2-tert-butylcyclohexyl ethyl carbonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 30 August, 2011 to 2 october, 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD Guideline 422 with minor deviations on an analogue considered in a read-across approach.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

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:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

10 weeks premating exposure in males and females

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

cis-2-tert-butylcyclohexyl acetate

EC Number:

243-718-1

EC Name:

cis-2-tert-butylcyclohexyl acetate

Cas Number:

20298-69-5

IUPAC Name:

2-tert-butylcyclohexyl acetate

Test material form:

liquid

Details on test material:

- Physical state: Clear, colorless liquid
- Storage condition of test material: Ambient temperature
- Expiration date of the lot/batch: 31 July 2013

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han) strain

Details on species / strain selection:

The rat was used because this species is considered one of the most suitable species for this type of study, and is usually required by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Female 6 weeks, Male 7 weeks
- Weight at study initiation: (P) Males: 175.18 ± 3.25 g to 182.16 ± 1.13 g; Females: 123.96 ± 2.13 g to 127.48 ± 1.23 g
- Fasting period before study: None
- Housing: Animals were housed in macrolon cages with a bedding of wood shavings (Lignocel, Type ¾), strips of paper (Enviro-dri) and (from 15 November 2011 onwards) 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
- Acclimation period: 1 week

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

IN-LIFE DATES: From: 21 September 2011 To: 2 January 2012

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- 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.

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), afterstorage 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 the diets was considered to be stable when stored at ambient temperature in the animal room in an open container for one day for the low and high dose group. For the mid dose level, however, test substance was considered to be unstable in the diet at ambient temperature in the animal room in an open container for one day as the decrease was 12%.
Upon storage in the freezer (at < -18 °C) in a closed container for 5 weeks, the relative decrease of the test substance concentration was less than 10% for all dose levels.

Duration of treatment / exposure:

Male animals received these diets during a 10-week premating period and during mating up to the day of sacrifice.
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

Dose / conc.:

800 mg/kg diet

Remarks:

nominal expected dose level: 75 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

2 500 mg/kg diet

Remarks:

nominal expected dose level: 200 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

7 500 mg/kg diet

Remarks:

nominal expected dose level: 500 mg/kg bw/day

No. of animals per sex per dose:

12

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose range finding study was conducted to select the dose levels for OECD 422 study. In a dose-range finding study, Wistar rats (4 rats/sex/dose) received diets containing 0, 1155, 2310, 7700 and 15400 mg/kg diet (intended dose of 0, 75, 150, 500 and 1000 mg/kg bw/day) for two weeks. 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/kg diet (corresponding with a nominal expected dose level of 0, 75, 200 and 500 mg/kg bw) for the animals of the low-, mid- and high-dose groups, respectively.

- Rationale for animal assignment: One day before the start of the premating period, the animals (males and females separately) were allocated to the various groups by computer randomization proportionately to body weight. Surplus animals were kept in the animal room as sentinel animals. These rats were not used in the study.

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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.

BODY WEIGHT: Yes
- Time schedule for examinations: 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 AND COMPOUND INTAKE (if feeding study):
- 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. The results in this report are expressed in g per animal per day and g per kg body weight per day.
- 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.

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the premating period (week 10)
- Anaesthetic used for blood collection: Yes (CO2/O2 anaesthesia)
- Animals fasted: Yes; animals were fasted overnight (water was freely available) and blood was taken by orbita punction whilst under CO2/O2 anaesthesia
- How many animals: 5 rats/sex/group
- K3-EDTA was used as anticoagulant (haematology). Blood was collected in heparinised plastic tubes and plasma was prepared by centrifugation (clinical chemistry).
- Haematology parameters: 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 parameters: 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).
- Parameters checked in table [No.?] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 5 animals/sex/group prior to the end of the premating period (week 9)- Dose groups that were examined:
- Battery of functions tested: 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.

IMMUNOLOGY: No

OTHER:
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.

Sacrifice and pathology:

GROSS PATHOLOGY
- 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.

HISTOPATHOLOGY / ORGAN WEIGHTS
- 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 and urinary bladder.

Organ weights: The following organs were weighed (paired organs together) as soon as possible after dissection to avoid drying: adrenals, brain, heart, kidneys, liver, spleen, and thymus
Histopathology: 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.
Microscopic examination was performed on the collected organs of all animals of the control and highdose group. Since histopathological effects were observed in the kidneys of the male animals of the high-dose group histopathological examination of the kidneys was extended to male animals of the low- and mid-dose groups.
Furthermore, in the kidneys of the male animals, the α-globuline protein was detected immunocytochemically to confirm α-hydrocarbon nephropathy. Hereto, sections of the kidneys of the male animals were processed for immunocytochemical staining of α-2-microglobuline using a mouse-anti-rat alpha 2-microglobuline antibody (SSI, Copenhagen, Denmark).

Other examinations:

Sperm parameters (parental animals) Parameters examined in male parental generations:
Epididymal sperm motility, count and morphology: At scheduled necropsy, epididymal sperm was derived from the left cauda epididymis of 5 males/group. 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 highdose 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 contr ol 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.

Reproductive indices:
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

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).

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- 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.

Mortality:

no mortality observed

Description (incidence):

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.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 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 and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

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:
- 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.

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- 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 biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- In the high dose group males, the concentration of ASAT was decreased and not considered to be treatment-related. In males the creatine concentration 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.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The functional observational battery observations and motor activity assessment did not reveal any treatment-related effects on neurobehaviour.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- 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.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- At necropsy no treatment related 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.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- 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 mid-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.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

- Sperm parameters: No treatment-related effects were observed on sperm-parameters (epidydimal sperm motility, sperm count and morphology and testicular sperm count).
- Fertility and reproductive performance: No effects on fertility (pre-coital time, duration of gestation and mating index were comparable in all groups).

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Remarks on result:

other: equivalent to an overall intake of at least 505 mg/kg bw/day for males and at least 437 mg/kg bw/ day for females

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 7.8.1/1 : Test substance intake

Males/females	Mean (range) substance intake (mg/kg bw/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

Daily 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. One animal in the low dose was sacrificed moribund during lactation. Neurobehavioural observations and motor activity assessment did not indicate any neurotoxic potential of the test substance.

A lower body weight and body weight gain was observed in the males of the high dose group. 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 and, for that reason, considered of marginal toxicological relevance. Also in the female animals of the mid- and high-dose groups, a slightly lower body weight gain was observed in the first week of dosing only. No effects on food consumption were observed for the males. In the females, food consumption was lower in the mid and high dose group during the first week of dosing only.

The mean test substance intake (mg Verdox/kg body weight/day), calculated from the nominal dietary concentration, feed consumption and the body weight was 56 for the males and 52 – 59 for the females of the low dose group, 168 for the males and 151 – 177 for the females of the mid dose group and 505 for the males and 437 – 554 for the females of the high dose group. However, the actual test substance intake was lower since the test substance concentration was not met in the low dose group (-11%) and was not stable in the diet in the mid dose group (-12%). For that reason the actual test substance intake ranged between 50 – 56 for males and 46 – 59 for the females of the low dose group. For the mid dose group the actual test substance concentration ranged from 148 – 168 for the males and 133 – 177 for the females.

No treatment related effects were observed on pre-coital time, mating index, male and female fertility indices, female fecundity index, gestation index, duration of gestation, pre- and postimplantation loss, number of corpora lutea, number of implantation sites and number of pups delivered.

No effects were observed on litter size, pup sex and weight and pup survival.

No treatment-related effects were observed on sperm-parameters (epidydimal sperm motility, sperm count and morphology and testicular sperm count).

At autopsy the males of the high dose group showed an increased relative liver weight. Since no adverse effects were observed on histopathology of the liver and on clinical chemistry parameters the increased liver weight as observed in the male animals is considered as an adaptive response to increased physiological demand and of no toxicological relevance. A dose related increase in relative kidney weight was observed in the mid and high dose males. The increased weight of the kidneys were related to α2u-microglobulin nephropathy (see next paragraph) and considered of no toxicological relevance. No effects on organ weights were observed in females.

The male animals showed a dose-dependent increase of hyalin droplet nephropathy that resembled that of chemically induced α2u-globulin nephropathy. Immunohistochemical staining confirmed the increased accumulation of α2u-globulin in the cortical tubular epithelial cells. The discrepancy between the incidence of hyalin droplet nephropathy observed in the HE stained slides and the incidence of increased α2u-globulin staining was caused by the different approach of the evaluation. In the HE stained slides the kidneys were examined with high magnification necessary to establish the presence of the intracellular hyalin droplets. The immunohisto- chemically stained slides were examined for the presence of α2u-globulin which was done by judging the general positive staining intensity which has to be assessed with low magnification.

The proposed mechanism of hyalin droplet nephropathy is that the test substance or its metabolite(s) bind(s) to the α2u-globulin protein. This protein/chemical complex is filtered at the glomerulus and is reabsorbed into the proximal tubule cells. The binding of the chemical inhibits the lysosomal breakdown of the α2u-globulin and the protein/chemical complex builds up in the lysosomes (hyalin droplets formation). Ultimately the lysosomes rupture and the cells die as a result (eosinophilic debris in the tubular lumen). The α2u-globulin is produced in the liver and androgen controlled, hence not present in female rats. α2u-Globulin nephropathy is a well known phenomenon. It is male rat specific and considered of no toxicological relevance.

For male and female animals the NOAEL for Verdox 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.

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the NOAEL for male and female animals in this study was ≥500 mg/kg bw/d. A NOAEL of 500 mg/kg bw/d in the diet is equivalent to an overall intake of at least 505 mg/kg bw/day for males and at least 437 mg/kg body weight/day for females.

Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, Wistar rats were treated with the test substance (0, 75, 200, and 500 mg/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. Dose levels were selected on the basis of results of two-weeks dose-range finding study (0, 1155, 2310, 7700 and 15400 mg/kg diet (intended dose of 0, 75, 150, 500 and 1000 mg/kg bw/day).

The following observations and examinations were evaluated: mortality, clinical signs, functional observations, body weight, food consumption, haematology, clinical chemistry, organ weights, gross and histopathological examination and reproduction/developmental parameters. Diet preparations were analyzed once during the study to assess accuracy and homogeneity.

 

The mean test substance intake (mg/kg bw/day), calculated from the nominal dietary concentration, feed consumption and the body weight was 56 for the males and 52-59 for the females of the low dose group, 168 for the males and 151-177 for the females of the mid dose group and 505 for the males and 437-554 for the females of the high dose group. However, the actual test substance intake was lower since the test substance concentration was not met in the low dose group (-11%) and was not stable in the diet in the mid dose group (-12%). For that reason the actual test substance intake ranged between 50-56 for males and 46-59 for the females of the low dose group. For the mid dose group the actual test substance concentration ranged from 148-168 for the males and 133-177 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. One animal in the low dose was sacrificed moribund during lactation. Neurobehavioural observations and motor activity assessment did not indicate any neurotoxic potential of the test substance.

 

No toxicological relevant effects on body weight and body weight gain of the male and female animals were observed. No toxicological relevant effects on food consumption were observed for males and females throughout the study.

 

No effects on red blood cell variables or clotting potential. 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). No treatment related effects were observed on pre-coital time, mating index, male and female fertility indices, female fecundity index, gestation index, duration of gestation, pre- and postimplantation loss.

 

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.

 

Macroscopic and microscopic examination did not reveal any treatment-related effects.

 

Under the test conditions, the NOAEL for male and female animals in this study was ≥500 mg/kg bw/d in the diet. A NOAEL of 500 mg/kg bw/d in the diet is equivalent to an overall intake of at least 505 mg/kg bw/day for males and at least 437 mg/kg body weight/day for females.