Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From December 01, 2014 to March 09, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3550
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EC No 440/2008 B.7: "Repeated Dose (28 days) Toxicity (oral)", May 2008
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Trioctylphosphine oxide
EC Number:
201-121-3
EC Name:
Trioctylphosphine oxide
Cas Number:
78-50-2
Molecular formula:
C24H51OP
IUPAC Name:
trioctylphosphine oxide
Test material form:
other: Solid
Details on test material:
- Identification: Tri-n-octylphosphine oxide
- Appearance: White solid candle
- Batch Lot no.: WE8091351
- Purity/Composition: 96.2%
- Storage: At room temperature
- Stability: Stable under storage conditions until September 15, 2015 (expiry date)
- pH (1% in water, indicative range): 4.3 – 3.8

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 11 weeks (males) and 12 weeks (females),
- Housing:
- 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.
- Pre-mating: Animals were housed in groups of 5 animals/sex/cage 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.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water.
- Acclimation period: At least 5 d prior to start of treatment.

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

IN-LIFE DATES: From: December 01, 2014, To: March 09, 2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
specific gravity 1.036
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 5 h prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test substance.
- Administration: Method oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
- Dose volume: 5 mL/kg bw. Actual dose volumes were calculated according to the latest body weight.
Details on mating procedure:
Following a minimum of 14 d 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. A maximum of 14 d was allowed for mating.
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. The samples were analyzed to assess accuracy of preparation (all groups), homogeneity (lowest and highest concentration) and stability in vehicle over 7 h at room temperature under normal laboratory light conditions (lowest and highest concentrations). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
Males were exposed for 29 d, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 40-47 d, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 d of lactation (up to the day prior to scheduled necropsy).
Frequency of treatment:
Once daily for 7 d per week, approximately the same time each day with a maximum of 6 h difference between the earliest and latest dose.
Details on study schedule:
- Parturition: 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.
- Number of pups: 433 pups.
- Identification of pups: On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink.
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1,000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a 10 d dose range finding study.
- Rationale for animal assignment: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily from treatment onwards up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter.

FOOD CONSUPTION: Yes
- Time schedule: Weekly, except for males and females which were housed together for mating and for females without evidence of mating.

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters examined: Red Blood Cell (erythrocyte) count (RBC), White Blood Cell (leukocyte) count (WBC), Differential leucocyte count (DLC), Haemoglobin concentration (Hgb), Haematocrit (Hct), Mean Corpuscular (erythrocyte) Volume (MCV), Mean Corpuscular (erythrocyte) Haemoglobin (MCH), Mean Corpuscular (erythrocyte) Haemoglobin Concentration (MCHC), Reticulocyte count, Red blood cell distribution width (RDW)
- BLOOD CLOTTING TIMES: including Activated Partial Thromboplastin Time (APTT), Prothrombin Time (PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period on the day of scheduled necropsy
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters examined: Glucose, Total Bilirubin, Urea, Cholesterol, Creatinine, Sodium, Potassium, Calcium, Chloride, Total Protein, Albumin, Aspartate aminotransferase, Alanine aminotransferase, Alkaline. phosphatase, Inorganic phosphate, Bile acids.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period from lactation Day 4 onwards (all before blood sampling).
- Dose groups that were examined: all.
- Battery of functions tested: - Hearing ability, Pupillary reflex, Static righting reflex,
- 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 h 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.

GENERAL REPRODUCTION OBSERVATIONS: Yes.
- Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Palpation was used to aid in confirmation of pregnancy. 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.
Litter observations:
Each litter was examined to determine the following, if practically possible:
- Mortality / Viability: 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 pups.
- 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):
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
- All males and the selected 5 females/group were deprived of food overnight (with a maximum of 24 h) prior to planned necropsy, but water was provided.
- The following tissues and organs were retained from all animals:
- Adrenal glands, (Aorta), brain - cerebellum, mid-brain, cortex, caecum, cervix, clitoral gland, colon, coagulation gland, duodenum, epididymides, eyes (with optic nerve (if detectable) and Harderian gland), female mammary gland area, femur including joint, heart, ileum, jejunum, kidneys, (Lacrimal gland, exorbital), (larynx), liver, lung infused with formalin, lymph nodes - mandibular, mesenteric, ovaries, (pancreas), Peyer's patches [jejunum, ileum] if detectable, pituitary gland, preputial gland, prostate gland, rectum, (salivary glands - mandibular, sublingual), sciatic nerve, seminal vesicles, Skeletal muscle, (skin), spinal cord -cervical, midthoracic, lumbar, spleen, sternum with bone marrow, stomach, testes, thymus, thyroid including parathyroid if detectable, (tongue), trachea, urinary bladder, uterus, vagina.
- Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

ORGAN WEIGHTS:
- Adrenal glands, uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus, ovaries, thyroids with parathyroids, spleen.

HISTOPATHOLOGY:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4
- Slides from gross lesions
Postmortem examinations (offspring):
- Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation.
- Pups found dead during the weekend were fixed in identified containers containing 70% ethanol (from Klinipath, Duiven, The Netherlands).
- 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:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. 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). Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
Following indices were calculated: Mating index (%), Fertility index (%), Conception index (%), Gestation index (%),
Offspring viability indices:
Following indices were calculated: Percentage live males at First Litter Check, Percentage live females at First Litter Check, Percentage of postnatal loss, Viability index.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was a trend towards lower body weights and body weight gains for females at 1,000 mg/kg bw/day during post-coitum and lactation, reaching statistical significance for body weights during lactation only.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was a trend towards lower body weights and body weight gains for females at 1,000 mg/kg bw/day during post-coitum and lactation, reaching statistical significance for body weights during lactation only.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Higher alanine aminotransferase (ALAT) activity at 100 mg/kg bw/day (females only; not statistically significant), 300 mg/kg bw/day (both sexes; statistically significant in females only) and 1,000 mg/kg bw/day (both sexes).
- Higher aspartate aminotransferase (ASAT) activity at 1,000 mg/kg bw/day (both sexes).
- Higher alkaline phosphatase (ALP) at 300 and 1,000 mg/kg bw/day (males only).
- Lower total protein at 300 mg/kg bw/day (females only) and 1,000 mg/kg bw/day (both sexes), which was caused by lower albumin levels.
- Higher cholesterol at 1,000 mg/kg bw/day in both sexes (statistically significant in males only).
- Higher potassium at 300 mg/kg bw/day (not statistically significant) and 1,000 mg/kg bw/day in both sexes.
- Higher chloride at 1,000 mg/kg bw/day (females only).
- Lower calcium at 1,000 mg/kg bw/day (females only).
- Higher inorganic phosphate at 1,000 mg/kg bw/day (males only).
All remaining clinical biochemistry parameters in the treated groups remained within the normal range of biological variation.
At the individual level, a relatively high value for bile acids was recorded for one female (no. 55) at 100 mg/kg bw/day. As no females in the higher dose groups were affected and in the absence of any corroborative finding at the microscopic level, it was considered not to be toxicologically relevant.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Adverse test substance-related findings were noted in:
- The mesenteric lymph nodes starting at 100 mg/kg bw/day, consisting of vacuolation / vacuolar degeneration of macrophages. This finding was regarded adverse, due to the unusual nature, the unknown mechanism and the degenerative nature of this change of the macrophages.
- The liver starting at 300 mg/kg bw/day. The hepatocellular hypertrophy, statistical significant higher organ weights and statistical significant increases in liver enzymes were suspected to be correlated and considered to be adverse in nature.
- The statistical significant higher liver weights in females treated at 100 mg/kg bw/day were not accompanied by histopathologic or liver enzyme alterations. Therefore at this dose level, the change in liver weight was considered not to be adverse.
- The forestomach starting at 100 mg/kg bw/day in females and at 1,000 mg/kg bw/day in males. At 1,000 mg/kg bw/day, the combination of findings (erosion, hyperplasia, inflammation, edema) may reflect a response to damage of the forestomach epithelium by the test substance, including an interruption of the protective forestomach epithelium. Although only one single female was affected at 100 mg/kg bw/day, findings were considered as possible adverse in nature. The reason for this was that the edema was mostly submucosal located (similar as in the 1,000 mg/kg bw/day group), present at slight degree, and absent in the concurrent control rats.
Possible adverse test substance-related findings were noted in:
- The spleen (females) at 1,000 mg/kg bw/day consisting of decreased hematopoiesis. While, there were no changes in hematology (such as decreased haemoglobin, haematocrit or increased reticulocytes) this finding was regarded as possible adverse.

Reproductive function / performance (P0)

Reproductive performance:
no effects observed

Details on results (P0)

MORTALITY: No mortality occurred during the study period that was considered to be related to treatment with the test substance.

CLINICAL SIGNS: There were no adverse findings up to 1,000 mg/kg bw/day. Two females at 1,000 mg/kg bw/day had swelling and/or focal erythema of the ear(s) from treatment Week 3 onwards. At the low incidence and without any other corroborative finding, it was considered not to be toxicologically relevant. Piloerection was noted for two females each at 300 and 1,000 mg/kg bw/day towards the end of the treatment period. In the absence of any dose-related trend this finding was considered not to be toxicologically relevant.
Salivation seen after dosing among animals of the 100, 300 and 1,000 mg/kg bw/day dose groups was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). Incidental findings that were noted included scabs on the flews, alopecia and rales. 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.

BODY WEIGHT AND WEIGHT GAIN: There was a trend towards lower body weights and body weight gains for females at 1,000 mg/kg bw/day during post-coitum and lactation, reaching statistical significance for body weights during lactation only. Body weights and body weight gain of females treated at 100 and 300 mg/kg bw/day remained in the same range as controls over the treatment period. No toxicologically relevant changes in body weights and body weight gain were noted for males up to 1,000 mg/kg bw/day. The significantly higher mean body weight gain recorded for males at 100 mg/kg bw/day on mating Day 8 was considered to be caused by a relatively low control value rather than to be treatment related.

FOOD CONSUMPTION: Food consumption (absolute and relative to body weight) was initially lower for treated females (Groups 3 and 4) as compared to controls but recovered after the first week of treatment and even surpassed control levels in the high dose group (1,000 mg/kg bw/day) during post-coitum. Changes were relatively slight and reached statistical significance for relative food consumption of females at 1,000 mg/kg bw/day from Days 4-7 post-coitum only. As such, they were considered not to be toxicologically relevant. A trend towards lower food consumption (absolute and relative to body weight; not statistically significant) was noted for females at 1,000 mg/kg bw/day during lactation. There were no treatment related effects on food consumption for males up to 1,000 mg/kg bw/day. For high dose males a relatively high food intake was recorded for one cage during the first week of mating. There was no apparent reason for this finding.

FUNCTIONAL OBSERVATIONS: No toxicologically relevant effects on hearing ability, pupillary reflex, static righting reflex, grip strength and motor activity were observed. Motor activity data showed a similar habituation profile for all groups with high activity in the first interval that decreased over the duration of the test period. The mean values for ambulations were higher in males at 100, 300 and 1,000 mg/kg bw/day. There was also a trend towards higher mean values for total movements in treated males (Groups 2-4; not statistically significant), but no dose-related distribution could be established. The higher motor activity noted in males was considered to be due to a relatively low level of activity in the control group (both total movements and ambulations) at the beginning of the test period and hence not treatment-related. The relatively high variation in the group mean value for total movements and ambulations in females at 1,000 mg/kg bw/day was attributed to the relatively high motor activity noted for a single female (no. 72).

HAEMATOLOGY: Haematological parameters of treated rats were considered not to have been affected by treatment. The statistically significant difference seen between controls and females at 1,000 mg/kg bw/day in mean corpuscular haemoglobin (MCH) values was not considered to be toxicologically relevant as the change was relatively slight and no other related parameters were affected.

CLINICAL CHEMISTRY: The following changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Higher alanine aminotransferase (ALAT) activity at 100 mg/kg bw/day (females only; not statistically significant), 300 mg/kg bw/day (both sexes; statistically significant in females only) and 1,000 mg/kg bw/day (both sexes).
- Higher aspartate aminotransferase (ASAT) activity at 1,000 mg/kg bw/day (both sexes).
- Higher alkaline phosphatase (ALP) at 300 and 1,000 mg/kg bw/day (males only).
- Lower total protein at 300 mg/kg bw/day (females only) and 1,000 mg/kg bw/day (both sexes), which was caused by lower albumin levels.
- Higher cholesterol at 1,000 mg/kg bw/day in both sexes (statistically significant in males only).
- Higher potassium at 300 mg/kg bw/day (not statistically significant) and 1,000 mg/kg bw/day in both sexes.
- Higher chloride at 1,000 mg/kg bw/day (females only).
- Lower calcium at 1,000 mg/kg bw/day (females only).
- Higher inorganic phosphate at 1,000 mg/kg bw/day (males only).
All remaining clinical biochemistry parameters in the treated groups remained within the normal range of biological variation.
At the individual level, a relatively high value for bile acids was recorded for one female (no. 55) at 100 mg/kg bw/day. As no females in the higher dose groups were affected and in the absence of any corroborative finding at the microscopic level, it was considered not to be toxicologically relevant.

ORGAN WEIGHTS: Mean terminal body weights were statistically significantly lower at 1,000 mg/kg bw/day in females (relative difference from control value: 8%).
The following changes in organ weights (absolute and relative to body weight) distinguished treated animals from control animals:
- Higher liver weights at 100 mg/kg bw/day (females only; relative weights 13% higher than in concurrent controls), 300 mg/kg bw/day (relative weights 12% higher for males, 21% higher for females) and 1,000 mg/kg bw/day (relative weights 16% higher for males, 40% higher for females).
- Lower thymus weight at 1,000 mg/kg bw/day (females only; relative weights 34% lower).
- Lower spleen weight at 1,000 mg/kg bw/day (females only; relative weights 21% lower).
All remaining organ weights and organ to body weight ratios among the dose groups were similar to control levels.

GROSS PATHOLOGY: Macroscopic observations at necropsy did not reveal any toxicologically relevant alterations. Discoloration (reddish, dark-red or red-brown) of the mesenteric lymph nodes was noted for two females each at 300 mg/kg bw/day (nos. 61 and 63) and 1,000 mg/kg bw/day (nos. 74 and 75). It correlated to erythrocytes, intrasinusoidal / erythrophagocytosis (minimal to slight) at the microscopic level which was also noted for another selected high dose female (no. 78). No toxicological relevance was attached to this finding as also two selected control females (nos. 47 and 50) had the same microscopic finding. The incidence of all remaining findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a doserelated incidence trend.

HISTOPATHOLOGY: NON-NEOPLASTIC: Adverse test substance-related findings were noted in:
- The mesenteric lymph nodes starting at 100 mg/kg bw/day, consisting of vacuolation / vacuolar degeneration of macrophages. This finding was regarded adverse, due to the unusual nature, the unknown mechanism and the degenerative nature of this change of the macrophages.
- The liver starting at 300 mg/kg bw/day. The hepatocellular hypertrophy, statistical significant higher organ weights and statistical significant increases in liver enzymes were suspected to be correlated and considered to be adverse in nature.
- The statistical significant higher liver weights in females treated at 100 mg/kg bw/day were not accompanied by histopathologic or liver enzyme alterations. Therefore at this dose level, the change in liver weight was considered not to be adverse.
- The forestomach starting at 100 mg/kg bw/day in females and at 1,000 mg/kg bw/day in males. At 1,000 mg/kg bw/day, the combination of findings (erosion, hyperplasia, inflammation, edema) may reflect a response to damage of the forestomach epithelium by the test substance, including an interruption of the protective forestomach epithelium. Although only one single female was affected at 100 mg/kg bw/day, findings were considered as possible adverse in nature. The reason for this was that the edema was mostly submucosal located (similar as in the 1,000 mg/kg bw/day group), present at slight degree, and absent in the concurrent control rats.
Possible adverse test substance-related findings were noted in:
- The spleen (females) at 1,000 mg/kg bw/day consisting of decreased hematopoiesis. While, there were no changes in hematology (such as decreased haemoglobin, haematocrit or increased reticulocytes) this finding was regarded as possible adverse.

REPRODUCTION FINDINGS: No toxicologically relevant effects on reproductive parameters were noted. Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment. The slightly lower group means for corpora lutea and implantation sites in high dose females were considered not to be toxicologically relevant as values remained within the normal range of biological variation.
For females nos. 50 (Group 1) and 78 (Group 4), the number of pups was slightly higher than the number of implantations and/or corpora lutea. This was considered caused by normal resorption of these areas as these enumerations were performed on respectively Days 5 and 6 of lactation.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproductive toxicity was observed with treatment up to 1,000 mg/kg bw/day.
Dose descriptor:
LOAEL
Remarks:
maternal toxicity
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: mensenteric lymph nodes toxicity at 100 mg/kg bw /day and above in both sexes

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
An increase in postnatal loss and a correspondingly lower viability index were seen for females at 1,000 mg/kg bw/day. The number of pups that were found dead or missing during lactation was 5(3) and 17(3) pups (litter) in the control and 1,000 mg/kg bw/day groups. In the high dose group, the majority of the pups came from two litters: 6/11 pups in one litter were missing on Day 3 and all 10 pups in another litter were found dead or missing on Day 2. Pups missing were most likely cannibalised. In addition, 3/5 remaining pups in the first litter that survived until scheduled necropsy had a developmental delay (lean appearance). Many of these dead pups had no milk in their stomach. No abnormalities were seen in the mammary glands of the dams. There were no signs of ill health in other pups from this high dose level. No pups died or went missing in the 100 and 300 mg/kg bw/day groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Lower body weights of pups were recorded at 1,000 mg/kg bw/day on Days 1 and 4 of lactation. No effects on body weights of pups were noted at 100 and 300 mg/kg bw/day.
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

- An increase in postnatal loss and a correspondingly lower viability index were seen for females at 1,000 mg/kg bw/day. The number of pups that were found dead or missing during lactation was 5(3) and 17(3) pups (litter) in the control and 1,000 mg/kg bw/day groups. In the high dose group, the majority of the pups came from two litters: 6/11 pups in one litter were missing on Day 3 and all 10 pups in another litter were found dead or missing on Day 2. Pups missing were most likely cannibalised. In addition, 3/5 remaining pups in the first litter that survived until scheduled necropsy had a developmental delay (lean appearance). Many of these dead pups had no milk in their stomach. No abnormalities were seen in the mammary glands of the dams. There were no signs of ill health in other pups from this high dose level. No pups died or went missing in the 100 and 300 mg/kg bw/day groups.

- Lower body weights of pups were recorded at 1,000 mg/kg bw/day on Days 1 and 4 of lactation. No effects on body weights of pups were noted at 100 and 300 mg/kg bw/day.
- No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study i.e. gestation index and duration, parturition, and maternal care, clinical signs and macroscopic abnormalities for pups surviving until scheduled necropsy.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental toxicity (increased post natal loss and lower pup body weights) was seen at 1,000 mg/kg bw/day only

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Hepatic toxicity was noted for animals of both sexes at 300 and 1,000 mg/kg bw/day. This was supported by changes in clinical pathology endpoints (increase of ALAT, ASAT, ALP, cholesterol and decrease of albumin, total protein, among others), increased liver weights (relative weights 12% and 21% higher for males and females at 300 mg/kg bw/day, and 16% and 40% higher for males and females at 1,000 mg/kg bw/day) and hepatocellular hypertrophy at the microscopic level. Taken together, the findings at 300 and 1,000 mg/kg bw/day were considered to be adverse. The statistical significant higher liver weights in females treated at 100 mg/kg bw/day were not accompanied by changes at the enzymatic or morphological level. Therefore at this low dose level, the change in liver weight was considered not to be adverse.

 

 

In addition, adverse findings were noted in the forestomach starting at 100 mg/kg bw/day in females and at 1,000 mg/kg bw/day in males. At 1,000 mg/kg bw/day, the combination of findings (erosion, hyperplasia, inflammation, edema) may reflect a response to damage of the forestomach epithelium by the test substance, including an interruption of the protective forestomach epithelium. Although only one single female was affected at 100 mg/kg bw/day, findings were considered as possible adverse in nature. The reason for this was that the edema was mostly submucosal located (similar as in the 1,000 mg/kg bw/day group), present at slight degree, and absent in the concurrent control rats.

 

 

Another target organ were the mesenteric lymph nodes starting at 100 mg/kg bw/day in both sexes. The finding of vacuolation/vacuolar degeneration of the macrophages was regarded adverse due to the unusual nature, the unknown mechanism and the degenerative nature of this change. Possible adverse test substance-related findings were noted in the spleen (females) at 1,000 mg/kg bw/day consisting of lower organ weight correlated with decreased hematopoiesis at the microscopic level. While, there were no corroborative changes in hematology (such as decreased haemoglobolin and haematocrite, or increased numbers of reticulocytes) this finding was regarded as possible adverse.

 

 

Non-adverse test substance-related findings were noted in the thyroid glands starting at 300 mg/kg bw/day in both sexes. The noted increased incidence and severity of follicular cell hypertrophy may reflect an increase in thyroxin production in response to feedback mechanisms as a result of increased turnover of thyroxin by hypertrophic hepatocytes. The lower thymus weight observed in females at 1,000 mg/kg bw/day with correlating atrophy at the microscopic level was considered to be secondary to stress caused by the treatment with the test substance.

 

 

No treatment-related toxicologically significant changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance, functional observations, haematology, macroscopic examination).

Applicant's summary and conclusion

Conclusions:
The reproduction NOAEL was established at 1,000 mg/kg bw/day, while developmental NOAEL was established at 300 mg/kg bw/day based on the findings of increased post natal loss and lower pup body weights seen at 1,000 mg/kg bw/day only.
Executive summary:

study was conducted to assess the reproductive toxicity of the test substance following repeated administration according to OECD Guideline 422, in compliance with GLP. Based on the results of a 10 d dose range finding study, male and female SPFbred Wistar Han rats received dose levels of 100, 300 and 1,000 mg/kg bw/day through oral gavage. One control group and three treated groups were tested, each consisting of 10 males and 10 females. Males were exposed for 29 d, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 40-47 d, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 d of lactation. The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), clinical pathology (end of treatment), macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, pre-coital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy). No reproduction toxicity was observed up to the highest dose level tested (1,000 mg/kg bw/day). No treatment-related toxicologically significant changes were noted in any of the following reproductive parameters investigated in this study i.e. mating, fertility and conception indices, pre-coital time, and numbers of corpora lutea and implantation sites, spermatogenic profiling, and histopathological examination of reproductive organs. An increase in postnatal loss and a correspondingly lower viability index were seen for females at 1,000 mg/kg bw/day. The number of pups that were found dead or missing during lactation was 5(3) and 17(3) pups (litter) in the control and 1,000 mg/kg bw/day groups. In the high dose group, the majority of the pups came from two litters: 6/11 pups in one litter were missing on Day 3 and all 10 pups in another litter were found dead or missing on Day 2. Pups missing were most likely cannibalised. In addition, 3/5 remaining pups in the first litter that survived until scheduled necropsy had a developmental delay (lean appearance). Many of these dead pups had no milk in their stomach. No abnormalities were seen in the mammary glands of the dams. There were no signs of ill health in other pups from this high dose level. No pups died or went missing in the 100 and 300 mg/kg bw/day groups. However, lower body weights of pups were recorded at 1,000 mg/kg bw/day on Days 1 and 4 of lactation. No effects on body weights of pups were noted at 100 and 300 mg/kg bw/day. No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study i.e. gestation index and duration, parturition, and maternal care, clinical signs and macroscopic abnormalities for pups surviving until scheduled necropsy. Based on the above information, the reproduction NOAEL was established at 1,000 mg/kg bw/day, while developmental NOAEL was established at 300 mg/kg bw/day based on the findings of increased post natal loss and lower pup body weights seen at 1,000 mg/kg bw/day only (Peter B, 2015)