explanation for the premature death of this individual could be given after pathological examinations and no other animal died ahead of schedule, its occurrence was assessed to be spontaneous in nature and not related to treatment. However, having the results of the range-finding study in mind together with the occurrence of the mentioned findings and the premature death of one animal during the first days of application, a reduction of the dose level seemed to be necessary. Thus, it was reduced to 240 mg/kg bw/d from study day 10 onwards.
- Fertility indices for male and female animals were not impaired by test-substance administration even at a dose level 240 mg/kg bw/d (formerly 375 mg/kg bw/d). In addition, live birth indices of pups in all test groups were not influenced. The viability index as indicator for pup mortality was not altered.
- Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose level of the compound of 375 and 240 mg/kg bw/d.
- Regarding pathology, no treatment-related findings were identified. All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Number of abortions:

not examined

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

POSTIMPLANTATION LOSS:
- The postimplantation loss was 6.7% in control group 0, 5.9% in test group 1 (40 mg/kg bw/d), 6.1% in test group 2 (120 mg/kg bw/d) and 6.9% in test group 3 (375 and 240 mg/kg bw/d).
- These values were within the normal range of biological variation inherent in the strain of rats used for this study.

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

not examined

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

- The mean duration of gestation was 22.2 days in test group 0 (control), and 22.1 days in test groups 1 (40 mg/kg bw/d), 2 (120 mg/kg bw/d) and 3 (375 and 240 mg/kg bw/d).

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

FEMALE FERTILITY NDEX:
- All sperm positive rats of test groups 0 to 2 (control; 40 and 120 mg/kg bw/d) delivered pups. Thus, the female fertility index was 100% in test groups 0 and 2. In test group 3 (375 and 240 mg/kg bw/d), all but one (No. 134) female
animals were sperm positive. As female animal No. 139 was sperm positive but had no implants and did not generate pups, the female fertility index was 77.8%.
- These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

GESTATION INDEX:
- The gestation index was 100% in all test groups.

Other effects:

no effects observed

Details on maternal toxic effects:

- The test substance was administered daily by gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 40 mg/kg bw/d (test group 1), 120 mg/kg bw/d (test group 2) and 375 mg/kg bw/d (test group 3). Deionized water served as vehicle, control animals were dosed daily with the vehicle only.
- The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.
- Regarding clinical examinations, obvious signs of general systemic toxicity were observed in female animals of test group 3 (375 mg/kg bw/d) during the premating period when treated at 375 mg/kg bw/d. Food consumption was significantly reduced and body weight loss occurred during the first week of treatment. Respiration sounds were observed in 2 male and 2 female animals of the same test group. Male animal No. 31 of test group 3 showed respiration sounds between study days 28 to 31. These changes were assessed to be related to treatment and adverse. Furthermore, one female animal of test group 3 was found dead on study day 5. Because no explanation for the premature death of this individual could be given after pathological examinations and no other animal died ahead of schedule, its occurrence was assessed to be spontaneous in nature and not related to treatment. However, having the results of the range-finding study in mind together with the occurrence of the mentioned findings and the premature death of one animal during the first days of application, a reduction of the dose level seemed to be necessary. Thus, it was reduced to 240 mg/kg bw/d from study day 10 onwards.
- Fertility indices for male and female animals were not impaired by test-substance administration even at a dose level 240 mg/kg bw/d (formerly 375 mg/kg bw/d). In addition, live birth indices of pups in all test groups were not influenced. The viability index as indicator for pup mortality was not altered.
- Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose level of the compound of 375 and 240 mg/kg bw/d.
- Regarding pathology, no treatment-related findings were identified. All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed at this dose.

Dose descriptor:

LOAEL

Remarks:

general systemic toxicity

Effect level:

240 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Dose descriptor:

NOAEL

Remarks:

reproductive performance and fertility

Effect level:

240 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed up to the highest tested dose.

Abnormalities:

no effects observed

Fetal body weight changes:

not examined

Reduction in number of live offspring:

not examined

Changes in sex ratio:

not examined

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

not examined

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Description (incidence and severity):

PUB NUMBER AND STATUS AT DELIVERY:
- The mean number of delivered F1 pups per dam was 12.3 in control group 0, 11.3 in test group 1 (40 mg/kg bw/d), 12.6 in test group 2 (120 mg/kg bw/d) and 11.4 in test group 3 (375 and 240 mg/kg bw/d).

PUB VIABILITY INDEX/MORTALITY:
- The rate of liveborn pups in all test groups was not affected by the test substance, as indicated by live birth indices of 100% in test groups 0, 2 and 3 (control, 120 as well as 375 and 240 mg/kg bw/d). The live birth index in test group 1 (40 mg/kg bw/d) was 89.3% because of two pups of female animal No. 117 died on the day of birth.
- The viability index indicating pup mortality between PND 0 and 4 was 100% in test groups 0 (control) and 1 (40 mg/kg bw/d), 98.3% in test group 2 (120 mg/kg bw/d) and 95.3% in test group 3 (375 and 240 mg/kg bw/d).
- The survival index indicating pup mortality between PND 4 and 13 was 100% in all test groups including test group 3 (375 and 240 mg/kg bw/d).
- The values for test groups 0, 1, 2 and 3 reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

PUB SEX RATIO:
- In test groups 0, 1, 2 and 3 (control, 40, 120 as well as 375 and 240 mg/kg bw/d, respectively), the sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups. Slight differences were regarded to be spontaneous in nature.

PUB CLINICAL OBSERVATIONS:
- In each test group including control group some pups died during the lactation period.
- All other F1 pups in test groups 0 to 3 did not show clinical signs up to scheduled sacrifice on PND 4 and PND 13.

PUB BODY WEIGHT DATA:
- Mean pup body weights and pup body weight change values were similar in all test groups.
- Eleven male runts and seven female runts were found in one litter of test group 2 (120 mg/kg bw/d; female animal No. 125). Two female runts were found in different litters of test groups 0 (control; pup Nos. 109-14 and 110-11) and in one litter of test group 3 (375 and 240 mg/kg bw/d; pup Nos. 133-05 and 133-06).
- A relation to dosing was not observed, test substance-related effects did not occur.

ANOGENITAL DISTANCE:
- In test groups 1 - 3 (40, 120 as well as 375 and and 240 mg/kg bw/d, respectively), the apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.

PUB NECROPSY OBSERVATION:
- One female pup (No. 119-07) of test group 1 (40 mg/kg bw/d) showed a dilated renal pelvis in the right kidney and one female pup (No. 121-10) of test group 2 (120 mg/kg bw/d) showed a dilated renal pelvis in the left kidney. These findings were assessed to be incidental and not related to the test substance.
- One male (No. 133-07) and one female pup (No. 135-12) of test group 3 (375 and 240 mg/kg bw/d) were cannibalized and could not be found in the respective cage on PND 1.

THYROID HORMONES:
- In pubs of all dosing groups sacrificed at PND 13, no treatment-related alterations of T4 and TSH levels were observed.

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to the highest tested dose.

Remarks on result:

other: fetus analysis is not applicable for OECD TG 422

Abnormalities:

not examined

Developmental effects observed:

no

Table 6: Male fertility indices for F0 males

 

	Test group 0 (0 mg/kg bw/d)	Test group 1 (40 mg/kg bw/d)	Test group 2 (120 mg/kg bw/d)	Test group 3 (375 and 240 mg/kgbw/d)
Male fertility index [%]	100.0	100.0	100.0	77.8

* p≤0.05; ** p≤0.01

 

Table 7:Female fertility index

 

	Test group 0 (0 mg/kg bw/d)	Test group 1 (40 mg/kg bw/d)	Test group 2 (120 mg/kg bw/d)	Test group 3 (375 and 240 mg/kgbw/d)
Female fertility index [%]	100.0	100.0	100.0	77.8

* p≤0.05; ** p≤0.01

 

Table 8: Sex ratio of live F1 pups

 

PND 0	Test group 0 (0 mg/kg bw/d)	Test group 1 (40 mg/kg bw/d)	Test group 2 (120mg/kg bw/d)	Test group 3 (375 and 240 mg/kgbw/d)
Live males [%]	52.8	51.3	52.2	55.8
Live females [%]	47.2	48.7	47.8	44.2
PND 13	Test group 0 (0 mg/kg bw/d)	Test group 1 (40 mg/kg bw/d)	Test group 2 (120mg/kg bw/d)	Test group 3 (375 and 240 mg/kgbw/d)
Live males [%]	48.8	50.0	51.2	57.1
Live females [%]	51.2	50.0	48.8	42.7

 

Table 9:Relative organ weights

 

	Female animals
Test group (mg/kg bw/d)	1 (40)	2 (120)	3 (375/240)
Spleen	97%	84%*	90%

* p≤0.05; ** p≤0.01

 

Table 10:Summary - Clinical Observation, males, pre-mating

 

			Test group 0 (0 mg/kg bw/d)	Test group 1 (40 mg/kg bw/d)	Test group 2 (120 mg/kg bw/d)	Test group 3 (375 and 240 mg/kgbw/d)
Day 0àday 31	Animals examined	N	10	10	10	10
	Animals with signs	N	0	0	0	8
	Headsalivation	N	0	0	0	8
	Dead Sacrificed scheduled	N	10	10	10	10
	Normal NAD	N	10	10	10	10
	Reapiration sounds	N	0	0	0	3

 

Table 11:Summary - Clinical Observation, females, pre-mating

 

			Test group 0 (0 mg/kg bw/d)	Test group 1 (40 mg/kg bw/d)	Test group 2 (120 mg/kg bw/d)	Test group 3 (375 and 240 mg/kgbw/d)
Day 0àday 13	Animals examined	N	10	10	10	10
	Animals with signs	N	0	0	0	2
	Dead Found dead	N	0	0	0	1
	Normal NAD	N	10	10	10	10
	Mass Palpable through skin	N	0	0	1	0
	Reapiration sounds	N	0	0	0	2

 

 

Table 12:Summary – Food consumption, females, in-life

 

		Test Group0/F 0 mg/kgbw/d	Test Group1/F 40 mg/kgbw/d	Test Group2/F 120mg/kgbw/d	Test Group3/F 375/240 mg/kgbw/d
d 0 -> 7	Mean [g]	16.1 n	15.4	16.0	13.3**
	S.d.	1.2	1.3	0.4	1.4
	N	5	5	5	5
	Deviation Vs Control [%]		-4.7	-0.9	-17.6
d 7 -> 13	Mean [g]	15.2 n	14.7	14.2	12.8*
	S.d.	1.6	1.4	0.2	1.0
	N	5	5	5	5
	Deviation Vs Control [%]		-3.2	-6.9	-16.1

Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

d= day; n=DUNNETT

 

Table 13:Summary – Body Weights - BW / Body Weights [g], females, in-life

 

		Test Group0/F 0 mg/kgbw/d	Test Group1/F 40 mg/kgbw/d	Test Group2/F 120mg/kgbw/d	Test Group3/F 375/240 mg/kgbw/d
d 0	Mean [g]	231.6n	227.5	230.2	119.2
	S.d.	13.0	15.8	14.5	10.1
	N	10	10	10	10
	Deviation Vs Control [%]		-1.8	-0.6	-1.0
d 7	Mean [g]	230.9n	225.7	227.7	219.3
	S.d.	12.1	10.5	11.6	6.1
	N	10	10	10	9
	Deviation Vs Control [%]		-2.3	-1.4	-5.0
d 13	Mean [g]	234.1n	228.5	231.7	225.0
	S.d.	14.5	9.9	10.5	7.1
	N	10	10	10	9
	Deviation Vs Control [%]		-2.4	-1.0	-3.9

Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

n=DUNNETT

 

Table 14: Summary Mating Report

 

		Test Group 0/F 0 mg/kgbw/d		Test Group 1/F 40 mg/kgbw/d	Test Group 2/ F 120 mg/kgbw/d		Test Group 3/F 240 mg/kgbw/d
No. of females mated	N	10		10	10	9
- Inseminated	N	10	f-	10	10	8
Female mating index	%	100.0		100.0	100.0	88.9
-- Pregnant	N	10	f-	10	10	7
Female fertility index	%	100.0		100.0	100.0	87.5
No. of males mated	N	10		10	10	9
- With inseminated females	N	10	f-	10	10	8
Male mating index	%	100.0		100.0	100.0	88.9
- With pregnant females	N	10	f-	10	10	7
Male fertility index	%	100.0		100.0	100.0	77.8
Females with defined Day 0 pc	N	10		10	10	8
Mating days until Day 0 pc	Mean	2.8	x+	3.1	2.5	2.5
	S.d.	1.4		1.5	1.1	1.7
	N	10		10	10	8
Days 0 To 4	N	9		9	10	7
	%	90.0		90.0	100.0	87.5
Days 5 To 9	N	1		1	0	1
	%	10.0		10.0	0.0	12.5
Days 10 To 14	N	0		0	0	0
	%	0.0		0.0	0.0	0.0

Statistic Profile = Fisher's exact test (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics f=FISHER-EXACT; x=WILCOX

 

 

Table 15: Summary Pregnancy Status Report – Reproduction

 

		Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 40 mg/kgbw/d	Test Group 2/ F 120 mg/kgbw/d		Test Group 3/F 240 mg/kgbw/d
No. of females at start	N	10	10	10	9
No. of females at mated	N	10	10	10	9
Without evidence og mating	N	0	0	0	1
- Pregnant	N	0	0	0	0
- Not pregnant	N	0	0	0	1
Females with defined Day 0 pc	N	10	10	10	8
Pregnant	N	10	10	10	7
- sacrificed scheduled	N	10	10	10	7
Not pregnant	N	0	0	0	2
- sacrificed scheduled	N	0	0	0	2
Pregnant, not delivering	N	0	0	0	0
Delivering	N	10	10	10	7
-- With liveborn pubs	N	10	10	10	7
	%	100.0	100.0	100.0	100.0
-- With all pubs stillborn	N	0	0	0	0
	%	0.0	0.0	0.0	0.0

 

 

Table 16: Summary Delivery Report

 

		Test Group 0/F 0 mg/kgbw/d		Test Group 1/F 40 mg/kgbw/d	Test Group 2/ F 120 mg/kgbw/d		Test Group 3/F 240 mg/kgbw/d
No. of females at start	N	10		10	10	9
No. of females at mated	N	10	f-	10	10	9
	%	100.0		100.0	100.0	100.0
Pregnant	N	10	f-	10	10	7
	%	100.0		100.0	100.0	77.8
Dead	N	10		10	10	9
Without Delivery	N	0		0	0	2
- Pregnant	N	0		0	0	0
- Not pregnant	N	0		0	0	2
-- Delivering	N	10	f-	10	10	7
	%	100.0		100.0	100.0	100.0
-- With liveborn pubs	N	10	f-	10	10	7
Gestation Index	%	100.0		100.0	100.0	100.0
Gestation days	Mean	22.2	n	22.1	22.1	22.1
	S.d.	0.4		0.3	0.6	0.4
	%	10		10	10	7
-- With stillborn pubs	N	0		1	0	0
	%	0.0	f+	10.0	0.0	0.0
-- With all pubs stillborn	N	0		0	0	0
	%	0.0	f+	0.0	0.0	0.0

Statistic Profile = Fisher's exact test (one-sided-), Dunnett test (two-sided), Fisher's exact test (one-sided+), *p<=0.05, ** p <=0.01, X = Group excluded from statistics

F=FISHER-EXACT; n=DUNNETT

 

 

Table 17: Summary Litter Report

 

		Test Group 0/F 0 mg/kgbw/d		Test Group 1/F 40 mg/kgbw/d	Test Group 2/ F 120 mg/kgbw/d		Test Group 3/F 240 mg/kgbw/d
Total Number of Pregnant Females	N	10		10	10	7
Total number of litters	N	10		10	10	7
With liveborn pubs	N	10	f-	10	10	7
	%	100.0		100.0	100.0	100-0
With stillborn pubs	N	0	f+	1	0	0
	%	0.0		10.0	10.0	0.0
With all pubs stillborn	N	0	f+	0	0	0
	%	0.0		0.0	0.0	0.0
Implantation Sites	N	131		122	134	86
	Mean	13.1	x-	12.2	13.4	12.3
	S.d.	1.9		1.8	1.8	1.5
	N	10		10	10	7
Pubs delivered	N	122		115	126	80
	Mean	12.2	x-	11.5	12.6	11.4
	S.d.	2.2		2.1	2.3	2.3
	N	10		10	10	7
Postimplantation Loss	Mean%	6.7	x+	5.9	6.1	6.9
	S.d.	11.0		7.4	9.4	15.5
	N	10		10	10	7
Pubs liveborn	N	122		113	126	80
	%%	100.0		98.3	100.0	100.0
	Mean	12.2	x-	11.3	12.6	11.4
	S.d.	2.2		2.2	2.3	2.3
	N	10		10	10	7

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-),Fisher's exact test (one-sided+), *p<=0.05, ** p <=0.01, X = Group excluded from statistics

F=FISHER-EXACT; n=DUNNETT

 

Table 18: Summary Litter Report – Pup status

 

		Test Group 0/F 0 mg/kgbw/d		Test Group 1/F 40 mg/kgbw/d	Test Group 2/ F 120 mg/kgbw/d		Test Group 3/F 240 mg/kgbw/d
Pubs stillborn	N	0		2	0	0
	%	0.0		1.7	0.0	0.0
	Mean	0.0	x+	0.2	0.0	0.0
	S.d.	0.0		0.6	0.0	0.0
	N	10		10	10	7
Perinatal Loss	Mean%	0.0	x+	1.8	0.0	0.0
	S.d.	0.0		5.7	0.0	0.0
	N	10		10	10	7

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-),Fisher's exact test (one-sided+), *p<=0.05, ** p <=0.01, X = Group excluded from statistics

F=FISHER-EXACT; n=DUNNETT

 

 

Table 19: Summary Litter Report - Dead Pups

 

		Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 40 mg/kgbw/d	Test Group 2/ F 120 mg/kgbw/d		Test Group 3/F 240 mg/kgbw/d
Litters with liveborn pups	N	10	10	10	7
Pubs delivered	N	122	115	126	8
found dead [pup] / Dead	N	0	0	2	1
	%	0.0	0.0	1.6	1.2
Stillborn / Dead	N	0	2	0	0
	%	0.0	1.7	0.0	0.0
Alive / Alive	N	122	113	126	80
	%	100.0	98.3	100.0	100.0
cannibalizing [pup] / Dead	N	1	0	0	2
	%	0.8	0.0	0.0	2.5
sacrificed scheduled [pup] / Dead	N	79	80	80	54
	%	64.8	69.6	63.5	67.5
Culled / Dead	N	42	33	44	23
	%	34.4	28.7	34.9	28.8
Litters not surviving Day 13	N	0	0	0	0
	%	0.0	0.0	0.0	0.0

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-),Fisher's exact test (one-sided+), *p<=0.05, ** p <=0.01, X = Group excluded from statistics

 

 

Table 20: Summary Litter Report - Pups Died

 

		Test Group 0/F 0 mg/kgbw/d		Test Group 1/F 40 mg/kgbw/d	Test Group 2/ F 120 mg/kgbw/d	Test Group 3/F 240 mg/kgbw/d
Litters with liveborn pups	N	10		10	10	7
Pubs delivered	N	122		115	126	80
Day 0 To 0	N	0		0	0	0
	%	0		0	0	0
Day 1 To 4	N	0		0	2	3
	%	0		0	1.6	3.8
Day 5 To 7	N	0		0	0	0
	%	0		0	0	0
Day 8 To 13	N	0		0	0	0
	%	0		0	0	0
Pups surviving days 0 To 4	N	123		113	124	77
Viability Index	Mean%	100.0	x-	100	98.3	95.3
	S.d.	0.0		0.0	3.8	6.1
	N	10		10	10	7
Pups surviving days 4 To 13	N	81		80	80	54
Viability Index	Mean%	100.0	NA	100.0	100.0	100.0
	S.d.	0.0		0.0	0.0	0.0
	N	10		10	10	7

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-),Fisher's exact test (one-sided+), *p<=0.05, ** p <=0.01, x =WILCOX; NA = No Test Applicable

 

 

Table 21: Summary Pup Clinical Observation during Lactation

 

Males			Test Group 0/M 0 mg/kgbw/d	Test Group 1/M 40 mg/kgbw/d	Test Group 2/ M 120 mg/kgbw/d		Test Group 3/M 240 mg/kgbw/d
Day 0 -->13	Animals examined	N	65	60	66	42
	normal NAD	N	65	59	66	42
Females			Test Group 0/ F 0 mg/kg bw/d	Test Group 1/ F 40 mg/kg bw/d	Test Group 2/ F 120 mg/kg bw/d	Test Group 3/ F 240 mg/kg bw/d
Day 0 -->13	Animals examined	N	57	55	60	38
	Animals with signs	N	1	1	1	2
	normal NAD	N	57	54	60	38
	reproduction not assessed (died)	N	1	1	1	2

 

 

Table 22: Summary Pup Report Body Weights - BW / Body Weights [g]

 

		Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 40 mg/kgbw/d	Test Group 2/ F 120 mg/kgbw/d	Test Group 3/F 240 mg/kgbw/d
day 1 Runt	Males	0	0	11	2
	Females	2	0	7	0
day 1 Males	Mean	7.0 n	6.9	6.8	6.8
	S.d.	0.6	0.4	1.0	0.7
	N	10	10	10	7
	Deviation Vs Control [%]		-0.7	-3.0	-2.5
day 1 Females	Mean	6.5 n	6.5	6.3	6.7
	S.d.	0.7	0.4	1.0	0.7
	N	10	10	10	6
	Deviation Vs Control [%]		0.0	-3.0	2.5
day 1 Males + Females	Mean	6.8 n	6.7	6.5	6.7
	S.d.	0.7	0.4	1.0	0.7
	N	10	10	10	7
	Deviation Vs Control [%]		-0.4	-3.3	-1.0
day 4 Males	Mean	10.7 n	10.6	10.3	10.4
	S.d.	1.3	0.8	1.7	1.1
	N	10	10	10	7
	Deviation Vs Control [%]		-0.8	-3.9	-3.1
day 4 Females	Mean	10.1 n	10.2	9.7	10.4
	S.d.	1.4	0.9	1.7	1.2
	N	10	10	10	6
	Deviation Vs Control [%]		1.0	-3.8	2.2
day 4 Males + Females	Mean	10.4 n	10.4	10.0	10.3
	S.d.	1.3	0.8	1.7	1.1
	N	10	10	10	7
	Deviation Vs Control [%]		-0.1	-4.2	-1.5
	Mean	17.4 n	17.1	16.7	16.5
day 7 Males	S.d.	1.5	1.3	2.8	1.8
	N	10	10	10	7
	Deviation Vs Control [%]		-1.3	-3.7	-4.9
	Mean	16.2 n	16.4	16.0	16.4
day 7 Females	S.d.	1.6	1.4	2.4	1.7
	N	10	10	10	6
	Deviation Vs Control [%]		1.8	-1.0	1.3
	Mean	16.8 n	16.8	16.4	16.3
day 7 Males + Females	S.d.	1.5	1.3	2.6	1.7
	N	10	10	10	7
	Deviation Vs Control [%]		0.2	-2.3	-2.6
	Mean	32.3 n	31.9	31.1	31.2
day 13 Males	S.d.	1.9	1.8	3.6	2.1
	N	10	10	10	7
	Deviation Vs Control [%]		-1.0	-3.5	-3.2
	Mean	30.7 n	31.0	30.2	30.8
day 13 Females	S.d.	2.4	1.9	2.8	2.0
	N	10	10	10	6
	Deviation Vs Control [%]		1.0	-1.4	0.5
	Mean	31.5 n	31.5	30.7	31.0
day 13 Males + Females	S.d.	2.1	1.8	3.2	2.0
	N	10	10	10	7
	Deviation Vs Control [%]		0.0	-2.4	-1.6

Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

n=DUNNETT

 

 

Table 23: Summary Pup Report AG Distance + AG Index of males

 

		Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 40 mg/kgbw/d	Test Group 2/ F 120 mg/kgbw/d	Test Group 3/F 240 mg/kgbw/d
AG Distance	Mean	2.97 n	2.99	2.91	2.91
[mm]	S.d.	0.29	0.27	0.16	0.15
day 1 Males	N	10	10	10	7
AG Index Cubic Root	Mean	1.56 n	1.57	1.54	1.54
[---]	S.d.	0.14	0.16	0.06	0.07
day 1 Males	N	10	10	10	7
AG Distance	Mean	1.47 n	1.45	1.44	1.51
[mm]	S.d.	0.08	0.04	0.08	0.14
day 1 Females	N	10	10	10	6
AG Index Cubic Root	Mean	0.79 n	0.78	0.78	0.80
[---]	S.d.	0.04	0.02	0.02	0.07
day 1 Females	N	10	10	10	6

Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

n=DUNNETT

 

 

Table 24: Summary Pup Report Nipple development

 

		Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 40 mg/kgbw/d	Test Group 2/ F 120 mg/kgbw/d	Test Group 3/F 240 mg/kgbw/d
Nipple development	Passed
[lncidence]	-N	25	21	31	16
day 13 Males	-%	64	52	76	53
	Failed
	-N	14	19	10	14
	-%	36	48	24	47
Nipple development [%]	Mean	62.50 x+	51.17	75.00	52.38
[%]	S.d.	37.73	39.85	23.57	38.70
day 13 Males	N	10	10	10	7
Nipple Number	Mean	1.30 x+	1.08	1.46	1.19
[%]	S.d.	1.09	1.11	0.70	0.90
day 13 Males	N	10	10	10	7

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

x=WILCOX

 

 

Table 25: Summary - Pup Necropsy Observation

 

Males			Test Group 0/M 0 mg/kgbw/d	Test Group 1/M 40 mg/kgbw/d	Test Group 2/ M 120 mg/kgbw/d		Test Group 3/M 240 mg/kgbw/d
Day 0 -->13 With unscheduled data	Animals examined	N	65	60	66	42
	Animals with signs	N	0	0	0	1
		%	0.0	0.0	0.0	2.4
	General Not assessed	N	0	0	0	1
		%	0.0	0.0	0.0	2.4
	normal NAD	N	65	60	66	41
		%	100.0	100.0	100.0	97.6
Females			Test Group 0/ F 0 mg/kg bw/d	Test Group 1/ F 40 mg/kg bw/d	Test Group 2/ F 120 mg/kg bw/d	Test Group 3/ F 240 mg/kg bw/d
Day 0 -->13 With unscheduled data	Animals examined	N	57	55	60	38
	Animals with signs	N	0	1	1	1
		%	0.0	1.8	1.7	2.6
	Renal pelvis dilated	N	0	1	1	0
		%	0.0	1.8	1.7	0.0
	General Not assessed	N	0	0	0	1
		%	0.0	0.0	0.0	2.6
	normal NAD	N	57	55	60	37
		%	100.0	100.0	100.0	97.4

 

 

Conclusions:

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of the test substance to male and female Wistar rats revealed signs of systemic toxicity at a dose level of 375 mg/kg bw/d taking clinical findings in male and female animals into account.
Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 120 mg/kg bw/d for male for female Wistar rats.
The NOAEL for reproductive performance and fertility was also set to 240 mg/kg bw/d for male and female Wistar rats.
The NOAEL for developmental toxicity was 240 mg/kg bw/d.

Executive summary:

In a GLP-compliant OECD TG 422-study, the test substancewas administered daily by gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 40 mg/kg bw/d (test group 1), 120 mg/kg bw/d (test group 2) and 375 mg/kg bw/d (test group 3). Deionized waterserved as vehicle, control animals were dosed daily with the vehicle only.

The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

 During the course of the premating period, reduced food consumption and body weight loss occurred in female animals of test group 3 (375 mg/kg bw/d). In addition, one female animal of test group 3 was found dead on study day 5 and respiration sounds were observed in 2 male and 2 female animals of the same test group starting on study day 9. Therefore, the dose level was reduced to 240 mg/kg bw/d from study day 10 onwards.

 

Observations

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances.

F0 animals were mated for a maximum of two weeks after the beginning of treatment to produce a litter (F1 generation pups). As soon as sperm was detected in the vaginal smear, mating was discontinued. F0 animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the postimplantation loss in all F0 females.

A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals.

Food consumption of the F0 parents was determined regularly once weekly before and after the mating period, as well as in dams during gestation days 7, 14 and 20 and lactation days 4, 7, 10 and 13.

In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, and on postnatal days (PND) 0, 4, 7, 10 and 13.

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PNDs 1, 4, 7 and on PND 13 and their viability was recorded. On day 1 after birth the anogenital distance (AGD) was determined on all live male, female and uncertain pups. On PND 4, the individual litters were standardized in such a way that, whenever possible, each litter contains 4 male and 4 female pups (as a rule, the first 4 surviving pups/sex in each litter were taken for further rearing). On PND 13, all male F1 pups were examined for retention of nipples/areolae. The number of nipples/areolae anlagen were counted.

At necropsy on PNDs 4 and 13, all pups were sacrificed with CO₂under isoflurane anesthesia and examined macroscopically for external and visceral findings. Blood samples were taken from all surplus pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally, blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and transferred to the Laboratory Pathology for possible further processing.

Towards the end of the administration period, a functional observational battery was performed, and motor activity was measured in 5 parental animals per sex and test group. Clinico-chemical and hematological examinations were performed in 5 parental animals per sex and group.

All F0 parental animals were sacrificed by decapitation under isoflurane anesthesia and were assessed by gross pathology. Weights of selected organs were recorded, and a histopathological examination was performed.

 

Results

Analyses:

The various analyses confirmed

• the stability of the test-substance preparations for a period of at least 7 days at room temperature,
• the homogeneous distribution of the test substance in the vehicle,
• the correctness of the prepared concentrations.

Effects:

The following test substance-related, relevant findings were noted:

 

Test group 3: 375 mg/kg bw/d (from premating day 0 to 9) and 240 mg/kg bw/d (from premating day 10 onwards)

 F0 PARENTAL ANIMALS

Clinical Examinations

• Starting on premating day 9, respiration sounds were observed in 2 male animals (finding was observable until study days 17 and 18). The same was true for 2 female animals between study days 9 to 13. In addition, another male animal showed respiration sounds between study days 28 to 31.
• Significantly reduced food consumption was observed in female animals during the first week of application, i.e. -18%, as well as between premating days 7-13, i.e. -16%.
• Body weight loss by -10 g (maximum value) was observed in female animals during the first week of application. Between premating days 7-13, the animals gained weight again but were still below their starting weight at premating day 0.

Reproductive Performance, Clinical Pathology and Pathology

• No treatment-related, adverse effects were observed¹⁾.

F1 PUPS

Clinical Examinations/ Gross Findings

•  No treatment-related, adverse effects were observed.

 Testgroup 2: 120 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical Examination, Reproductive Performance, Clinical Pathology and Pathology

• No treatment-related, adverse effects were observed.

F1 PUPS

Clinical Examinations/ Gross Findings

• No treatment-related, adverse effects were observed.

Test group 1: 40 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical Examination, Reproductive Performance, Clinical Pathology and Pathology

•  No treatment-related, adverse effects were observed.

F1 PUPS

Clinical Examinations/ Gross Findings

• No treatment-related, adverse effects were observed.

Conclusion

Under the conditions of thisCombined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage ofN,N-Dimethylisopropanolamine to male and female Wistar ratsrevealed signs of systemic toxicity at a dose level of 375 mg/kg bw/d taking clinical findings in male and female animals into account.

Thus,the no observed adverse effect level (NOAEL) forgeneral systemic toxicitywas 120 mg/kg bw/d for male for female Wistar rats.

The NOAELforreproductive performanceandfertilitywas also set to 240 mg/kg bw/d formale and female Wistar rats.

TheNOAELfordevelopmental toxicitywas 240 mg/kg bw/d.

 

¹⁾Note: One female animal of test group 3 was found dead on study day 5. Because no explanation for the premature death of this individual could be given after pathological examinations and no other animal died during the administration period, its occurrence was assessed to be spontaneous in nature and not related to treatment

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

240 mg/kg bw/day

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The study is described in detail under "Effects on Fertility: Additional information".

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is not considered to be classified for reproductive toxicity under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.