2-tert-pentylcyclohexyl acetate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Remarks:

Extended to 10 weeks OECDTG 422 study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 30 August, 2011 to 2 october, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The information from Verdox is used to assess the repeated dose toxicity for Coniferan.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 30 August, 2011 to 2 october, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at initiation of treatment: Female 6 weeks, Male 7 weeks
- Average weight at study initiation: Males: 179 g, Females: 126g
- Housing: In macrolon cages with a bedding of wood shavings (Lignocel, Type ¾), strips of paper (Enviro-dri) and a wooden block as environmental enrichment. During the premating period, the animals were housed in groups of 4 per sex. For mating, one male and one female were housed together. Mated females were housed individually in macrolon cages, which were placed in a separate cage rack. After delivery, the cages containing the dam with litter were transferred to another cage rack.
- Diet: Cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, Witham, England), ad libitum.
- Water: Domestic mains tap-water, ad libitum. In the last week of gestation and during lactation a bottle containing glucose water (5%) was added to the cages in which the females were housed, in addition to the domestic tap water supply.
- Acclimation period: One week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2. During several short periods, temperature was outside the limits with a minimum of 19.8 ºC and a maximum of 24.9 ºC.
- Humidity (%): 45% and not exceeding 65%. Relative humidity was outside the limits during several short periods (a maximum value of 78.1% was recorded and in one occasion a minimum of 44% was recorded).
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

The test substance was incorporated in the basal diet by mixing in a mechanical blender. The experimental diets were prepared shortly before the start of the study and subsequently every ca. 4 or 5 weeks. After preparation, the experimental diets were divided into daily amounts of diets that were stored in plastic bags in a freezer (<-18°C). Each day, one bag per group was removed from the freezer to feed the animals.

Details on mating procedure:

At the end of the premating period each female was caged with one male from the same group. Every morning during the mating period, vaginal smears were made for determination of the presence of sperm. The day on which sperm was detected in the vaginal smear was considered as gestation Day 0. Upon evidence of copulation, the females were caged individually for the birth and rearing of their pups. Sperm positive females that turned out to be non-pregnant (females C61 and C67 of the mid dose group) were sacrificed 31 and 30 days after copulation, respectively. All other dams were allowed to raise their litter until sacrifice on day 4 of lactation or shortly thereafter.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Immediately after preparation of the first batch of the experimental diets, samples were taken and stored in the freezer until analysis. The stability of the test substance under simulated experimental conditions was demonstrated as follows: samples of control diet, low-dose diet, mid-dose diet and high-dose diet were prepared on 19 September 2011. After preparation samples were stored at <-18°C and analyzed on 7 October 2011 (t = 0), after storage at ambient temperature in an open container in the animal room for one day and after storage in the freezer (at < -18 °C) in a closed container for at least 5 weeks. The 5 week storage stability samples (< -18 °C) were analyzed on 26 October 2011. The homogeneity and content (achieved concentration) of the test substance in the experimental diets were demonstrated in the same batch of diets by analyzing five samples (taken at different locations in the feed container) of each test diet; one sample of the control diet was analyzed in the same series.
Result: The test substance was considered to be homogeneously distributed in the diets. The concentration of the test substance was close to intended for all dose levels (relative difference from the intended concentration was <10%), except for the low dose group (-11%). The test substance in diet was considered to be stable when stored in the freezer (at < -18 °C) in a closed container for 5 weeks (relative decrease of the Verdox concentration was less than 10% for all dose levels) and when stored at ambient temperature in an animal room in an open container for one day for the low and high dose groups. For the mid dose group the test substance was considered unstable as the relative decrease was 12%.

Duration of treatment / exposure:

Male animals received the diets during a 10-week premating period and during mating up to the day of sacrifice. The female animals were given the diets with the test substance during a 10-week premating period, during mating, gestation and lactation, up to the day of sacrifice (approx. day 4 of lactation).

Frequency of treatment:

continuously

Dose / conc.:

75 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

800 mg/kg diet

Dose / conc.:

2 500 mg/kg diet

Dose / conc.:

7 500 mg/kg diet

No. of animals per sex per dose:

12

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale main study: selection based on 2-week range-finding study.

Parental animals: Observations and examinations:

- General clinical observations: Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. All cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. All abnormalities, signs of ill health or reactions to treatment were recorded.
- Neurobehavioural testing: Functional Observational Battery (FOB) tests, including assessment of grip strength and sensory reactivity to stimuli of different types, and spontaneous Motor Activity Assessment (MAA) were performed in 5 animals/sex/group prior to the end of the premating period (week 9). During neuro-behavioural testing, the observer was unaware of the treatment of the animals.
- Body weight: Body weights of male and female rats were recorded at randomization one day before the start of administration of the test substance and at the start of the study (Day 0). Males were then weighed once per week until sacrifice. Females were then weighed once per week during the premating and mating period. Mated females were weighed on Days 0, 7, 14 and 21 during presumed gestation and on Days 1 and 4 of lactation. After the mating period, nonmated females were weighed once per week. In addition, the animals were weighed on their scheduled necropsy date in order to calculate the correct organ to body weight ratios.
- Food consumption: Except during the mating period, food consumption was measured from day 0 onwards on the same days as body weight was measured. Nevertheless during the last week of the premating period where body weight was measured at day 63 while food was given on day 64 because some of the animals were fasted for one night for haematology and clinical chemistry.
- Intake of test substance: The intake of the test substance per kg body weight per day was calculated from the nominal dietary concentration of the test substance in the diet, the food consumption and the mean body weight measured at the beginning and the end of the pertaining period.
- Haematology: At the end of the premating period (week 10), 5 rats/sex/group were fasted overnight (water was freely available) and blood was taken by orbita punction whilst under CO2/O2 anaesthesia. K3-EDTA was used as anticoagulant. In each sample the following determinations were carried out according: haemoglobin, packed cell volume, red blood cell count, reticulocytes,
total white blood cell count, differential white blood cell counts (neutrophils, lymphocytes, eosinophils, basophils, monocytes), prothrombin time, thrombocyte count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration.
- Clinical chemistry: At the end of the premating period (week 10), 5 rats/sex/group were fasted overnight (water was freely available) and blood was taken by orbital punction whilst under CO2/O2 anaesthesia. Blood was collected in heparinised plastic tubes and plasma was prepared by centrifugation. The following measurements were made in the plasma: alkaline phosphatase activity, aspartate aminotransferase activity, alanine aminotransferase activity, gamma glutamyl transferase activity, total protein, albumin, ratio albumin to globulin, urea, creatinine, bilirubin (total), cholesterol (total), triglycerides, phospholipids, calcium, sodium, potassium, chloride, inorganic phosphate, glucose (fasting).

Sperm parameters (parental animals):

- Epididymal sperm motility, count and morphology: At scheduled necropsy, epididymal sperm was derived from the left cauda epididymis of 5 males/group. For this purpose the left cauda epididymis was dissected and weighed and thereafter minced in M199 medium containing 0.5% bovine serum albumin. Sperm motility and, after sonification and DNA staining, the cauda epididymal sperm reserves (sperm count) were measured for these males, using the Hamilton Thorne Integrated Visual Optical System (IVOS). In addition, a smear of the sperm solution was prepared and stained and 200 spermatozoa of the smear of males of the control group and of the high-dose group were examined for morphology.
- Testicular sperm count: At necropsy, the left testis of the same males as used for epididymal sperm analysis was placed on dry ice and subsequently stored in a freezer (<-70°C) for later determination of the number of homogenization-resistant spermatids. Testicular sperm count was conducted in the control and high-dose group. The testes were thawed just before further processing. Following removal of the tunica albuginea, the testicular parenchyma was weighed, minced and homogenized in Saline Triton X-100 solution. Following DNA-staining, the homogenizationresistant sperm heads were enumerated using the IVOS. The daily sperm production was calculated.

Litter observations:

- Parturition and litter evaluation: At the end of the gestation period (gestation day 21), females were examined twice daily for signs of parturition. Any difficulties occurring during parturition were recorded. To keep nest disturbance to a minimum the litters were examined only once daily for dead pups.
- Litter size, sexes and weight: The total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups and grossly malformed pups were evaluated on Days 1 and 4 of lactation. The pups were individually weighed on Days 1 and 4 of lactation. Mean pup weight was calculated per sex and for both sexes combined.

Postmortem examinations (parental animals):

- Gross necropsy and histology of parental animals: All male and female parent rats were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia at necropsy and then examined grossly for pathological changes. Male animals were sacrificed after the mating period. Female animals were sacrificed at or shortly after day 4 of lactation. Furthermore, organs showing gross lesions of animals of all groups were examined microscopically. Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes which was preserved in Bouin's fixative: Ovaries (after counting of the corpora lutea), Uterus (after counting of the implantation sites), testes, epididymides, seminal vesicles, prostate, all gross lesions. In addition, of 5 animals/sex/group the following organs were preserved: adrenals, bone marrow (femur), brain (including sections of cerebrum, cerebellum, medulla/pons), heart, small and large intestines (including Peyer’s patches), kidneys, liver, lungs, lymph nodes (mesenterial and axillary), peripheral nerve (tibial), spinal cord (cervical, mid-thoracic, and lumbar), spleen, stomach, thymus, thyroid, trachea, urinary bladder.
The following organs were weighed (paired organs together): testes, epididymides, adrenals, brain, heart, kidneys, liver, spleen, thymus.
For sperm analysis, the left cauda epididymis and left testis of the 5 selected males were used.
Tissues for microscopic examination were embedded in paraffin wax, sectioned at 5 mm, and stained with haematoxylin and eosin, except for sections of the testes which were stained with PAS haematoxylin. The kidneys of the male rats were also stained immunocytochemically (using a mouse-anti-rat alpha 2-microglobuline antibody from SSI, Copenhagen, Denmark) to confirm alpha-hydrocarbon nephropathy. Microscopic examination was performed on the collected organs of all animals of the control and high-dose group. Microscopy of the kidneys was extended to all male animals of the low- and mid-dose groups.

Postmortem examinations (offspring):

Pathology of pups: A necropsy was performed on stillborn pups and on pups that died during the study. At necropsy of the dams, at or shortly after day 4 of lactation, all other pups were examined externally for gross abnormalities and killed by appropriate techniques.

Statistics:

The resulting data were analyzed using the methods given below. P < 0.05 was considered as the level of significance. Clinical findings were evaluated by Fisher's exact probability test. Body weight, body weight gain, food consumption and organ weights data were subjected to one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests. Fisher's exact probability test was used to evaluate the number of mated and pregnant females, the number of pregnant females with implants but no pups, females with live pups, females with stillborn pups, live and dead fetuses or pups and the numbers of litters lost entirely.Pre-coital time (mean number of days), the duration of gestation, the number of corpora lutea and implantation sites, the total number of pups delivered (mean), the mean number of live pups per litter and pre- and post-implantation loss (%) were evaluated by Kruskal-Wallis nonparametric analysis of variance and by the Mann-Whitney U test. Haematology and clinical chemistry parameters were subjected to one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests. Mortality data and data of the pathology of parent animals were evaluated by the Fisher’s exact probability test. Sperm parameters were evaluated by one-way analysis of variance followed by Dunnett’s multiple comparison test (epididymal and testicular sperm count and numerical sperm motility parameters) or by Kruskal-Wallis non parametric analysis of variance and by Mann-Whitney U test (motility parameters expressed as a percentage and sperm morphology).

Reproductive indices:

number of females placed with males, number of males mated with females, number of successful copulations (= number of females mated), number of males that became sire, number of pregnant females as demonstrated by the presence of implantation sites observed at necropsy, number of females surviving delivery, number of females with liveborn and (all) stillborn pups, number of pups delivered (live- and stillborn), number of live pups on day 1 and 4, number of pups lost, number of litters lost entirely, number of male pups on day 1 and 4, number of corpora lutea, number of implantation sites, number of lost implantations, litter size.
The following parameters were calculated: pre-coital time = time between the start of mating and successful copulation, duration of gestation = time between gestation day 0 and day of delivery, mating index= (number of females mated/number of females placed with males) x 100, male fertility index = (number of males that became sire/number of males placed with females) x 100, female fertility index = (number of pregnant females/number of females placed with males) x 100, female fecundity index = (number of pregnant females/number of females mated) x 100, gestation index = (number of females with live pups/number of females pregnant) x 100, pre-implantation loss = [(number of corpora lutea – number of implantation sites)/number of corpora lutea] x 100.

Offspring viability indices:

Live birth index = (number of pups born alive/number of pups born) x 100, viability index day n-m = (number of pup surviving m days/number of liveborn on day n) x 100, pup mortality day n = (number of dead pups on day n/total number of pups on day n) x 100, sex ratio day n = (number of live male pups on day n/ number of live pups on day n) x 100, number of lost implantations = number of implantations sites - number of pups born alive, post-implantation loss = [(number of implantation sites - number of pups born alive)/number of implantation sites] x 100.

- Mortalities and clinical signs: One animal from the low dose group was sacrificed moribund during lactation, showing hunched posture, lethargy, piloerection, encrustations around nose and eyes and haemorrhagic discharge around the vagina. In the males one animal in the control group showed red ears and one animal in the high dose group showed sparsely haired skin and encrustations. In the females one animal in the high dose group showed cateract of the eyes throughout all study periods. One control animal showed sparsely haired skin during gestation and lactation. One animal in the control group and one animal in the high dose group showed piloerection during lactation. Based on the incidence and distribution the observed clinical signs are considered not to be treatment-related.
- Neurobehavioural testing: The functional observational battery observations and motor activity assessment did not reveal any treatment-related effects on neurobehaviour.
- Body weight and body weight change: Males in the high dose group showed a slightly lower body weight as compared to the control animals, which reached the level of statistically significance on days 28, 35 and 56 of the study. Body weight gain was also slightly lower in this group, mainly in the first 4 weeks of the study, and reached the level of statistically significance in the periods from day 0-7, 21-28 and 49-56 of the study. As a consequence, the total body weight gain of the male animals between days 0-77 was also slightly, but statistically significantly, decreased. Since the effect on body weight gain was most clear in the first weeks of the study, the effect may be (partly) due to the palatability of the test substance in the diets. For that reason, the effect on body weight gain of the male animals is considered of marginal toxicological relevance. No effects on body weight or body weight change were observed in the low dose and mid dose groups for the males. In females, no effects on body weight were observed during premating, gestation and lactation. In the high-dose group females body weight gain was only statistically significantly lower in the first week of premating and recovered thereafter.
- Food consumption: In males, no effects on food consumption were observed throughout the study. In females, a slight, but statistically significant lower food consumption (g/animal/day) was observed in the females of the mid dose group and high dose group in the first week of the premating phase, which returned to normal thereafter. No effects on food consumption were observed during the further premating phase or during gestation or lactation.
- Test substance intake: Test substance intake (mg Verdox/kg body weight/day) was calculated over the premating period (males and females), gestation period (females) and lactation period from day 1-4 (females) and is summarized in the table under: Any other information on results incl. tables. The intake of the test substance per kg body weight per day was calculated from the nominal dietary concentration, the feed consumption and the body weight in the pertaining week. Since the concentration of test substance was lower than intended in the low dose group (relative difference from intended concentration -11%) and since the test substance in diet was not stable when stored at ambient temperature in an open container for 24h in the mid dose group (relative decrease -12%), the actual test substance intake was lower than indicated in the table above for animals in the low dose and mid dose groups.
- Fertility and reproductive performance: In each group 12 females were placed with a male from the same dose group. The mean precoital time was comparable in all groups and ranged from 2.25 days in the low dose group to 2.75 days in the high dose group. The mating index was 100% in all groups and the number of pregnant females was 12/12 for the control, low dose and high dose group and 10/12 in the mid dose group. No differences were observed on male and female fertility indices, female fecundity index, gestation index or duration of gestation. No effects were observed on the mean number of implantation sites, pre-implantation loss and post-implantation loss. All pregnant females survived delivery, but one female in the Low Dose group was sacrifice moribund during lactation. Two animals in the control group, and four animals in both the mid dose and high dose groups delivered both live and stillborn pups. In addition, one female in the control group and two females in the low dose group delivered stillborn pups only.
- Haematology: In the male animals no statistically significant findings were observed. In the female animals an incidental, not dose-related, decrease was observed on the absolute number of lymphocytes in group 2 and 3. There were no statistically significant changes in red blood cell variables or clotting potential.
- Clinical chemistry: In the males in from the high dose group the concentration of ASAT was decreased. This is not considered to be treatment-related because in case of an effect of treatment an increase in the concentration of ASAT would be expected to occur instead of the observed decrease. In males the concentration creatine was increased in the low dose and mid dose group, but not in the high dose group. In females the concentration of phosphate was decreased in the low dose and mid dose groups and the concentration of potassium was decreased in the mid dose group. These findings, not confirmed at the high-dose levels, are considered incidental findings and not related to treatment. In males of group 2, 3 and 4 a statistically significant increase in the urea-concentration was found when compared to the control group. This finding might be indicative of an effect on the kidneys.
- Sperm analysis: Epididymal sperm motility: No statistically significant effects on sperm motility parameters (motile cells, static cells and progressive cells) were observed among the groups. Furthermore, no differences in the derived parameters describing sperm motility were found between the groups. Epididymal sperm count: No statistically significant effects were observed on epidydimal sperm count parameters between the groups. Epididymal sperm morphology: No differences were observed on sperm morphology between the control group and the highdose group. Testicular sperm count: No effects were observed on the testicular parenchyma weight, the number of spermatozoa per gram testicular parenchyma or on the daily sperm production between the control and the high dose group.
- Parental necropsy observations: Organ weights of parental animals: The terminal mean body weight was decreased in the males of the high dose group. The absolute and relative weights of the spleen were statistically significantly increased in males of the low and mid dose groups, but this finding was not confirmed at the high-dose level and was therefore not considered toxicologically relevant. The relative weight of the liver was statistically significantly increased (circa 15%) in males of the high-dose group. A dose related increase in relative kidney weight was observed in the males of the mid and high dose groups. No effects on organ weights were observed in female animals. Macroscopic examination of parental animals: At necropsy no treatment realted macroscopic changes were observed. Animal 27 of the low-dose group was killed in moribund condition on day 96 of the study. At necropsy the most important finding was adhesions of the liver with the stomach and pancreas. Microscopic examination of parental animals: A dose-dependent increase of hyalin droplet nephropathy was found in the male animals, characterised by an abundant presence of eosinophilic hyalin droplets in the proximal tubular cells, which in several cases was accompanied by dilated tubuli filled with eosinophilic debris in the corticomedullary area. In agreement with the nephropathy, a dose dependent increase in basophilic tubuli was observed. Involution of the thymus in females is a normal phenomenon during pregnancy. At microscopical examination of animal 27 of the low-dose group (killed in moribund condition on day 96 of the study) severe peritonitis was observed, which was related to perforative ulcerative duodenitis, ulcerative gastritis and severe lobular necrotizing hepatitis. The reason for this condition could not be established The other histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to treatment. Immunohistochemistry: The kidneys of male animals were stained for the presence of α2u-globulin using specific antibodies. Microscopical examination revealed a dose-dependent increase of α2u-globulin staining of the cortical tubular epithelial cells and staining of the observed corticomedullary tubular cell debris.

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

>= 437 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

>= 437 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

- Litter data: Litter size and sex: Sex ratio was comparable in all goups on lactation day 1 and lactation day 4. The mean number of pups delivered was comparable in all groups (134, 132, 111 and 125 for the control, low dose, mid dose and high dose groups, respectively). The relative number of liveborn pups was lower in the low dose group (120 of 134, 102 of 132, 97 of 111 and 112 of 125 for the control, low dose, mid dose and high dose groups, in 12, 12, 10 and 12 litters respectively) and the number of stillborn pups was increased in the low dose group (14, 30, 14 and 13 for the control, low dose, mid dose and high dose group, respectively). The number of pups that were lost between postnatal days 1-4 was statistically significantly lower in the low dose group (34, 2, 34 and 19 in the control group, low dose, mid dose and high dose groups, respectively). The number of litters lost entirely was comparable in all groups (4 in the control group, 2 in the low dose group, 3 in the mid dose group and 2 in the high dose group). The incidence of litter loss as was observed in this study was relatively high but in the same order as was observed in other reproductive toxicity studies performed at this test site, in the same time period with this strain of rats of this supplier. In none of these studies, the observed litter loss was related to treatment. There were no treatment-related effects on litter size and pup survival.
- Pup observations: One litter in the control group, one litter in the low dose group, two litters in the mid dose group and one litter in the high dose group comprised cold pups. In addition to this observation, the litters in the mid dose group showed small pups and pups that had no milk in the stomach. Although the incidence of clinical signs was statistically significantly increased in the mid dose group, all fetuses showing clinical signs came from two litters. In addition, no dose response relationship was observed. Based on the incidende and distribution of litters comprising pups showing clinical signs the observed clinical signs are not considered to be treatment-related. Pup body weight: There were no effects on mean pup body weight or body weigh changes for pups on lactation days 1 and 4. Macroscopic observations of stillborn pups or pups that died during lactation: One pup in the contol group and one pup in the low dose group showed a dilated urinary bladder. Other observations included no presence of milk in the stomach and no distention of the lungs and these observations are related to the stillborn or moribund status of the pups. There were no treatment-related macroscopic observations in pups that were stillborn or that died during lactation.

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

>= 437 other: mg/kg bw/day (actual dose received by parental animals)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Reproductive effects observed:

not specified

Test substance intake:

	Mean (range) substance intake (mg Verdox/kg body weight/day)
	Low dose	Mid dose	High dose
Premating males	55.65 (45.85 – 69.47)	167.74 (140.66 – 209.10)	504.58 (415.47 – 616.50)
Premating females	59.12 (50.21 – 70.66)	177.44 (154.81 – 209.25)	554.02 (471.33 – 671.55)
Gestation females	52.03 (42.22 – 58.40)	165.82 (128.61 – 185.02)	471.43 (380.64 – 528.21)
Lactation females	58.34	151.44	437.18

Conclusions:

The NOAEL for fertility and developmental toxicity is 437 mg/kg bw in rats as determined in a GLP compliant combined oral repeated dose toxicity study and reproduction/developmental toxicity screening test.

Executive summary:

In a GLP compliant combined repeated dose and reproduction / developmental screening study, performed according to OECD guideline 422, Wistar rats were treated with the test substance (0, 75, 200, and 500 mg verdox/kg bw/day, expected nominal dose levels) in the diet,during a premating period of 10 weeks and during mating (1 week), gestation and lactation until postnatal day 4. The actual test substance intake ranged between 46-69 for males and 42-71 for the females of the low dose group. For the mid dose group the actual test substance concentration ranged from 141-209 for the males and 129-209 for the females. For the high dose group the actual test substance concentration ranged from 415-617 for the males and ranged between 381-672 for the females. Clinical observations during the premating, gestation and lactation period did not reveal any treatment-related changes in the animal’s appearance, general condition or behaviour. Neurobehavioural observations and motor activity assessment did not indicate any neurotoxic potential of the test substance. No toxicological relevant effects on body weight and food consumption were observed in both sexes. No treatment related effects were observed on pre-coital time, mating index, duration of gestation, pre- and postimplantation loss, number of corpora lutea, number of implantation sites and number of live pups on lactation. No treatment related clinical signs in pups, no effect on sex ratio in pups on day 1 and 4 of lactation, no effects on the mean pup body weight and body weight gain on lactation days 1 and 4 and no treatment-related macroscopic observations in pups that were stillborn or that died during lactation were observed. No effects on red blood cell variables or clotting potential was observed. A statistically significant increase in urea-concentration was observed in males of all dosing groups which might be related to the observed kidney effects (α2u-microglobulin nephropathy). No treatment-related effects were observed on sperm-parameters (epidydimal sperm motility, sperm count and morphology and testicular sperm count). An increased relative liver weight was observed in high dose males which was considered as an adaptive response to increased physiological demand. A dose related increase in relative kidney weight was observed in mid and high dose males which was related to the observed α2u-microglobulin nephropathy. No effects on organ weights were observed in females. Effects observed on kidney weights and urea-concentrations were considered to be related to α2u-microglobulin nephropathy which was confirmed by immunocytochemical staining of the α2u-microglobulin protein in the cortical tubular epithelial cells. This effect observed in rats is generally regarded as of no toxicological relevance for humans. Macrosopic and microscopic examination did not reveal any treatment-related effects. In conclusion, the NOAEL for Verdox for males and females in this study was ≥ 500 mg/kg body weight in the diet. Based on the absence of effects on fertility parameters and developmental parameters, the NOAEL for Verdox for fertility and developmental toxicity in this study was ≥ 500 mg/kg body weight in the diet. A NOAEL of 500 mg/kg body weight in the diet is equivalent to an overall intake of at least 505 mg/kg body weight/day for males and at least 437 mg/kg body weight/day for females.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 30 August, 2011 to 2 october, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study performed with read across substance

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at initiation of treatment: Female 6 weeks, Male 7 weeks
- Average weight at study initiation: Males: 179 g, Females: 126g
- Housing: In macrolon cages with a bedding of wood shavings (Lignocel, Type ¾), strips of paper (Enviro-dri) and a wooden block as environmental enrichment. During the premating period, the animals were housed in groups of 4 per sex. For mating, one male and one female were housed together. Mated females were housed individually in macrolon cages, which were placed in a separate cage rack. After delivery, the cages containing the dam with litter were transferred to another cage rack.
- Diet: Cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, Witham, England), ad libitum.
- Water: Domestic mains tap-water, ad libitum. In the last week of gestation and during lactation a bottle containing glucose water (5%) was added to the cages in which the females were housed, in addition to the domestic tap water supply.
- Acclimation period: One week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2. During several short periods, temperature was outside the limits with a minimum of 19.8 ºC and a maximum of 24.9 ºC.
- Humidity (%): 45% and not exceeding 65%. Relative humidity was outside the limits during several short periods (a maximum value of 78.1% was recorded and in one occasion a minimum of 44% was recorded).
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

The test substance was incorporated in the basal diet by mixing in a mechanical blender. The experimental diets were prepared shortly before the start of the study and subsequently every ca. 4 or 5 weeks. After preparation, the experimental diets were divided into daily amounts of diets that were stored in plastic bags in a freezer (<-18°C). Each day, one bag per group was removed from the freezer to feed the animals.

Details on analytical verification of doses or concentrations:

Immediately after preparation of the first batch of the experimental diets, samples were taken and stored in the freezer until analysis. The stability of the test substance under simulated experimental conditions was demonstrated as follows: samples of control diet, low-dose diet, mid-dose diet and high-dose diet were prepared on 19 September 2011. After preparation samples were stored at <-18°C and analyzed on 7 October 2011 (t = 0), after storage at ambient temperature in an open container in the animal room for one day and after storage in the freezer (at < -18 °C) in a closed container for at least 5 weeks. The 5 week storage stability samples (< -18 °C) were analyzed on 26 October 2011. The homogeneity and content (achieved concentration) of the test substance in the experimental diets were demonstrated in the same batch of diets by analyzing five samples (taken at different locations in the feed container) of each test diet; one sample of the control diet was analyzed in the same series.
Result: The test substance was considered to be homogeneously distributed in the diets. The concentration of the test substance was close to intended for all dose levels (relative difference from the intended concentration was <10%), except for the low dose group (-11%). The test substance in diet was considered to be stable when stored in the freezer (at < -18 °C) in a closed container for 5 weeks (relative decrease of the Verdox concentration was less than 10% for all dose levels) and when stored at ambient temperature in an animal room in an open container for one day for the low and high dose groups. For the mid dose group the test substance was considered unstable as the relative decrease was 12%.

Details on mating procedure:

At the end of the premating period each female was caged with one male from the same group. Every morning during the mating period, vaginal smears were made for determination of the presence of sperm. The day on which sperm was detected in the vaginal smear was considered as gestation Day 0. Upon evidence of copulation, the females were caged individually for the birth and rearing of their pups. Sperm positive females that turned out to be non-pregnant (females C61 and C67 of the mid dose group) were sacrificed 31 and 30 days after copulation, respectively. All other dams were allowed to raise their litter until sacrifice on day 4 of lactation or shortly thereafter.

Duration of treatment / exposure:

Male animals received the diets during a 10-week premating period and during mating up to the day of sacrifice. The female animals were given the diets with the test substance during a 10-week premating period, during mating, gestation and lactation, up to the day of sacrifice (approx. day 4 of lactation).

Frequency of treatment:

continuously

Duration of test:

10-weeks

Dose / conc.:

75 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

800 mg/kg diet

Dose / conc.:

2 500 mg/kg diet

Dose / conc.:

7 500 mg/kg diet

No. of animals per sex per dose:

12

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale main study: selection based on 2-week range-finding study.

Maternal examinations:

- General clinical observations: Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. All cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. All abnormalities, signs of ill health or reactions to treatment were recorded.
- Neurobehavioural testing: Functional Observational Battery (FOB) tests, including assessment of grip strength and sensory reactivity to stimuli of different types, and spontaneous Motor Activity Assessment (MAA) were performed in 5 animals/sex/group prior to the end of the premating period (week 9). During neuro-behavioural testing, the observer was unaware of the treatment of the animals.
- Body weight: Body weights were recorded at randomization one day before the start of administration of the test substance and at the start of the study (Day 0). Females were then weighed once per week during the premating and mating period. Mated females were weighed on Days 0, 7, 14 and 21 during presumed gestation and on Days 1 and 4 of lactation. After the mating period, nonmated females were weighed once per week. In addition, the animals were weighed on their scheduled necropsy date in order to calculate the correct organ to body weight ratios.
- Food consumption: Except during the mating period, food consumption was measured from day 0 onwards on the same days as body weight was measured. Nevertheless during the last week of the premating period where body weight was measured at day 63 while food was given on day 64 because some of the animals were fasted for one night for haematology and clinical chemistry.
- Intake of test substance: The intake of the test substance per kg body weight per day was calculated from the nominal dietary concentration of the test substance in the diet, the food consumption and the mean body weight measured at the beginning and the end of the pertaining period.
- Haematology: At the end of the premating period (week 10), 5 rats/sex/group were fasted overnight (water was freely available) and blood was taken by orbita punction whilst under CO2/O2 anaesthesia. K3-EDTA was used as anticoagulant. In each sample the following determinations were carried out according: haemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell counts (neutrophils, lymphocytes, eosinophils, basophils, monocytes), prothrombin time, thrombocyte count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration.
- Clinical chemistry: At the end of the premating period (week 10), 5 rats/sex/group were fasted overnight (water was freely available) and blood was taken by orbital punction whilst under CO2/O2 anaesthesia. Blood was collected in heparinised plastic tubes and plasma was prepared by centrifugation. The following measurements were made in the plasma: alkaline phosphatase activity, aspartate aminotransferase activity, alanine aminotransferase activity, gamma glutamyl transferase activity, total protein, albumin, ratio albumin to globulin, urea, creatinine, bilirubin (total), cholesterol (total), triglycerides, phospholipids, calcium, sodium, potassium, chloride, inorganic phosphate, glucose (fasting).
- Gross necropsy and histology of parental animals: All parent rats were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia at necropsy and then examined grossly for pathological changes. Female animals were sacrificed at or shortly after day 4 of lactation. Furthermore, organs showing gross lesions of animals of all groups were examined microscopically. Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde: Ovaries (after counting of the corpora lutea), Uterus (after counting of the implantation sites), all gross lesions. In addition, of 5 animals/sex/group the following organs were preserved: adrenals, bone marrow (femur), brain (including sections of cerebrum, cerebellum, medulla/pons), heart, small and large intestines (including Peyer’s patches), kidneys, liver, lungs, lymph nodes (mesenterial and axillary), peripheral nerve (tibial), spinal cord (cervical, mid-thoracic, and lumbar), spleen, stomach, thymus, thyroid, trachea, urinary bladder. The following organs were weighed (paired organs together): adrenals, brain, heart, kidneys, liver, spleen, thymus. Tissues for microscopic examination were embedded in paraffin wax, sectioned at 5 mm, and stained with haematoxylin and eosin. Microscopic examination was performed on the collected organs of all animals of the control and high-dose group.

Fetal examinations:

- Parturition and litter evaluation: At the end of the gestation period (gestation day 21), females were examined twice daily for signs of parturition. Any difficulties occurring during parturition were recorded. To keep nest disturbance to a minimum the litters were examined only once daily for dead pups.
- Litter size, sexes and weight: The total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups and grossly malformed pups were evaluated on Days 1 and 4 of lactation. The pups were individually weighed on Days 1 and 4 of lactation. Mean pup weight was calculated per sex and for both sexes combined.
- Pathology of pups: A necropsy was performed on stillborn pups and on pups that died during the study. At necropsy of the dams, at or shortly after day 4 of lactation, all other pups were examined externally for gross abnormalities and killed by appropriate techniques.

Statistics:

The resulting data were analyzed using the methods given below. P < 0.05 was considered as the level of significance. Clinical findings were evaluated by Fisher's exact probability test. Body weight, body weight gain, food consumption and organ weights data were subjected to one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests. Fisher's exact probability test was used to evaluate the number of mated and pregnant females, the number of pregnant females with implants but no pups, females with live pups, females with stillborn pups, live and dead fetuses or pups and the numbers of litters lost entirely.Pre-coital time (mean number of days), the duration of gestation, the number of corpora lutea and implantation sites, the total number of pups delivered (mean), the mean number of live pups per litter and pre- and post-implantation loss (%) were evaluated by Kruskal-Wallis nonparametric analysis of variance and by the Mann-Whitney U test. Haematology and clinical chemistry parameters were subjected to one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests. Mortality data and data of the pathology of parent animals were evaluated by the Fisher’s exact probability test.

Indices:

- Reproductive: number of females placed with males, number of males mated with females, number of successful copulations (= number of females mated), number of pregnant females as demonstrated by the presence of implantation sites observed at necropsy, number of females surviving delivery, number of females with liveborn and (all) stillborn pups, number of pups delivered (live- and stillborn), number of live pups on day 1 and 4, number of pups lost, number of litters lost entirely, number of male pups on day 1 and 4, number of corpora lutea, number of implantation sites, number of lost implantations, litter size. The following parameters were calculated: pre-coital time = time between the start of mating and successful copulation, duration of gestation = time between gestation day 0 and day of delivery, mating index= (number of females mated/number of females placed with males) x 100, female fertility index = (number of pregnant females/number of females placed with males) x 100, female fecundity index = (number of pregnant females/number of females mated) x 100, gestation index = (number of females with live pups/number of females pregnant) x 100, pre-implantation loss = [(number of corpora lutea – number of implantation sites)/number of corpora lutea] x 100.
- Offspring viability: Live birth index = (number of pups born alive/number of pups born) x 100, viability index day n-m = (number of pup surviving m days/number of liveborn on day n) x 100, pup mortality day n = (number of dead pups on day n/total number of pups on day n) x 100, sex ratio day n = (number of live male pups on day n/ number of live pups on day n) x 100, number of lost implantations = number of implantations sites - number of pups born alive, post-implantation loss = [(number of implantation sites - number of pups born alive)/number of implantation sites] x 100.

Details on maternal toxic effects:

Details on maternal toxic effects:
- Mortalities and clinical signs: One animal from the low dose group was sacrificed moribund during lactation, showing hunched posture, lethargy, piloerection, encrustations around nose and eyes and haemorrhagic discharge around the vagina. One animal in the high dose group showed cateract of the eyes throughout all study periods. One control animal showed sparsely haired skin during gestation and lactation. One animal in the control group and one animal in the high dose group showed piloerection during lactation. Based on the incidence and distribution the observed clinical signs are considered not to be treatment-related.
- Neurobehavioural testing: The functional observational battery observations and motor activity assessment did not reveal any treatment-related effects on neurobehaviour.
- Body weight and body weight change: No effects on body weight were observed during premating, gestation and lactation. In the high-dose group females body weight gain was only statistically significantly lower in the first week of premating and recovered thereafter.
- Food consumption: A slight, but statistically significant lower food consumption (g/animal/day) was observed in the females of the mid dose group and high dose group in the first week of the premating phase, which returned to normal thereafter. No effects on food consumption were observed during the further premating phase or during gestation or lactation.
- Test substance intake: Test substance intake (mg Verdox/kg body weight/day) was calculated over the premating period, gestation period and lactation period from day 1-4 and is summarized in the table under: Any other information on results incl. tables. The intake of the test substance per kg body weight per day was calculated from the nominal dietary concentration, the feed consumption and the body weight in the pertaining week. Since the concentration of test substance was lower than intended in the low dose group (relative difference from intended concentration -11%) and since the test substance in diet was not stable when stored at ambient temperature in an open container for 24h in the mid dose group (relative decrease -12%), the actual test substance intake was lower than indicated in the table above for animals in the low dose and mid dose groups.
- Fertility and reproductive performance: In each group 12 females were placed with a male from the same dose group. The mean precoital time was comparable in all groups and ranged from 2.25 days in the low dose group to 2.75 days in the high dose group. The mating index was 100% in all groups and the number of pregnant females was 12/12 for the control, low dose and high dose group and 10/12 in the mid dose group. No differences were observed on female fertility indices, female fecundity index, gestation index or duration of gestation. No effects were observed on the mean number of implantation sites, pre-implantation loss and post-implantation loss. All pregnant females survived delivery, but one female in the Low Dose group was sacrifice moribund during lactation. Two animals in the control group, and four animals in both the mid dose and high dose groups delivered both live and stillborn pups. In addition, one female in the control group and two females in the low dose group delivered stillborn pups only.
- Haematology: An incidental, not dose-related, decrease was observed on the absolute number of lymphocytes in group 2 and 3. There were no statistically significant changes in red blood cell variables or clotting potential.
- Clinical chemistry: The concentration of phosphate was decreased in the low dose and mid dose groups and the concentration of potassium was decreased in the mid dose group. These findings, not confirmed at the high-dose levels, are considered incidental findings and not related to treatment.
- Parental necropsy observations: Organ weights of parental animals: No effects on organ weights were observed. Macroscopic examination of parental animals: At necropsy no treatment realted macroscopic changes were observed. Animal 27 of the low-dose group was killed in moribund condition on day 96 of the study. At necropsy the most important finding was adhesions of the liver with the stomach and pancreas. Microscopic examination of parental animals: Involution of the thymus in females is a normal phenomenon during pregnancy. At microscopical examination of animal 27 of the low-dose group (killed in moribund condition on day 96 of the study) severe peritonitis was observed, which was related to perforative ulcerative duodenitis, ulcerative gastritis and severe lobular necrotizing hepatitis. The reason for this condition could not be established The other histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 437 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: Based on no maternal adverse effects observed at tested concentrations

Key result

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
- Litter data: Litter size and sex: Sex ratio was comparable in all goups on lactation day 1 and lactation day 4. The mean number of pups delivered was comparable in all groups (134, 132, 111 and 125 for the control, low dose, mid dose and high dose groups, respectively). The relative number of liveborn pups was lower in the low dose group (120 of 134, 102 of 132, 97 of 111 and 112 of 125 for the control, low dose, mid dose and high dose groups, in 12, 12, 10 and 12 litters respectively) and the number of stillborn pups was increased in the low dose group (14, 30, 14 and 13 for the control, low dose, mid dose and high dose group, respectively). The number of pups that were lost between postnatal days 1-4 was statistically significantly lower in the low dose group (34, 2, 34 and 19 in the control group, low dose, mid dose and high dose groups, respectively). The number of litters lost entirely was comparable in all groups (4 in the control group, 2 in the low dose group, 3 in the mid dose group and 2 in the high dose group). The incidence of litter loss as was observed in this study was relatively high but in the same order as was observed in other reproductive toxicity studies performed at this test site, in the same time period with this strain of rats of this supplier. In none of these studies, the observed litter loss was related to treatment. There were no treatment-related effects on litter size and pup survival.
- Pup observations: One litter in the control group, one litter in the low dose group, two litters in the mid dose group and one litter in the high dose group comprised cold pups. In addition to this observation, the litters in the mid dose group showed small pups and pups that had no milk in the stomach. Although the incidence of clinical signs was statistically significantly increased in the mid dose group, all fetuses showing clinical signs came from two litters. In addition, no dose response relationship was observed. Based on the incidende and distribution of litters comprising pups showing clinical signs the observed clinical signs are not considered to be treatment-related. Pup body weight: There were no effects on mean pup body weight or body weigh changes for pups on lactation days 1 and 4. Macroscopic observations of stillborn pups or pups that died during lactation: One pup in the contol group and one pup in the low dose group showed a dilated urinary bladder. Other observations included no presence of milk in the stomach and no distention of the lungs and these observations are related to the stillborn or moribund status of the pups. There were no treatment-related macroscopic observations in pups that were stillborn or that died during lactation.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 437 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on no adverse developmental toxicity effects observed at tested concentrations.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Test substance intake:

	Mean (range) substance intake (mg Verdox/kg body weight/day)
	Low dose	Mid dose	High dose
Premating females	59.12 (50.21 – 70.66)	177.44 (154.81 – 209.25)	554.02 (471.33 – 671.55)
Gestation females	52.03 (42.22 – 58.40)	165.82 (128.61 – 185.02)	471.43 (380.64 – 528.21)
Lactation females	58.34	151.44	437.18

 

Conclusions:

The NOAEL for maternal toxicity and developmental toxicity is 437 mg/kg bw in rats as determined in this GLP compliant extended combined oral repeated dose toxicity study and reproduction/developmental toxicity screening test.

Executive summary:

In a GLP compliant extended combined repeated dose and reproduction / developmental screening study, performed according to OECD guideline 422, Wistar rats were treated with the test substance (0, 75, 200, and 500 mg verdox/kg bw/day, expected nominal dose levels) in the diet,during a premating period of 10 weeks and during mating (1 week), gestation and lactation until postnatal day 4. The actual test substance intake ranged between 42-71, 129 -209, and 381-672 for the low, mid, and high dose group, respectively. Clinical observations during the premating, gestation and lactation period did not reveal any treatment-related changes in the animal’s appearance, general condition or behaviour. Neurobehavioural observations and motor activity assessment did not indicate any neurotoxic potential of the test substance. No toxicological relevant effects on body weight and food consumption were observed. No treatment related effects were observed on pre-coital time, mating index, duration of gestation, pre- and postimplantation loss, number of corpora lutea, number of implantation sites and number of live pups on lactation. No treatment related clinical signs in pups, no effect on sex ratio in pups on day 1 and 4 of lactation, no effects on the mean pup body weight and body weight gain on lactation days 1 and 4 and no treatment-related macroscopic observations in pups that were stillborn or that died during lactation were observed. No effects on red blood cell variables or clotting potential was observed. No effects on organ weights were observed in females. Macrosopic and microscopic examination did not reveal any treatment-related effects. In conclusion, the NOAEL for maternal toxicity in this study was ≥ 500 mg/kg body weight in the diet. Based on the absence of effects on developmental parameters, the NOAEL for Verdox for developmental toxicity in this study was ≥ 500 mg/kg body weight in the diet. A NOAEL of 500 mg/kg body weight in the diet is equivalent to an overall intake of at least 437 mg/kg body weight/day for females.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

The OECD TG 422 study was carried out for at least 10 weeks considered to be similar to OECD TG 408

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Verdox Cas no 20298-69-5

IUPAC Name:

Verdox Cas no 20298-69-5

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Verdox

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at initiation of treatment: Female 6 weeks, Male 7 weeks
- Average weight at study initiation: Males: 179 g, Females: 126g
- Housing: In macrolon cages with a bedding of wood shavings (Lignocel, Type ¾), strips of paper (Enviro-dri) and a wooden block as environmental enrichment. During the range finding study, the animals were housed pairwise (2 animals of the same sex and dosing group per cage). In the main study, during the premating period, the animals were housed in groups of 4 per sex. For mating, one male and one female were housed together. Mated females were housed individually in macrolon cages, which were placed in a separate cage rack. After delivery, the cages containing the dam with litter were transferred to another cage rack.
- Diet: Cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, Witham, England), ad libitum.
- Water: Domestic mains tap-water, ad libitum. In the last week of gestation and during lactation a bottle containing glucose water (5%) was added to the cages in which the females were housed, in addition to the domestic tap water supply.
- Acclimation period: One week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2. During several short periods, temperature was outside the limits with a minimum of 19.8 ºC and a maximum of 24.9 ºC.
- Humidity (%): 45% and not exceeding 65%. Relative humidity was outside the limits during several short periods (a maximum value of 78.1% was recorded and in one occasion a minimum of 44% was recorded).
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

The test substance was incorporated in the basal diet by mixing in a mechanical blender.
In the main study, the experimental diets were prepared shortly before the start of the study and subsequently every ca. 4 or 5 weeks. After preparation, the experimental diets were divided into daily amounts of diets that were stored in plastic bags in a freezer (<-18°C). Each day, one bag per group was removed from the freezer to feed the animals.
The report of the range-finding study gives no information on diet preparation and refreshing frequency and storage conditions.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

RANGE FINDING STUDY:
The conclusion of the diet analysis was that the test substance was not stable in the diets when stored in the animal room.
MAIN STUDY:
Immediately after preparation of the first batch of the experimental diets, samples were taken and stored in the freezer until analysis. The stability of the test substance under simulated experimental conditions was demonstrated as follows: samples of control diet, low-dose diet, mid-dose diet and high-dose diet were prepared on 19 September 2011. After preparation samples were stored at <-18°C and analyzed on 7 October 2011 (t = 0), after storage at ambient temperature in an open container in the animal room for one day and after storage in the freezer (at < -18 °C) in a closed container for at least 5 weeks. The 5 week storage stability samples (< -18 °C) were analyzed on 26 October 2011. The homogeneity and content (achieved concentration) of the test substance in the experimental diets were demonstrated in the same batch of diets by analyzing five samples (taken at different locations in the feed container) of each test diet; one sample of the control diet was analyzed in the same series.
Result: The test substance was considered to be homogeneously distributed in the diets. The concentration of the test substance was close to intended for all dose levels (relative difference from the intended concentration was <10%), except for the low dose group (-11%). The test substance in diet was considered to be stable when stored in the freezer (at < -18 °C) in a closed container for 5 weeks (relative decrease of the Verdox concentration was less than 10% for all dose levels) and when stored at ambient temperature in an animal room in an open container for one day for the low and high dose groups. For the mid dose group the test substance was considered unstable as the relative decrease was 12%.

Duration of treatment / exposure:

Range finding study: two weeks
Main study: Male animals received the diets during a 10-week premating period and during mating up to the day of sacrifice. The female animals were given the diets with the test substance during a 10-week premating period, during mating, gestation and lactation, up to the day of sacrifice (approx. day 4 of lactation).

Frequency of treatment:

continuously

Doses / concentrations

Doses / concentrations 1

Remarks:

Dose range finding study: 75, 150, 500, 1000 mg/kg bw/day
Intended dose for animals receiving 1155, 2310, 7700 and 15400 mg/kg diet

Doses / concentrations 2

Remarks:

Main study: 75, 200, 500 mg/kg bw/day
Intended dose for animals receiving 800, 2500, 7500 mg/kg diet

No. of animals per sex per dose:

Range finding study: 4
Main study: 12

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale main study: selection based on 2-week range-finding study.

Examinations

Observations and examinations performed and frequency:

DOSE RANGE FINDING STUDY:
- Clinical observations: Each animal was observed daily in the morning hours. On working days, all cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. All abnormalities, signs of ill health or reactions to treatment were recorded.
- Body weight: The body weight of each animal was recorded twice weekly.
- Feed consumption: Feed consumption was recorded twice weekly.
MAIN STUDY:
- General clinical observations: Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. All cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. All abnormalities, signs of ill health or reactions to treatment were recorded.
- Neurobehavioural testing: Functional Observational Battery (FOB) tests, including assessment of grip strength and sensory reactivity to stimuli of different types, and spontaneous Motor Activity Assessment (MAA) were performed in 5 animals/sex/group prior to the end of the premating period (week 9). During neuro-behavioural testing, the observer was unaware of the treatment of the animals.
- Body weight: Body weights of male and female rats were recorded at randomization one day before the start of administration of the test substance and at the start of the study (Day 0). Males were then weighed once per week until sacrifice. Females were then weighed once per week during the premating and mating period. Mated females were weighed on Days 0, 7, 14 and 21 during presumed gestation and on Days 1 and 4 of lactation. After the mating period, nonmated females were weighed once per week. In addition, the animals were weighed on their scheduled necropsy date in order to calculate the correct organ to body weight ratios.
- Food consumption: Except during the mating period, food consumption was measured from day 0 onwards on the same days as body weight was measured. Nevertheless during the last week of the premating period where body weight was measured at day 63 while food was given on day 64 because some of the animals were fasted for one night for haematology and clinical chemistry.
- Intake of test substance: The intake of the test substance per kg body weight per day was calculated from the nominal dietary concentration of the test substance in the diet, the food consumption and the mean body weight measured at the beginning and the end of the pertaining period.
- Mating: At the end of the premating period each female was caged with one male from the same group. Every morning during the mating period, vaginal smears were made for determination of the presence of sperm. The day on which sperm was detected in the vaginal smear was considered as gestation Day 0. Upon evidence of copulation, the females were caged individually for the birth and rearing of their pups. Sperm positive females that turned out to be non-pregnant (females C61 and C67 of the mid dose group) were sacrificed 31 and 30 days after copulation, respectively. All other dams were allowed to raise their litter until sacrifice on day 4 of lactation or shortly thereafter.
- Parturition and litter evaluation: At the end of the gestation period (gestation day 21), females were examined twice daily for signs of parturition. Any difficulties occurring during parturition were recorded. To keep nest disturbance to a minimum the litters were examined only once daily for dead pups.
- Litter size, sexes and weight: The total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups and grossly malformed pups were evaluated on Days 1 and 4 of lactation. The pups were individually weighed on Days 1 and 4 of lactation. Mean pup weight was calculated per sex and for both sexes combined.
- Haematology: At the end of the premating period (week 10), 5 rats/sex/group were fasted overnight (water was freely available) and blood was taken by orbita punction whilst under CO2/O2 anaesthesia. K3-EDTA was used as anticoagulant. In each sample the following determinations were carried out : haemoglobin, packed cell volume, red blood cell count, reticulocytes,
total white blood cell count, differential white blood cell counts (neutrophils, lymphocytes, eosinophils, basophils, monocytes), prothrombin time, thrombocyte count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration.
- Clinical chemistry: At the end of the premating period (week 10), 5 rats/sex/group were fasted overnight (water was freely available) and blood was taken by orbital punction whilst under CO2/O2 anaesthesia. Blood was collected in heparinised plastic tubes and plasma was prepared by centrifugation. The following measurements were made in the plasma: alkaline phosphatase activity, aspartate aminotransferase activity, alanine aminotransferase activity, gamma glutamyl transferase activity, total protein, albumin, ratio albumin to globulin, urea, creatinine, bilirubin (total), cholesterol (total), triglycerides, phospholipids, calcium, sodium, potassium, chloride, inorganic phosphate, glucose (fasting)

Sacrifice and pathology:

DOSE RANGE FINDING STUDY:
- At autopsy all animals were examined macroscopically. Testes, kidney, liver and spleen were weighed.
MAIN STUDY:
- Gross necropsy and histology of parental animals: All male and female parent rats were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia at necropsy and then examined grossly for pathological changes. Male animals were sacrificed after the mating period. Female animals were sacrificed at or shortly after day 4 of lactation. Furthermore, organs showing gross lesions of animals of all groups were examined microscopically. Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes which was preserved in Bouin's fixative: Ovaries (after counting of the corpora lutea), Uterus (after counting of the implantation sites), testes, epididymides, seminal vesicles, prostate, all gross lesions. In addition, of 5 animals/sex/group the following organs were preserved: adrenals, bone marrow (femur),
brain (including sections of cerebrum, cerebellum, medulla/pons), heart, small and large intestines (including Peyer’s patches), kidneys, liver, lungs, lymph nodes (mesenterial and axillary), peripheral nerve (tibial), spinal cord (cervical, mid-thoracic, and lumbar), spleen, stomach, thymus, thyroid, trachea, urinary bladder.
The following organs were weighed (paired organs together): testes, epididymides, adrenals, brain, heart, kidneys, liver, spleen, thymus.
For sperm analysis, the left cauda epididymis and left testis of the 5 selected males were used.
Tissues for microscopic examination were embedded in paraffin wax, sectioned at 5 mm, and stained with haematoxylin and eosin, except for sections of the testes which were stained with PAS haematoxylin. The kidneys of the male rats were also stained immunocytochemically (using a mouse-anti-rat alpha 2-microglobuline antibody from SSI, Copenhagen, Denmark) to confirm alpha-hydrocarbon nephropathy. Microscopic examination was performed on the collected organs of all animals of the control and high-dose group. Microscopy of the kidneys was extended to all male animals of the low- and mid-dose groups.
Epididymal sperm motility, count and morphology: At scheduled necropsy, epididymal sperm was derived from the left cauda epididymis of 5 males/group. For this purpose the left cauda epididymis was dissected and weighed and thereafter minced in M199 medium containing 0.5% bovine serum albumin. Sperm motility and, after sonification and DNA staining, the cauda epididymal sperm reserves (sperm count) were measured for these males, using the Hamilton Thorne Integrated Visual Optical System (IVOS). In addition, a smear of the sperm solution was prepared and stained and 200 spermatozoa of the smear of males of the control group and of the high-dose group were examined for morphology.
Testicular sperm count: At necropsy, the left testis of the same males as used for epididymal sperm analysis was placed on dry ice and subsequently stored in a freezer (<-70°C) for later determination of the number of homogenization-resistant spermatids. Testicular sperm count was conducted in the control and high-dose group. The testes were thawed just before further processing. Following removal of the tunica albuginea, the testicular parenchyma was weighed, minced and homogenized in Saline Triton X-100 solution. Following DNA-staining, the homogenizationresistant sperm heads were enumerated using the IVOS. The daily sperm production was calculated.
- Pathology of pups: A necropsy was performed on stillborn pups and on pups that died during the study. At necropsy of the dams, at or shortly after day 4 of lactation, all other pups were examined externally for gross abnormalities and killed by appropriate techniques.

Other examinations:

Fertility and reproductive performance: For each mating the following data are presented for each group: number of females placed with males, number of males mated with females, number of successful copulations (= number of females mated), number of males that became sire, number of pregnant females as demonstrated by the presence of implantation sites observed at necropsy, number of females surviving delivery, number of females with liveborn and (all) stillborn pups, number of pups delivered (live- and stillborn), number of live pups on day 1 and 4, number of pups lost, number of litters lost entirely, number of male pups on day 1 and 4, number of corpora lutea, number of implantation sites, number of lost implantations, litter size. The following parameters were calculated: pre-coital time = time between the start of mating and successful copulation, duration of gestation = time between gestation day 0 and day of delivery, mating index= (number of females mated/number of females placed with males) x 100, male fertility index = (number of males that became sire/number of males placed with females) x 100, female fertility index = (number of pregnant females/number of females placed with males) x 100, female fecundity index = (number of pregnant females/number of females mated) x 100, gestation index = (number of females with live pups/number of females pregnant) x 100, live birth index = (number of pups born alive/number of pups born) x 100, viability index day n-m = (number of pup surviving m days/number of liveborn on day n) x 100, pup mortality day n = (number of dead pups on day n/total number of pups on day n) x 100, sex ratio day n = (number of live male pups on day n/ number of live pups on day n) x 100, pre-implantation loss = [(number of corpora lutea – number of implantation sites)/number of corpora lutea] x 100, number of lost implantations = number of implantations sites - number of pups born alive, post-implantation loss = [(number of implantation sites - number of pups born alive)/number of implantation sites] x 100.

Statistics:

DOSE RANGE FINDING STUDY:
Clinical findings and parental necropsy observations were evaluated by Fisher's exact probability test. Body weight (gain), organ weights and feed consumption data were subjected to one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests. All analyses were two-sided. Group mean differences with an associated probability of less than 0.05 were considered to be statistically significant.
MAIN STUDY:
P < 0.05 was considered as the level of significance. Clinical findings were evaluated by Fisher's exact probability test. Body weight, body weight gain, food consumption and organ weights data were subjected to one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests. Fisher's exact probability test was used to evaluate the number of mated and pregnant females, the number of pregnant females with implants but no pups, females with live pups, females with stillborn pups, live and dead fetuses or pups and the numbers of litters lost entirely.Pre-coital time (mean number of days), the duration of gestation, the number of corpora lutea and implantation sites, the total number of pups delivered (mean), the mean number of live pups per litter and pre- and post-implantation loss (%) were evaluated by Kruskal-Wallis nonparametric analysis of variance and by the Mann-Whitney U test. Haematology and clinical chemistry parameters were subjected to one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests. Mortality data and data of the pathology of parent animals were evaluated by the Fisher’s exact probability test. Sperm parameters were evaluated by one-way analysis of variance followed by Dunnett’s multiple comparison test (epididymal and testicular sperm count and numerical sperm motility parameters) or by Kruskal-Wallis non parametric analysis of variance and by Mann-Whitney U test (motility parameters expressed as a percentage and sperm morphology).

Results and discussion

Results of examinations

Details on results:

DOSE RANGE FINDING STUDY:
- Clinical signs and survival: No treatment-related clinical signs were observed.
- Body weights: Mean body weights were statistically significantly decreased in females of the middose group on day 10 and in females of the top-dose group from day 3 to 14. Mean body weight changes were statistically significantly reduced in male animals of the high-dose group from day 10-14 and in males animals of the top-dose group from day 0 to 3 and 0 to 14. Mean body weight changes were statistically significantly reduced in female animals of the top-dose group from day 0 to 7.
- Feed consumption: Food consumption (g/animal/day) of the male animals of the high- and top-dose groups was statistically significantly decreased from day 0 to 3. Food consumption (g/kg bw/day) of the male animals of the top-dose group was statistically significantly decreased from day 0 to 3. Food consumption (g/animal/day) of the female animals of the high- and top-dose groups was statistically significantly decreased from day 7 to 10 and 10 to 14.
- Mean substance intake from day 0-14 of male animals was 95, 173, 581 and 1160 mg test substance/kg bw/day for the low-, mid-, high- and top-dose groups, respectively. Mean substance intake from day 0-14 of the female animals was 93, 183, 571 and 1121 mg test substance/kg bw/day for the low-, mid-, high- and top-dose groups, respectively.
- Necropsy observations: At macroscopic examination of the animals at sacrifice, the kidneys of all males of all groups were pale. Kidneys of the female animals did not show this observation. Except for a statistically significant increased weight of the livers of male and female animals of the top-dose group, no statistically significant effects were observed on organ weights.
MAIN STUDY:
- Mortalities and clinical signs: One animal from the low dose group was sacrificed moribund during lactation, showing hunched posture, lethargy, piloerection, encrustations around nose and eyes and haemorrhagic discharge around the vagina. In the males one animal in the control group showed red ears and one animal in the high dose group showed sparsely haired skin and encrustations. In the females one animal in the high dose group showed cateract of the eyes throughout all study periods. One control animal showed sparsely haired skin during gestation and lactation. One animal in the control group and one animal in the high dose group showed piloerection during lactation. Based on the incidence and distribution the observed clinical signs are considered not to be treatment-related.
- Neurobehavioural testing: The functional observational battery observations and motor activity assessment did not reveal any treatment-related effects on neurobehaviour.
- Body weight and body weight change: Males in the high dose group showed a slightly lower body weight as compared to the control animals, which reached the level of statistically significance on days 28, 35 and 56 of the study. Body weight gain was also slightly lower in this group, mainly in the first 4 weeks of the study, and reached the level of statistically significance in the periods from day 0-7, 21-28 and 49-56 of the study. As a consequence, the total body weight gain of the male animals between days 0-77 was also slightly, but statistically significantly, decreased. Since the effect on body weight gain was most clear in the first weeks of the study, the effect may be (partly) due to the palatability of the test substance in the diets. For that reason, the effect on body weight gain of the male animals is considered of marginal toxicological relevance. No effects on body weight or body weight change were observed in the low dose and mid dose groups for the males. In females, no effects on body weight were observed during premating, gestation and lactation. In the high-dose group females body weight gain was only statistically significantly lower in the first week of premating and recovered thereafter.
- Food consumption: In males, no effects on food consumption were observed throughout the study. In females, a slight, but statistically significant lower food consumption (g/animal/day) was observed in the females of the mid dose group and high dose group in the first week of the premating phase, which returned to normal thereafter. No effects on food consumption were observed during the further premating phase or during gestation or lactation.
- Test substance intake: Test substance intake (mg Verdox/kg body weight/day) was calculated over the premating period (males and females), gestation period (females) and lactation period from day 1-4 (females) and is summarized in the table under: Any other information on results incl. tables. The intake of the test substance per kg body weight per day was calculated from the nominal dietary concentration, the feed consumption and the body weight in the pertaining week. Since the concentration of test substance was lower than intended in the low dose group (relative difference from intended concentration -11%) and since the test substance in diet was not stable when stored at ambient temperature in an open container for 24h in the mid dose group (relative decrease -12%), the actual test substance intake was lower than indicated in the table above for animals in the low dose and mid dose groups.
- Fertility and reproductive performance: In each group 12 females were placed with a male from the same dose group. The mean precoital time was comparable in all groups and ranged from 2.25 days in the low dose group to 2.75 days in the high dose group. The mating index was 100% in all groups and the number of pregnant females was 12/12 for the control, low dose and high dose group and 10/12 in the mid dose group. No differences were observed on male and female fertility indices, female fecundity index, gestation index or duration of gestation. No effects were observed on the mean number of implantation sites, pre-implantation loss and post-implantation loss. All pregnant females survived delivery, but one female in the Low Dose group was sacrifice moribund during lactation. Two animals in the control group, and four animals in both the mid dose and high dose groups delivered both live and stillborn pups. In addition, one female in the control group and two females in the low dose group delivered stillborn pups only.
- Litter data: Litter size and sex: Sex ratio was comparable in all goups on lactation day 1 and lactation day 4. The mean number of pups delivered was comparable in all groups (134, 132, 111 and 125 for the control, low dose, mid dose and high dose groups, respectively). The relative number of liveborn pups was lower in the low dose group (120 of 134, 102 of 132, 97 of 111 and 112 of 125 for the control, low dose, mid dose and high dose groups, in 12, 12, 10 and 12 litters respectively) and the number of stillborn pups was increased in the low dose group (14, 30, 14 and 13 for the control, low dose, mid dose and high dose group, respectively). The number of pups that were lost between postnatal days 1-4 was statistically significantly lower in the low dose group (34, 2, 34 and 19 in the control group, low dose, mid dose and high dose groups, respectively). The number of litters lost entirely was comparable in all groups (4 in the control group, 2 in the low dose group, 3 in the mid dose group and 2 in the high dose group). The incidence of litter loss as was observed in this study was relatively high but in the same order as was observed in other reproductive toxicity studies performed at this test site, in the same time period with this strain of rats of this supplier. In none of these studies, the observed litter loss was related to treatment. There were no treatment-related effects on litter size and pup survival.
- Pup observations: One litter in the control group, one litter in the low dose group, two litters in the mid dose group and one litter in the high dose group comprised cold pups. In addition to this observation, the litters in the mid dose group showed small pups and pups that had no milk in the stomach. Although the incidence of clinical signs was statistically significantly increased in the mid dose group, all fetuses showing clinical signs came from two litters. In addition, no dose response relationship was observed. Based on the incidende and distribution of litters comprising pups showing clinical signs the observed clinical signs are not considered to be treatment-related. Pup body weight: There were no effects on mean pup body weight or body weigh changes for pups on lactation days 1 and 4. Macroscopic observations of stillborn pups or pups that died during lactation: One pup in the contol group and one pup in the low dose group showed a dilated urinary bladder. Other observations included no presence of milk in the stomach and no distention of the lungs and these observations are related to the stillborn or moribund status of the pups. There were no treatment-related macroscopic observations in pups that were stillborn or that died during lactation.
- Haematology: In the male animals no statistically significant findings were observed. In the female animals an incidental, not dose-related, decrease was observed on the absolute number of lymphocytes in group 2 and 3. There were no statistically significant changes in red blood cell variables or clotting potential.
- Clinical chemistry: In the males in from the high dose group the concentration of ASAT was decreased. This is not considered to be treatment-related because in case of an effect of treatment an increase in the concentration of ASAT would be expected to occur instead of the observed decrease. In males the concentration creatine was increased in the low dose and mid dose group, but not in the high dose group. In females the concentration of phosphate was decreased in the low dose and mid dose groups and the concentration of potassium was decreased in the mid dose group. These findings, not confirmed at the high-dose levels, are considered incidental findings and not related to treatment. In males of group 2, 3 and 4 a statistically significant increase in the urea-concentration was found when compared to the control group. This finding might be indicative of an effect on the kidneys.
- Sperm analysis: Epididymal sperm motility: No statistically significant effects on sperm motility parameters (motile cells, static cells and progressive cells) were observed among the groups. Furthermore, no differences in the derived parameters describing sperm motility were found between the groups. Epididymal sperm count: No statistically significant effects were observed on epidydimal sperm count parameters between the groups. Epididymal sperm morphology: No differences were observed on sperm morphology between the control group and the highdose group. Testicular sperm count: No effects were observed on the testicular parenchyma weight, the number of spermatozoa per gram testicular parenchyma or on the daily sperm production between the control and the high dose group.
- Parental necropsy observations: Organ weights of parental animals: The terminal mean body weight was decreased in the males of the high dose group. The absolute and relative weights of the spleen were statistically significantly increased in males of the low and mid dose groups, but this finding was not confirmed at the high-dose level and was therefore not considered toxicologically relevant. The relative weight of the liver was statistically significantly increased (circa 15%) in males of the high-dose group. A dose related increase in relative kidney weight was observed in the males of the mid and high dose groups. No effects on organ weights were observed in female animals. Macroscopic examination of parental animals: At necropsy no treatment realted macroscopic changes were observed. Animal 27 of the low-dose group was killed in moribund condition on day 96 of the study. At necropsy the most important finding was adhesions of the liver with the stomach and pancreas. Microscopic examination of parental animals: A dose-dependent increase of hyalin droplet nephropathy was found in the male animals, characterised by an abundant presence of eosinophilic hyalin droplets in the proximal tubular cells, which in several cases was accompanied by dilated tubuli filled with eosinophilic debris in the corticomedullary area. In agreement with the nephropathy, a dose dependent increase in basophilic tubuli was observed. Involution of the thymus in females is a normal phenomenon during pregnancy. At microscopical examination of animal 27 of the low-dose group (killed in moribund condition on day 96 of the study) severe peritonitis was observed, which was related to perforative ulcerative duodenitis, ulcerative gastritis and severe lobular necrotizing hepatitis. The reason for this condition could not be established The other histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to treatment. Immunohistochemistry: The kidneys of male animals were stained for the presence of α2u-globulin using specific antibodies. Microscopical examination revealed a dose-dependent increase of α2u-globulin staining of the cortical tubular epithelial cells and staining of the observed corticomedullary tubular cell debris.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 437 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on no adverse effects observed at tested concentrations.

Remarks on result:

other: Exposure period was at least 10 weeks

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Test substance intake:

	Mean (range) substance intake (mg Verdox/kg body weight/day) based on the nominal concentration of Verdox in the diet
	Low dose	Mid dose	High dose
Premating males	55.65 (45.85 – 69.47)	167.74 (140.66 – 209.10)	504.58 (415.47 – 616.50)
Premating females	59.12 (50.21 – 70.66)	177.44 (154.81 – 209.25)	554.02 (471.33 – 671.55)
Gestation females	52.03 (42.22 – 58.40)	165.82 (128.61 – 185.02)	471.43 (380.64 – 528.21)
Lactation females	58.34	151.44	437.18

Applicant's summary and conclusion

Conclusions:

A NOAEL of >= 437 in male/female rats, in a GLP compliant in an extended to at least 10 weeks OECD TG 422 study.

Executive summary:

In a dose-range finding study, rats received diets containing containing 0, 1155, 2310, 7700 and 15400 mg Verdox/kg diet (intented dose of 0, 75, 150, 500 and 1000 mg/kg body weight/day) for two weeks (4 rats/sex/dose). No treatment-related clinical signs were observed. Mean body weights in females from the mid- and top-dose groups were decreased. Body weight changes were reduced in males of the high- and top-dose groups and in female animals of the top-dose group. Food consumption was statistically significantly decreased in male and female animals of the high- and top-dose groups. At necropsy, kidneys of the male animals of all dosing groups showed a pale appearance. The weight of the livers of male and female animals of the top-dose group was statistically significantly increased. Diet analysis showed that the diets were not stable at (animal) room temperature. Therefore, in the main study, the diets had to be refreshed daily. Based on the result of the current study and in consultation with the sponsor, the following dose levels were proposed for the subsequent main study: 0, 800, 2500 and 7500 mg Verdox/kg diet (corresponding with a nominal expected dose level of 0, 75, 200 and 500 mg/kg body weight) for the animals of the low-, mid- and high-dose groups, respectively.

In the GLP compliant combined repeated dose and reproduction / developmental screening study, performed according to OECD guideline 422, Wistar rats were treated with the test substance (0, 75, 200, and 500 mg verdox/kg bw/day, nominal expected dose levels) in the diet, during a premating period of 10 weeks and during mating (1 week), gestation and lactation until postnatal day 4. The actual test substance intake ranged between 46-69 for males and 42-71 for the females of the low dose group. For the mid dose group the actual test substance concentration ranged from 141-209 for the males and 129-209 for the females. For the high dose group the actual test substance concentration ranged from 415-617 for the males and ranged between 381-672 for the females. Clinical observations during the premating, gestation and lactation period did not reveal any treatment-related changes in the animal’s appearance, general condition or behaviour. Neurobehavioural observations and motor activity assessment did not indicate any neurotoxic potential of the test substance. No toxicological relevant effects on body weight and food consumption were observed in both sexes. No treatment related effects were observed on pre-coital time, mating index, duration of gestation, pre- and postimplantation loss, number of corpora lutea, number of implantation sites and number of live pups on lactation. No treatment related clinical signs in pups, no effect on sex ratio in pups on day 1 and 4 of lactation, no effects on the mean pup body weight and body weight gain on lactation days 1 and 4 and no treatment-related macroscopic observations in pups that were stillborn or that died during lactation were observed. No effects on red blood cell variables or clotting potential was observed. A statistically significant increase in urea-concentration was observed in males of all dosing groups which might be related to the observed kidney effects (α2u-microglobulin nephropathy). No treatment-related effects were observed on sperm-parameters (epidydimal sperm motility, sperm count and morphology and testicular sperm count). An increased relative liver weight was observed in high dose males which was considered as an adaptive response to increased physiological demand. A dose related increase in relative kidney weight was observed in mid and high dose males which was related to the observed α2u-microglobulin nephropathy. No effects on organ weights were observed in females. Effects observed on kidney weights and urea-concentrations were considered to be related to α2u-microglobulin nephropathy which was confirmed by immunocytochemical staining of the α2u-microglobulin protein in the cortical tubular epithelial cells. This effect observed in rats is generally regarded as of no toxicological relevance for humans. Macrosopic and microscopic examination did not reveal any treatment-related effects. In conclusion, the NOAEL for Verdox for males and females in this study was ≥ 500 mg/kg body weight in the diet. Based on the absence of effects on fertility parameters and developmental parameters, the NOAEL for Verdox for fertility and developmental toxicity in this study was ≥ 500 mg/kg body weight in the diet. A NOAEL of 500 mg/kg body weight in the diet is equivalent to an overall intake of at least 505 mg/kg body weight/day for males and at least 437 mg/kg body weight/day for females.