Phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters, salts with 2,2'-iminodiethanol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Phosphoric acid, mixed decyl and octyl esters, compds. with diethanolamine

Cas Number:

68425-57-0

Molecular formula:

not applicable

IUPAC Name:

Phosphoric acid, mixed decyl and octyl esters, compds. with diethanolamine

Test material form:

solid: crystalline

Details on test material:

Name: Phosphoric acid, mixed decyl and octyl esters, compds. with diethanolamine
CAS No.: 68425-57-0
Physical state: white solid at 20 °C (turbid, non-pourable paste)

Specific details on test material used for the study:

Name: Phosphoric acid, mixed decyl and octyl esters, compds. with diethanolamine
CAS No.: 68425-57-0
Physical state: white solid at 20 °C (turbid, non-pourable paste)
Batch No.: PU61810016
Re-certification date of batch: 16 June 2018
Purity: 100 % (UVCB)
Moisture, % (KF) 1,2
pH, 10% in dW 7,2
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous
Age at the start of the treatment period: approx. 14-15 weeks old
Body weight at the allocation of the animals to the experimental groups:
males: 365 - 407 g (mean: 386.70 g, ± 20 % = 309.36 – 464.04 g)
females: 213 - 251 g (mean: 232.68 g, ± 20 % = 186.14 – 279.21 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to the German Act on Animal Welfare the animals were bred for experimental purposes. This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities. Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 +- 3 °C
- Relative humidity: 55  10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Animals were housed in groups of 5 animals / sex / cage in IVC cages (double decker, type GR1800) during the premating period for both males and females and during post-mating period for males depending on the mating status. During mating
period males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages and males were returned to their original cage. In each cage Altromin saw fibre was used as bedding.
- Makrolon tunnels will be provided for all males and for females until GD 18
- Nesting material were provided latest on GD 18 for all mated females
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Aqua ad injectionem

Details on mating procedure:

Mating was performed using a ratio of 1:1 (male to female) (if possible). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the mating. If the vaginal smear of a particular female was not found to be
sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation. The cages were arranged in such a way that possible effects due to cage placement were minimised.

Details on analytical verification of doses or concentrations:

Concentration analysis

Concentration analysis of formulation samples was determined at three concentrations, 5 mg/mL, 15 mg/mL and 50 mg/mL in study weeks 1, 3, 5 and in the last week of the study. The mean recoveries observed for the LD dose group was between 102.1 % and 115.0 % of the nominal value, between 102.0 % and 109.6 % for the MD dose group and between 104.0 % and 108.5 % of the nominal value for HD dose group. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 105.8 %, 106.1 %, and 106.7% of the nominal concentration, respectively. Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 15 %.

Homogeneity
Homogeneity of formulation samples was determined at three concentrations, 5 mg/mL, 15 mg/mL and 50 mg/mL, in study weeks 1, 3, 5 and the last week of the study. The coefficients of variation of the different sampling locations (top, middle, bottom) was between 0.3 % and 1.2 % in LD dose group, between 0.1 % and 1.3 % in MD dose group and between 1.1 % and 1.7 % in HD dose group. All samples were homogenous, as COV was below or equal 10 %.

Duration of treatment / exposure:

The test item is orally administered daily, i.e. 7 days per week in graduated doses to several groups of test animals (male and female), one dose level per group with a maximum exposure of 63 days in total in females (at least 14 days pre-mating, up to 14 days mating, approximately 22 days of gestation and up to post-natal day 12). Males are dosed for a minimum of four weeks until up to one day before the scheduled sacrifice (this includes a minimum of two weeks prior to mating, during the mating period and up to two weeks post-mating).

Frequency of treatment:

The test item is orally administered daily, i.e. 7 days per week in graduated doses to several groups of test animals (male and female).

Details on study schedule:

The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days, i.e. during 14 or 15 days of pre-mating and maximum 14 days of mating in both males and females. Thereafter, females were treated during the gestation period and up to post-natal day 12. Males were dosed after the mating period until the minimum total dosing period of 28 days is completed.

No. of animals per sex per dose:

100 animals (40 males and 60 females) were included in the study. 60 females were screened for regular estrous cycles for 14 days before the treatment initiation and only 40 females (10 females/ group) showing regular estrous cycles were continued in the study. Remaining not selected 20 females were discarded without any observations or used for other appropriate studies/procedures.

Control animals:

yes, concurrent vehicle

Details on study design:

The aim of this study was to assess the possible effects of Prüfmuster on male and female fertility and embryofetal development after repeated dose administration in Wistar rats. The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. Animals of an additional control group were handled identically as the dose groups but received the vehicle used in this study.

Positive control:

no positive control

Examinations

Parental animals: Observations and examinations:

Clinical Observations
General clinical observations were made at least once a day, preferably at the same time each day except for the pups on a single day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily. Once before the first exposure, and at least once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil
size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

Pathology
Vaginal smears were examined on the day of necropsy to determine the stage of estrous cycle. Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice. Non-pregnant females were sacrificed on study day 26 using the sperm-positive vaginal smear as an evidence of mating. All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), the thyroid/parathyroid glands and all organs showing macroscopic lesions of all adult animals were preserved in 4 % neutral-buffered formaldehyde, except for testes and epididymides which were preserved in modified Davidson’s Solution for 24 hours and then transferred to 70 % ethanol. The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. The number of corpora lutea and implantation sites was recorded for any females sacrificed 26 days after the end of the mating period with no evidence of mating and for any females sacrificed on day 26 post-coitum due to non-delivery.

Organ Weights
The wet weight of the organs of 5 randomly selected male and female animals (only lactating females were evaluated) from each group was recorded as soon as possible. Paired organs were weighed together. Organ weights of animals found dead or euthanised for animal welfare reasons were not recorded. Reproductive organs (testes, epididymides, prostate with seminal vesicles and coagulating glands, uterus with cervix and ovaries) were weighed from all animals. Thyroid/parathyroid glands from 1 pup/sex/litter/group (if possible) (sacrificed on PND 13) and from all adult males and females were preserved. Weight of thyroid/parathyroid glands was measured after fixation.

Organs to be Weighed at Necropsy: testes (paired weight), uterus with cervix, epididymides (paired weight), ovaries (paired weight), prostate, seminal vesicles and coagulating glands (complete weight) thymus,thyroid/parathyroid glands (from 1 pup/sex/litter/group and from all adult males and females) - will be weighed after fixation (complete weight), liver, kidneys (paired weight), spleen,adrenal (paired weight), brain, pituitary gland, heart.

The following tissues from the five randomly selected male and female animals were preserved in 4 % neutral-buffered formaldehyde except for eyes, testes and epididymides which were fixed in Modified Davidson fixative for approximately 24
hours before they will be transferred to 70 % ethanol.

Necropsy

Tissue / Preserved at Necropsy / Histopathological Examination
adrenal glands x x
all gross lesions x x
aorta x --
brain (incl. medulla/pons, cerebellar and cerebral
cortex) x x
caecum x x
colon x x
duodenum x x
epididymides x x
eyes x x
femur with knee joint x --
Harderian glands x --
heart x x
ileum (including Peyer´s patches) x x
jejunum x x
kidneys x x
liver x x
lungs x x
lymph nodes (axillary) x --
lymph nodes (mandibular) x x
lymph nodes (mesenteric) x x
mammary gland area (male and female) x --
oesophagus x --
optic nerves x --
ovaries x x
oviducts x --
pancreas x --
pituitary x --
prostate and seminal vesicles with coagulating
glands as a whole x x
rectum x x
salivary glands (sublingual, submandibular) x --
sciatic nerve x x
skeletal muscle x x
skin x --
spinal cord (cervical, thoracic and lumbar
segments) x x
spleen x x
sternum (with bone marrow) x x
stomach x x
testes x x
thymus x x
thyroid/parathyroid glands x x
tongue x --
trachea x x
ureters x --
urinary bladder x x
uterus with cervix and vagina x x

All animals found dead and/or intercurrently euthanised for animal welfare reasons were subjected to a gross necropsy and the organs preserved for a histopathological examination.

Oestrous cyclicity (parental animals):

Estrous cycles were monitored before treatment initiation to select for the study females with regular estrus cyclicity. Vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating.

Sperm parameters (parental animals):

For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.

Litter observations:

The duration of gestation was recorded and is calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after the delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted and sexed and litters weighed within 24 hours of littering (PND 0) and on PND 4 and PND 13. Live pups were identified by tattooing. In addition to the observations of the parent animals, any abnormal behaviour of the offspring were recorded. The anogenital distance (AGD) of each pup was measured on PND 0. Pup body weight measured on PND 0 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD/Cube root of pup weight). The number of nipples/areolae in male pups was counted on PND 12.

Postmortem examinations (parental animals):

Pathology: All surviving males were sacrificed any time after the completion of the mating period (after a minimum dosing period of 28 days) and females were sacrificed on the respective PND 13 by using anesthesia (ketamine/xylazine). Vaginal smears were examined on the day of necropsy to determine the stage of estrous cycle. Non-pregnant females were sacrificed on study day 26 using the sperm-positive vaginal smear as an evidence of mating. All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), the thyroid/parathyroid glands and all organs showing macroscopic lesions of all adult animals were preserved in 4 % neutral-buffered formaldehyde, except for testes and epididymides which were preserved in modified Davidson’s Solution for 24 hours and then transferred to 70 % ethanol. The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. The number of corpora lutea and implantation sites was recorded for any females sacrificed 26 days after the end of the mating period with no evidence of mating and for any females sacrificed on day 26 post-coitum due to non-delivery.

Organ Weights: The wet weight of the organs of 5 randomly selected male and female animals (only lactating females were evaluated) from each group was recorded as soon as possible. Paired organs were weighed together. Organ weights of animals found dead or euthanised for animal welfare reasons were not recorded. Reproductive organs (testes, epididymides, prostate with seminal vesicles and coagulating glands, uterus with cervix and ovaries) were weighed from all animals. All animals found dead and/or intercurrently euthanised for animal welfare reasons were subjected to a gross necropsy and the organs preserved for a histopathological examination.

Histopathology: A full histopathology was carried out on the preserved organs and tissues of all animals of the control and high dose groups which were sacrificed at the end of the treatment period. Thyroid/parathyroid glands from pups and from the adult animals were examined. A full histopathology was carried out on the preserved organs and tissues of all animals which died during the study or which were euthanised due to morbidity. Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and examined in control and HD animals and in non-pregnant female animals of the LD and MD animals. Testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland) were also examined in the mating partners of the non-pregnant female LD and MD animals. Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.

Postmortem examinations (offspring):

Pathology: Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice. All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

Organ Weights: Thyroid/parathyroid glands from 1 pup/sex/litter/group (if possible) (sacrificed on PND 13) and from all adult males and females were preserved. Weight of thyroid/parathyroid glands was measured after fixation.

Histopathology: Thyroid/parathyroid glands from pups and from the adult animals were examined.

Statistics:

A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry will be statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.1.3 software (p<0.05 was considered as statistically significant).

Reproductive indices:

Copulation Index (%), Fertility Index (%) and Delivery Index (%).

Offspring viability indices:

Mean viability Index, viability index (%) (PND 0-4) and viability index (%) (PND 4-13).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Some male and female animals showed test item-related clinical signs. Female no. 74 of the HD group was in a moribund condition on PMD 6. Clinical signs related to mortality in the HD group were abnormal breathing (animals no. 35, 79, 80) or regurgitation (animal no. 80). However, these signs were also observed in female animals of the LD (animal no. 52), MD (animals no. 61, 65, 66, 70) and HD (animal no. 76) groups that survived the treatment period. Occasionally, milder signs of discomfort, i.e.piloerection, moving the bedding, reduced spontaneous activity and salivation were noted in MD and HD groups – but also in animals of the LD group. One control animal was also transiently affected to a slight degree. The signs occurred either prior to death or were transient and occurred at any phase of the observation period (premating, mating, gestation, and lactation period) and are not considered adverse signs of systemic toxicity. Clinical symptoms like alopecia, erythema, and red nasal discharge were observed mostly transiently or on single days and within the normal background frequency.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality occurred at the high dose level in 1/10 male and 3/10 female animals. One male animal from the HD group (animal no. 35) died on day 9 of the premating period, one female from the HD group (animal no. 79) was found dead on day 5 of the premating period and two females from the HD group were euthanized due to animal welfare reasons on day 6 of the premating (animal no. 74) and day 6 of the gestation phase (animal no. 80), respectively. A fluid-filled lung or thoracic segment seen in animals no. 35, 79 and 80, and a lesion in the esophagus of animal no. 80 identified misgavaging as the cause of death of these animals. No mortalities were recorded at the other dose levels.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During premating and mating / postmating periods body weight was slightly but at times significantly lower in male animals of the HD group, when compared to the control group (with a maximum of 8 % below controls). In female animals of the HD group mean body weight was marginally lower than compared to the control (with a maximum of 8 % below controls) in the premating phase and reaching significance at the end of the gestation period (Gestation days 14) – however not during lactation. During the end of the lactation high inter individual differences were seen in all female animals. As a result body weight changes of the female animals showed high inter individual variations especially during the lactation period. However, at the end of the gestation phase (GD 14-20) the HD female animals were seen with a significant lower body weight gain than controls resulting in a significant lower mean body weight gain between GD 0 and GD 20. Besides the described changes, there were no considerable differences in mean body weight between dose groups and control group. One female of the HD group (animal no. 73) lost 82 g from day 4 to day 13 of the lactation.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

A marginal tendency towards lower food consumption was seen in male and female animals of the HD group during the entire treatment period. Besides, there were no considerable differences in food intake between dose groups and control group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Statistical significant changes in haematological parameters were observed in male animals at the MD and HD levels and in female animals at the HD level when compared to controls. Red cell parameters RBC, Hb and Hct of male animals were moderately, dose-dependently and statistically significantly lower from 60 mg/kg/d upwards, when compared to controls. This slight anemia was not regenerative as MCV and Ret were not elevated. Rather MCV value was slightly but statistically significantly lower at the HD level than in controls. The reason for this slight and dose-dependent anemic effect is unclear. This was not observed in female animals. Slightly but statistically significantly higher MCH in females of the HD group than in controls is not considered toxicologically relevant as all other red cell parameters were not considerably different in this group. A statistically significantly lower rate of neutrophils and higher rate of lymphocytes was seen in female animals of the HD group. Due to the lower WBC, absolute number of lymphocytes was not considerably changed. The reason for lower count of neutrophils is unclear. This was not observed in male animals. Besides, all haematological parameters were within the normal range of variation and are not assumed to be biologically relevant. A minimal but statistically significantly longer PT in male animals of the HD group is not considered toxicologically relevant. Values were in the range of historical control data.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Serum level of TBA was statistically significantly elevated in male animals of the HD group. As values were within the historical control data and there were no signs of hepatocellular or hepatobiliary toxicity, this is not considered adverse. This was not seen in female animals of this group. A slightly but statistically significantly increased serum ASAT level was found in male animals of the HD group. As levels were within the historical control data, this is not considered toxicologically relevant. Moreover, this was not associated with any histopathological findings in the liver. This was not seen in female animals of this group. Albumin level was slightly but statistically significantly higher in male animals of the HD group when compared to controls. As values were within the range of historical control
data this is not assumed to be toxicologically relevant. This was not seen in female animals of this group. Statistically significantly lower serum glucose level at the HD level is not considered toxicologically relevant, as values were within the range of historical control data. This was not seen in female animals of this group. A statistically significantly lower cholesterol level in male animals of the LD group is not considered biologically relevant. Besides, all remaining parameters of clinical chemistry of male animals were within the normal range of variation for this strain and are not assumed to be biologically relevant. There were no statistically significant effects of the test item on parameters of clinical chemistry in female animals.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The test item had no toxicologically relevant effect on urinary parameters analysed at the end of the treatment period of this study. One male of each dose group (animals no. 14, 23, 32) had a high erythrocyte value which might be due to blood contamination during urine sampling.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

In males and females, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period when compared with the controls. There were no biologically relevant differences in body temperature between the groups.

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):

Decedents:
Spleen, mesenteric lymph node, thymus – minimal to moderate lymphoid depletion in several decedents. This finding was considered incidental and most likely stress related.
Lung – minimal multifocal alveolar histiocytosis in all female decedents. This finding was considered incidental. Further, in female no. 79 slight mononuclear multifocal infiltration was also observed. This finding was deemed incidental and is most likely related to an ongoing inflammatory process in the lung.
Prostate gland - moderate mixed cell infiltrates were observed the decedent male. This finding was considered incidental and most likely related to an ongoing inflammatory process.
Esophagus – minimal multifocal hemorrhages were noticed in female no. 80. This finding was considered most likely of traumatic nature and deemed incidental. All other findings recorded in decedents were considered within the range of normal background alterations.

Survivors: Kidney–hyaline droplet accumulation in tubular epithelial cells was observed in some animals from both control and high dose animals. This renal change is a male rat specific phenomenon and of no toxicological relevance in humans. Stomach - in the forestomach a minor degree of mixed cell infiltrates, squamous hyperplasia and epithelial vacuolization was observed in some males and females from the high dose group. All above mentioned changes were deemed incidental and not test item related.
Lung – alveolar histiocytosis was noticed in some males and animals from the high dose and the control group. The above mentioned findings were considered incidental.
Thymus – slight multifocal hemorrhages were observed in the thymus of animal no. 53 from the low dose group. This finding was considered incidental and correlates with the red color of the thymus observed at necropsy. All other findings recorded in surviving animals were considered within the range of normal background alterations.

Reproductive System: Epididymides – a minor degree of mononuclear cells (mononuclear infiltrates) were observed in the head of the epididymis head in one male (animal no. 39) from the high dose group and was considered an incidental finding not test item related.
Sperm staging – The stages were checked on completeness of cell populations, completeness of stages and degenerative changes. No treatment-related effects on the testicular histomorphology were observed. Further, no treatment-related effect on interstitial cell structure was noticed.
Non-pregnant females and mating partners: The treatment with the test item did not induced histomorphological effects in the reproductive organs of the non-pregnant low dose group female (no. 58) and its pairing partner (no. 18) and the non-pregnant high dose group female 76 and their pairing partner 36.
All other findings recorded in the reproductive organs were considered within the range of normal background alterations.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related effects were observed.

Other effects:

no effects observed

Description (incidence and severity):

Thyroid Hormone (T4) Analysis and Thyroid Weight: The test item had no effect on pup thyroid gland weight and serum T4 level of PND 13 pup. However, T4 was statistically significantly increased in the male LD, MD and HD adults (21 %, 20 % and 25 % in the LD, MD and HD group, respectively. As this was not associated with a significantly increased thyroid gland weight and histopathological findings and values were within the normal range of historical control data, this is not assumed to be toxicologically relevant.

Haematology and Coagulation: Statistical significant changes in haematological parameters were observed in male animals at the MD and HD levels and in female animals at the HD level when compared to controls. Red cell parameters RBC, Hb and Hct of male animals were moderately, dose dependently and statistically significantly lower from 60 mg/kg/d upwards, when compared to controls. This slight anemia was not regenerative as MCV and Ret were not elevated. Rather MCV value was slightly but statistically significantly lower at the HD level than in controls. The reason for this slight and dose-dependent anemic effect is unclear. This was not observed in female animals. Slightly but statistically significantly higher MCH in females of the HD group than in controls is not considered toxicologically relevant as all other red cell parameters were not considerably different in this group. A statistically significantly lower rate of neutrophils and higher rate of lymphocytes was seen in female animals of the HD group. Due to the lower WBC, absolute number of lymphocytes was not considerably changed. The reason for lower count of neutrophils is unclear. This was not observed in male animals. Besides, all haematological parameters were within the normal range of variation and are not assumed to be biologically relevant. A minimal but statistically significantly longer PT in male animals of the HD group is not considered toxicologically relevant. Values were in the range of historical control data.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Test item had no biologically significant effect on the estrous cycle analysed during the 2 weeks premating period and after the first administration in treatment groups when compared to the controls. There were no considerable differences in the length or sequence of cycle stages between the treatment groups and the control group.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Sperm staging – The stages were checked on completeness of cell populations, completeness of stages and degenerative changes. No treatment-related effects on the testicular histomorphology were observed. Further, no treatment-related effect on
interstitial cell structure was noticed.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no test item related effects on the reproductive indices (copulation, viability, fertility, and delivery indices) in the dose groups when compared to the control group. However fertility ((No. of females pregnant / No. of females copulated) X 100) and delivery ((No. of dams with live newborns / No. of pregnant dams) X 100) indices were slightly lower in the HD (87.50 %, 85.71 %, respectively) group compared to the controls. This was considered to be a biological variation and not test item related.

Details on results (P0)

Precoital Interval and Duration of Gestation: There were no test item related or statistically significant effects observed on the duration of precoital interval and the duration of gestation in the dose groups when compared to the control group.

Litter Data: There was a significant effect on total number of pups on PND 0 (p<0.05) and related to this a significant lower mean number of live pups on PND 0 (p<0.01), PND 4 (p<0.01) and PND 13 (p<0.05) in the HD group compared to the control. This effect is considered to be test item related. In the sex ratio and number of still births, runts on PND 0 no considerable differences were noted in all dose groups compared to the control.

Litter Weight Data: There was no test item related effect on pup mean weight observed in treatment groups when compared with the controls. There was no statistically significant change in dose groups compared to corresponding controls. The total litter weight, female litter weight, male litter weight on PND 0, PND 4 and PND 13 were lower in the HD group achieving significance in total litter weight in the HD group on PND 0 (p<0.05), PND 4 (p<0.01) and PND 13. This effect is correlated to the lower number of live pups born in the HD group.

Pre- and Post-Natal Data: A slight but significantly lower mean number implantation sites (p<0.01) was seen in the HD group when compared to controls (24 % below controls). This effect cannot be attributed to single dams. It is assumed that this slightly lower number of implantation sites is associated with the slightly reduced health condition which is reflected in slightly attenuated body weight development in dams of the HD group. Moreover, the values were within the normal range of historical control data. The percent pre-implantation loss was slightly higher in the HD group compared to the control, without achieving significance due to high individual differences but all in the range of normal biological variation. No considerable difference was seen in post-implantation loss.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

food consumption and compound intake

haematology

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

haematopoietic

Organ:

blood

erythrocyte development

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No test item related gross external abnormalities of toxicological relevance on PND 0-12 were observed in the pups of any of the groups. However, few specific findings like alopecia, hindleg stiff (dam no. 59 pup 3) at the end of the treatment period or findings like dark snout, throat, abdomen and paws (dam no. 55, pup 15), crust on head (dam no. 62, pup 8), tail tag missing (dam no. 69 pup 7) during the treatment period were observed.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No test item related effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13 in treatment groups when compared to the control group. A marginally higher mean mortality of pups between PND 0 and PND 4 was observed in the LD group (0.85%) and HD (1.67 %) compared to the control (0.00 %) are related to two single dams which each lost one pups during this period, which is well in range of the biological variation. No further pup mortality was observed during the study period in all dose groups. These single findings were not considered to be test item related.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no test item related effect on pup mean weight observed in treatment groups when compared with the controls. There was no statistically significant change in dose groups compared to corresponding controls.

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:

no effects observed

Description (incidence and severity):

The test item had no effect on pup thyroid gland weight and serum T4 level of PND 13 pup.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item related gross external abnormalities of toxicological relevance on PND 0-12 were observed in the pups of any of the groups. However, few specific findings like alopecia, hindleg stiff (dam no. 59 pup 3) at the end of the treatment period or findings like dark snout, throat, abdomen and paws (dam no. 55, pup 15), crust on head (dam no. 62, pup 8), tail tag missing (dam no. 69 pup 7) during the treatment period were observed.

Histopathological findings:

not examined

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

yes

Applicant's summary and conclusion

Conclusions:

On the basis of this combined repeated dose oral toxicity and reproduction/developmental toxicity screening test with the test ietm in male and female Wistar rats with dose levels of 20, 60, and 200 mg/kg body weight day the following conclusions can be made: A slightly lower mean number of implantation sites, total number of pups and number of live pups found at the HD level when compared to controls are considered to be secondary to maternal toxicity observed at the highest dose level. In the absence of other correlating contributing factors for effect on developmental and reproductive toxicity in terms of effect on parental histopathology, pup weight, pup survival, reproductive and developmental indices, hormone analysis, estrus cyclicity, the NOAELfor this reproduction/developmental toxicity screening study may be considered to be 200 mg/kg bw/day.

Executive summary:

On the basis of this combined repeated dose oral toxicity and reproduction/developmental toxicity screening test with the test ietm in male and female Wistar rats with dose levels of 20, 60, and 200 mg/kg body weight day the following conclusions can be made: Mortality occurred at the high dose level in 1/10 male and 3/10 female animals. A relationship to the test item could be ruled out in one female animal of the HD group. No clinical signs of systemic toxicity were seen in the animals dosed with the test item. Mean body weight and food consumption were slightly lower in males and females of the HD group compared to the controls. A slight non-regenerative anemia was found in male animals of this group. Liver weights were moderately increased in these animals but without histomorphological correlate. In female animals low neutrophil count was noted. Based on mortality, a slight effect on body weight development and a slight anemia in male animals, the dose level of 200 mg/kg/d is considered in the toxic range. Therefore, the NOAEL for general toxicity is considered to be 60 mg/kg/d. A slightly lower mean number of implantation sites, total number of pups and number of live pups found at the HD level when compared to controls are considered to be secondary to maternal toxicity observed at the highest dose level. In the absence of other correlating contributing factors for effect on developmental and reproductive toxicity in terms of effect on parental histopathology, pup weight, pup survival, reproductive and developmental indices, hormone analysis, estrus cyclicity, the NOAELfor this reproduction/developmental toxicity screening study may be considered to be 200 mg/kg bw/day.