Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
March, 1996
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Version / remarks:
July, 2000
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July, 1995
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)
Version / remarks:
July, 2000
Qualifier:
according to guideline
Guideline:
other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
May, 2008
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3050 (Repeated dose 28-day oral toxicity study in rodents)
Version / remarks:
July, 2000
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1,6-Hexanediamine, N1,N1,N6,N6-tetramethyl-, propoxylated (>1 < 4,5 mol PO)
EC Number:
605-146-4
Cas Number:
158451-78-6
Molecular formula:
C16H38N2O2
IUPAC Name:
1,6-Hexanediamine, N1,N1,N6,N6-tetramethyl-, propoxylated (>1 < 4,5 mol PO)
Specific details on test material used for the study:
- Name of test material (as cited in study report): 1,6-Hexanediamine, N1,N1,N6,N6-tetramethyl-, propoxylated
- Appearance: clear yellowish liquid
- Batch: 0011784570
- Purity/composition: Water: 13.9% 1,6-Hexanediamine, N1,N1,N6,N6-tetramethyl-,propoxylated, 3.4% Propylene glycol, 82.7% Water
- Test substance storage: at room temperature
- Expiry date: 31 October 2015
- Specific gravity/density: 1.02 - 1.03 g/cm3 (20 *C)
- pH: 14

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
outbred, SPF-Quality
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 12 weeks
- Weight at study initiation: Males: 330 - 332 g; Females: 209 - 213 g
- Housing:
Pre-mating: Animals were housed in groups of 5 animals of the same sex in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
General: Sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hitemp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY: Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(Elix, Millipore S.A.S., Molsheim, France)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test substance (using a value of 1.025 g/cm3). No adjustment was made for specific gravity/density of the vehicle. No correction was made for the purity/composition of the test substance.

VEHICLE
- Amount of vehicle: 10 mL/kg
Details on mating procedure:
Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated.

The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of dose preparations were taken at the test facility on a single occasion during the treatment period (formulations were prepared and sampled on 10 March 2015). The samples were dispatched on dry ice to the test facility where they were analysed to assess accuracy of preparation, homogeneity and stability in vehicle over 5 hours at room temperature under normal laboratory light conditions. Analysis was performed using LC-MS/MS. The lower limit of quantitation was 1.00 mg/g of the validated method and the calibration curve ranged from 1.00 to 25.0 mg/L.


Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 41-55 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Some of the femaleswere not dosed on post-coitum Day 22 or Day 23 as these females were littering at the time of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.
Frequency of treatment:
Once daily for 7 days per week
Details on study schedule:
Not applicable
Doses / concentrationsopen allclose all
Dose / conc.:
63 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on a 14-day dose range finding study in which dose levels of 300 and 1000 mg/kg bw/day were tested. Animals given 1000 mg/kg bw/day had reduced body weight gain, most markedly at the start and end of the study, and slightly lower food consumption at the end of the study. They had no clinical signs of toxicity, macroscopic findings or organ weight changes. Some differences in clinical pathology values were noted, most of which were minor. No adverse findings were seen at 300 mg/kg bw/day.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. These clinical observations were made after dosing (at no specific time point as there was no peak occurrence of clinical signs after dosing in the dose range finding study.
- The time of onset, grade and duration of any observed sign were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

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

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day.
- Time schedule for examinations: weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE:
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

HAEMATOLOGY:
- Time schedule for collection of blood: at the end of the treatment period on the day of scheduled necropsy between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, overnight (maximum of 24 hours)
- How many animals: 5 animals/sex/group
- Parameters examined: White blood cells, Differential leukocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: at the end of the treatment period on the day of scheduled necropsy between 7.00 and 10.30 a.m.
- Animals fasted: Yes, overnight (maximum of 24 hours)
- How many animals: 5 animals/sex/group
- Parameters examined: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Bile acids, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate

NEUROBEHAVIOURAL EXAMINATION:
-The following functional observation tests were performed on each individual animal of the selected 5 animals/sex/group:
- hearing ability, pupillary reflex and static righting reflex (Score 0 = normal/present, score 1 = abnormal/absent).
- fore- and hind-limb grip strength were recorded as the mean of three measurements (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
- locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.
- The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period from lactation Day 4 onwards (all before blood sampling). These tests were performed after observation for clinical signs (incl. arena observation, if applicable) at no specific time point, but within a similar time period after dosing for the respective animals.

GENERAL REPRODUCTION DATA:
Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.
Sperm parameters (parental animals):
Parameters examined in male parental generation: testes (histologically), testis weight, epididymis weight
Litter observations:
- Mortality/viability: The numbers of live and dead pups were determined on Day 1 of lactation and daily thereafter. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made for all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals. Following completion of the mating period (a minimum of 28 days of dose administration)
- Maternal animals: All surviving animals. Females which delivered: lactation day 5-6; females which failed to deliver: post-coitum day 27 (female with evidence of mating); females with total litter loss: withiin 24 hours of litter loss

GROSS NECROPSY
After the animals were exsanguinated they were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Samples of the following tissues and organs were collected from all animals and fixed in 10% buffered formalin and examined: Adrenal glands, Peyer's patches [jejunum, ileum] if detectable, Brain (cerebellum, mid-brain, cortex), Pituitary gland, Caecum Preputial gland, Cervix Prostate gland, Clitoral gland Rectum, Colon, Coagulation gland, Ovaries, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides, Skeletal muscle, Eyes (with optic nerve (if detectable) and Harderian gland), Spinal cord (-cervical, midthoracic, lumbar), Female (and male) mammary gland area, Spleen, Femur including joint, Sternum with bone marrow, Heart, Stomach (forestomach and glandular stomach), Ileum, Testes, Jejunum, Thymus, Kidneys, Thyroid including parathyroid if detectable, Trachea, Liver, Urinary bladder, Lung (infused with formalin Uterus), Lymph nodes (- mandibular, mesenteric), Vagina, All gross lesions. The numbers of former implantation sites and corpora lutea were recorded for all paired females.

ORGAN WEIGHTS:
Terminal body weight was recorded from all surviving animals. The following organ weights were
recorded from the following surviving animals on the scheduled day of necropsy (from 5 animals/sex/group): Adrenal glands, Brain, Epididymides, Heart, Kidnesy, Liver, Ovaries, Spleen, Testes, Thymus, Uterus (including cervix), Prostate, Seminal vesicles including coagulating glands (Weighed when fixed for at least 24 hours), Thyroid including parathyroid

HISTOPATHOLOGY: tissues examined:
- From groups 1 and 4 (dose levels of 0 and 1000 mg/kg bw/day): Adrenal glands, Pancreas, Aorta, Peyer's patches [jejunum, ileum] if detectable, Brain (cerebellum, mid-brain, cortex), Pituitary gland, Caecum Preputial gland, Cervix Prostate gland, Clitoral gland Rectum, Colon (Salivary glands - mandibular, sublingual), Coagulation gland, Ovaries, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides, Skeletal muscle, Eyes (with optic nerve (if detectable) and Harderian gland), Skin, Spinal cord (-cervical, midthoracic, lumbar), Female (and male) mammary gland area, Spleen, Femur including joint, Sternum with bone marrow, Heart, Stomach (forestomach and glandular stomach), Ileum, Testes, Jejunum, Thymus, Kidneys, Thyroid including parathyroid if detectable, Lacrimal gland (exorbital), Tongue, Larynx, Trachea, Liver, Urinary bladder, Lung (infused with formalin Uterus), Lymph nodes (- mandibular, mesenteric), Vagina, Nasopharynx, Esophagus, All gross lesions
- The additional slides of the testes of all males of Groups 1 and 4 and all males that failed to sire to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The stomach of the selected males and females of Groups 2 and 3 (dose levels of 63 and 250 mg/kg bw/day), the heart, adrenal glands and mesenteric lymph nodes of the selected males of Groups 2 and 3, and the thymus, ileum, spleen
and caecum of the selected females of Groups 2 and 3, based on suspected treatment-related microscopic findings in these organs.
- The reproductive organs of all animals of Groups 1 and 4 and all males that failed to sire and all females that failed to deliver healthy pups and the mammary glands of females with total litter loss
Postmortem examinations (offspring):
Pups surviving to planned termination were killed by decapitation on Days 5-6 of lactation. Pups found dead during the weekend were fixed in identified containers containing 70% ethanol (Klinipath, Duiven, The Netherlands) if not necropsied on the same day. All pups were sexed and descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
Statistics:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.
Reproductive indices:
- Mating index (%) = number of females mated / number of females paired x 100
- Fertility index (%) = number of pregnant females / number of females paired x 100
- Conception index (%) = Number of pregnant females / number of females mated x 100
- Gestation index (%) = Number of females bearing live pups / number of pregnant females x 100
- Duration of gestation = number of days between confirmation of mating ant the beginning of parturition
Offspring viability indices:
- Percentage live males at First Litter Check = number of live male pups at First Litter check / number of live pups at First Litter Check x 100
- Percentage of live females at First Litter Check = number of live female pups at First Litter Check / Number of live pups at First Litter Check x 100
- Percentage of postnatal loss = Number of dead pups before planned necropsy / number of live pups at First Litter Check x 100
- Viability index = number of live pups before planned necropsy / number of pups born alive x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- No clinical signs of toxicity were noted up to 1000 mg/kg bw/day.
- Incidental findings that were noted included alopecia and salivation. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment with the test substance. Two females were sacrificed on Day 2 or 3 of lactation because of total litter loss (one from Group 1, one from Group 4).
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- Males at 1000 mg/kg bw/day had lower body weights and lower body weight gain from Day 1 of the mating period onwards. The differences from control values were statistically significant except for the lower mean body weight on Day 1 of the mating period. Towards the end of the study (Day 15 of the mating period), mean body weight of males at 1000 mg/kg bw/day was 7% lower than that of controls).
- Males at 250 mg/kg bw/day showed a statistically significantly lower body weight gain on Day 8 of the mating period. The difference from controls was slight and mean body weights of these males did not differ significantly from those of controls. Therefore, this finding was considered not to be toxicologically relevant.
- Females showed no treatment-related changes in body weight or body weight gain up to 1000 mg/kg bw/day. Statistically significantly higher body weight gain values were noted in females at 250 mg/kg bw/day on post-coitum Days 17 and 20. These findings were not attributed to treatment because there was no dose-related response.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Males at 1000 mg/kg bw/day had lower food consumption before allowance for body weight between Days 8-15 of the pre-mating and mating periods.
- Food consumption of these males after allowance for body weight was slightly lower between Days 8-15 of the pre-mating period. Food consumption of females before or after allowance for body weight was similar between treated and control animals throughout the study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- Haematological parameters of treated rats were not affected by treatment. Statistically significantly higher numbers of platelets were noted in males at 250 and 1000 mg/kg bw/day. These differences were not attributed to treatment because there was no dose-related response. Moreover, the concurrent control value was at the lower end of the normal range.
- A statistically significantly lower percentage of reticulocytes was noted in males at 250 mg/kg bw/day. In the absence of a dose-related response this finding was not attributed to treatment.
- Further it was noted that males at 1000 mg/kg bw/day had a higher mean value for neutrophils and lower mean values for lymphocytes and WBC. This was due to abnormal values of one male coming from Group 4. The other males treated at 1000 mg/kg bw/day had normal white blood cell values. Therefore, it was concluded that no toxicologically relevant changes occurred in haematology values of treated rats.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
- Clinical biochemistry parameters of treated rats were not affected by treatment. Males at 1000 mg/kg bw/day had a lower mean value for total protein and a higher mean value for bile acids. The differences from controls were not statistically significant and were particlularly due to a low (total protein) or high (bile acids) value in a single male animal. Therefore, these findings were considered not to be related to treatment.
- Further it was noted that the mean value for ALP was lower in males at 1000 mg/kg bw/day and that the mean glucose value was higher in females at this dose level. These findings were not attributed to treatment because the differences from controls were not statistically significant and in the normal range.
- A statistically significantly lower plasma level of inorganic phosphate noted in males treated at 250 mg/kg bw/day was not attributed to treatment because there was no dose-related response.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
- Hearing ability, pupillary reflex, static righting reflex and grip strength were not adversely affected by treatment. Mean forelimb grip strength and, to a lesser extent, mean hind limb grip strength values were lower in males at 1000 mg/kg bw/day. The differences from controls were not statistically significant and remained in the normal range for male rats of this age and strain. Therefore, these findings were considered not to be toxicologically relevant.
- The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period. The mean value for amubulations was statistically significantly higher in males at 250 mg/kg bw/day. As there was no dose-related response, this finding was not attributed to treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related microscopic findings were noted in forestomach (both sexes) and cecum (females).

Test item-related forestomach lesions were noted at 1000 mg/kg bw/day and consisted of:
- Erosion/ulceration in 3/8 males (moderate) and 2/6 females (marked). The erosion/ulceration was in all cases accompanied by the presence of significant submucosal granulation tissue.
- Lymphogranulocytic inflammation in 4/8 males (3 slight, 1 moderate) and 2/6 females (1 moderate, 1 marked).
- Edema in 4/8 males (1 minimal, 2 slight, 1 moderate) and 1/6 female (1 slight).
- Squamous cell hyperplasia in 5/8 males (2 minimal, 2 slight, 1 moderate) and 5/6 females (4 minimal, 1 slight).
- Hyperkeratosis at increased severity in 3/6 females (slight).

Test item-related findings were noted in the cecum of females at 1000 mg/kg bw/day and consisted of:
- Increased incidence of mucosal hypertrophy in 3/5 females (3 minimal) at 1000 mg/kg bw/day compared to 1/5 females at 250 mg/kg bw/day (1 minimal) and 0/5 at 63 and 0 mg/kg bw/day.

Findings of note were recorded in the thymus of females at 250 and 1000 mg/kg bw/day and consisted of:
- Increase in incidence of increased lymphocytolysis in 2/6 females at 1000 mg/kg bw/day compared to 1/5 females at 250 mg/kg bw/day and 0/5 at 63 and 0 mg/kg bw/day.

The remainder of the histologic changes were considered to be incidental findings. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Spermatogenic staging profiles were normal for all males examined.
Reproductive performance:
no effects observed
Description (incidence and severity):
- No toxicologically relevant effects on reproductive parameters were noted. The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment. The numbers of pregnant females were 10, 9, 10 and 10 at 0, 63, 250 and 1000 mg/kg bw/day. For one female (Group 2) the number of pups was slightly higher than the number of implantations. This was considered to be due to normal resorption of these areas as these enumerations were performed on Day 5 of lactation.
- There was one couple of the 63 mg/kg/day group with no offspring. Total litter loss was observed in one female of the control group and in one female of the 1000 mg/kg bw/day group. No abnormalities were seen in the reproductive organs and/or mammary glands, which could account for their lack of (healthy) offspring.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
other: overall systemic toxicity

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Pups that went missing or were found dead showed no clinical signs, except for one pup of 1 female (Group 4) that had no milk in the stomach and a lean appearance on Day 2 of lactation. Incidental findings among surviving pups included blue spot(s) on the nose, mouth, neck or snout, scabs on the snout, and pale appearance. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
At the first litter check, two pups of the control group (1 female had one dead pup only) and one pup at 1000 mg/kg were found dead. During lactation, three pups at 1000 mg/kg went missing (1 animal, total litter loss). Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were unaffected by treatment.
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:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic findings in pups that were found dead included beginning or advanced autolysis and absence of milk in the stomach. No macroscopic findings were noted among surviving pups. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

- At 1000 mg/kg bw/day, the sex ratio of the pups indicated more male than female pups (63% male pups versus 58% in the control group). This sex ratio was within the range observed in control groups of the laboratory (up to 64% males). Moreover, based on the number of pups there was no indication of selective elimination of females. Therefore, the sex ratio was considered not to be affected by treatment.
- No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed. The number of females with living pups on Day 1 of lactation was 9, 9, 10 and 10 at 0, 63, 250 and 1000 mg/kg bw/day.

GESTATION
The gestation index and duration of gestation were unaffected by treatment up to 1000 mg/kg bw/day.

PARTURITION/MATERNAL CARE
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

EARLY POSTNATAL PUP DEVELOPMENT
Number of dead and living pups at first litter check, postnatal loss and viability index were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

Effect levels (F1)

Key result
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

ANALYSIS OF DOSE PREPARATIONS

- No test substance was detected in the Group 1 formulations.

- The concentrations analysed in the formulations of Groups 2, 3 and 4 were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%).

- The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

- Formulations at the entire range were stable when stored at room temperature under normal Laboratory light conditions for at least 5 hours (i.e. relative difference ≤ 10%).

Applicant's summary and conclusion