cis-2-tert-butylcyclohexyl acetate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Fertility NOAEL: No adverse effects seen (>437 mg/kg bw in an >=10 weeks extended OECD TG 422)

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Aug 2011 to 2 Oct 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)

Remarks:

corresponds to 800 mg/kg diet

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

corresponds to 2500 mg/kg diet

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

corresponds to 7500 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. In this 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 (see details in the summary table below). 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 decreased in male and female animals of the high- and top-dose groups between 13 and 17%. These effects are expected to be due to the palatability of Verdox being a fragrance which may smell awkwardly for rats and therefore not tasty. At necropsy, kidneys of the male animals of all dosing groups showed a pale appearance. The relative weight of the livers of male and female animals of the top-dose group of 15400 mg/kg diet was statistically significantly increased up to 20 and 17%, respectively, which can be considered (close to or) at metabolic limit of saturation because the substance is expected to be fully absorbed based on its molecular weight and physico-chemical properties. At 7700 mg/kg diet the increases were 11 and 5%, respectively. 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. These level were expected no to affect the palatability of the diet and /or overloading metabolic saturation.

Summary of key effects in DRF

Mg/kg diet Body weight Body weight gain Food consumption / per animal Relative liver weight
Males
1155 Not applicable Not applicable Not applicable Not applicable
2310 Not applicable Not applicable Not applicable Not applicable
7700 No effect -18% -17% +11%
15400 No effect -25% -17% +20%
Females
1155 Not applicable Not applicable Not applicable Not applicable
2310 Not applicable Not applicable Not applicable Not applicable
7700 No effects -25% -13% +5%
15400 -6% -25% -15% +17%

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.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In females one 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. Additionally, 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.
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.
Based on the incidence and distribution the observed clinical signs are considered not to be treatment-related.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One animal from the low dose group was sacrificed moribund during lactation.

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 (partially) 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, non-treatment-related

Description (incidence and severity):

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 (mg substance/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 for animals in the low dose and mid dose groups.

Food efficiency:

not examined

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

not examined

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 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.

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

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 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.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

- 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.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

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.

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: Based on no adverse effects observed at tested concentrations.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

>= 473 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.

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

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 incidence and distribution of litters comprising pups showing clinical signs the observed clinical signs are not considered to be treatment-related.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

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.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no effects on mean pup body weight or body weigh changes for pups on lactation days 1 and 4.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

Sex ratio was comparable in all groups on lactation day 1 and lactation day 4. The mean number of pups delivered was comparable in all groups.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

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

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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:

Verdox was tested in a>=10 week OECDTG 422 study.

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 (see details in the summary table below). 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 decreased in male and female animals of the high- and top-dose groups between 13 and 17%. These effects are expected to be due to the palatability of Verdox being a fragrance which may smell awkwardly for rats and therefore not tasty. At necropsy, kidneys of the male animals of all dosing groups showed a pale appearance. The relative weight of the livers of male and female animals of the top-dose group of 15400 mg/kg diet was statistically significantly increased up to 20 and 17%, respectively, which can be considered (close to or) at metabolic limit of saturation because the substance is expected to be fully absorbed based on its molecular weight and physico-chemical properties. At 7700 mg/kg diet the increases were 11 and 5%, respectively. 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. These level were expected no to affect the palatability of the diet and /or overloading metabolic saturation.

Main study: 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.

Analysis of the test substance: 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 and 672 for females.

Clinical observationsduring the premating, gestation and lactation period did not reveal any treatment-related changes in the animal’s appearance, general condition or behaviour.Neurobehavioural observationsand 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.

Haematology: No effects on red blood cell variables or clotting potential were observed.

Clinical biochemistry: 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).

Organ effects: Increased relative liver weight (14%) 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 immune-cytochemical 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.

Fertility: 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, duration of gestation, pre- and postimplantation loss, number of corpora lutea, number of implantation sites and number of live pups on lactation.

Developmental toxicity: 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.

In conclusion on repeated dose toxicity: The NOAEL for Verdox for males and females in this study was ≥ 437 mg/kg body weight in the diet (based on the lowest intake of the test substance in lactating females).

In conclusion on fertility and developmental toxicity: 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 ≥ 437 mg/kg body weight in the diet, based on the lowest intake in lactating females. 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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Additional information

The executive summary of the >=10 weeks OECD TG 422 is presented below including repeated dose toxicity information and including the developmental toxicity information. Reports are attached in the repeated dose study record.

Verdox was tested in a>=10 week OECDTG 422 study.

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 decreased in male and female animals of the high- and top-dose groups between 13 and 17%. These effects are expected to be due to the palatability of Verdox being a fragrance which may smell awkwardly for rats and therefore not tasty. At necropsy, kidneys of the male animals of all dosing groups showed a pale appearance. The relative weight of the livers of male and female animals of the top-dose group of 15400 mg/kg diet was statistically significantly increased up to 20 and 17%, respectively, which can be considered (close to or) at metabolic limit of saturation because the substance is expected to be fully absorbed based on its molecular weight and physico-chemical properties. At 7700 mg/kg diet the increases were 11 and 5%, respectively. 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. These level were expected no to affect the palatability of the diet and /or overloading metabolic saturation.

Main study: 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.

Analysis of the test substance: 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 and 672 for females.

Clinical observationsduring the premating, gestation and lactation period did not reveal any treatment-related changes in the animal’s appearance, general condition or behaviour.Neurobehavioural observationsand 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.

Haematology: No effects on red blood cell variables or clotting potential were observed.

Clinical biochemistry: 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).

Organ effects: Increased relative liver weight (14%) 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 immune-cytochemical 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.

Fertility: 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, duration of gestation, pre- and postimplantation loss, number of corpora lutea, number of implantation sites and number of live pups on lactation.

Developmental toxicity: 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.

In conclusion on repeated dose toxicity: The NOAEL for Verdox for males and females in this study was ≥ 437 mg/kg body weight in the diet (based on the lowest intake of the test substance in lactating females).

In conclusion on fertility and developmental toxicity: 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 ≥ 437 mg/kg body weight in the diet, based on the lowest intake in lactating females. 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.

Verdox 90 -day study (OECD TG 408)

The male and female reproductive parameters were not affected in this 90 -day study further supporting the absence of fertility of Verdox, see for detailed information the repeated dose toxicity section. information.

Effects on developmental toxicity

Description of key information

In a prenatal developmental toxicity study (OECD TG 414) the NOAEL of >= 444 mg/kg bw/day was derived for both maternal and developmental toxicity, based on the absence of toxicological relevant effects.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Dec 2016 to 12 Jan 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 22nd January 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Triskelion B.V., Utrechtseweg 48, 3704 HE Zeist, The Netherlands

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han IGS (Crl:WI(Han))

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: The female rats were about 10 weeks old at arrival. Males for mating from the same strain and supplier were available at the test facility. At the time of mating these males were approximately 16 weeks old.
- Housing: Animals were housed in macrolon cages with a bedding of wood shavings (Lignocel) and strips of paper (Enviro-dri) and a wooden block as environmental enrichment. During the acclimatization period, the animals were housed in groups of 4/sex. For mating, one male and two females were housed together. Mated females were housed individually in macrolon cages, which were placed in another cage rack.
- Diet: From their arrival, the animals received a cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England), ab libitum. The feed was provided in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage.
- Water: Each cage was supplied with domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC), ab libitum. The water was given in polypropylene bottles, which were cleaned weekly and filled as needed.
- Acclimation period: At least 5 days prior to the start of the exposure.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 45 - 65
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
The test item was incorporated in the basal diet by mixing in a mechanical blender. The experimental diets were prepared on once. 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 test concentration was removed from the freezer to feed the animals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The concentrations of the test substance in the experimental diets were measured according to a validated UPLC-UV method (Sub-chronic (13-week) oral (dietary) toxicity study with Verdox in rats, D. van Berlo, 2017).
- It was shown that the test item was stable when the diets were stored in a freezer (<-18°C) but the test item was not stable in the diets stored at room temperature in the animal room. For that reason, the feed was replaced every day.
- The homogeneous distribution and achieved concentration of the test item in the VRF1 rat feed was analysed in the batch of diets prepared for this study. Directly after mixing of each diet, samples for the homogeneity experiments were taken from the mixer. Firstly, five homogeneity samples (about 50 g each) of each group were taken at left top, right top, middle, left bottom and right bottom of the container. Samples were taken in the order: top centre, middle centre, bottom centre, left centre, right centre. The samples taken for homogeneity experiments were also used for dose confirmation.

Details on mating procedure:

At the end of the acclimatisation period, males and females were placed together for mating (two females will be caged with one male). Animals were caged together until mating occurred and the required number of mated animals was achieved. Every consecutive morning during the mating period, vaginal smears were made for determination of the presence of sperm. The day on which sperm is detected in the vaginal smear was considered as gestation day 0 (GD 0). Upon evidence of copulation the females were caged individually. Females mated by the same male were placed in different groups.

Duration of treatment / exposure:

From gestation day (GD) 0 up to and including GD 21 (necropsy)

Frequency of treatment:

Continuously

Duration of test:

22 days

Dose / conc.:

800 mg/kg diet

Remarks:

Corresponding to an anticipated dose of 55 mg/kg bw/day

Dose / conc.:

2 500 mg/kg diet

Remarks:

Corresponding to an anticipated dose of 166 mg/kg bw/day

Dose / conc.:

7 700 mg/kg diet

Remarks:

Corresponding to an anticipated dose of 444 mg/kg bw/day

No. of animals per sex per dose:

Ninety-six female rats were mated and allocated to a control group and three treatment groups (24 mated females per group). Male rats were used for mating only.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
The dose levels were selected based on the results of a 14-day DRF for a previously performed 70-90-days extended OECD TG 422 study (Triskelion report V20063; A. Wolterbeek, 2012). In the DRF the dose of ca 15400 mg/kg diet (nominal 1000 mg/kg bw) was used. In this DRF at this high dose decrease in food consumption and body weight was seen and these effects were related to the palatability of the test item Verdox. Verdox is a fragrance which can smell awkwardly and is therefore not tasty either. At this 15400 mg/kg diet in the DRF the relative liver weight increased >=17% in males and females, which we considered to be due to metabolic saturation. This liver weight increase is also expected in combination with Verdox’s MW (196) and physico-characteristics, which indicate full and fast oral absorption.
The dietary 80-90-day Repeated dose / Reproscreen study with the highest dose of 7500 mg/kg diet (converted to nominal 500 mg/kg bw) showed decrease in body weight gain (ca. 15%), which was considered test item related but not adverse. Based on this the 7500 mg/kg diet was sufficiently high to present (absence of adverse) effects for the OECD TG 414 study and not muddle the outcome of the study due to potential palatability issues. In this Repeated dose / Reproscreen study the relative liver weights in males were increased around 14%, which we related to increased metabolic capacity and considered this sufficiently high to set the maximum dose levels at 7500 mg/kg diet in the follow up OECD TG 414 study.

Summary of key effects in DRF and extended OECD TG 422
DRF
Mg/kg diet Body weight Body weight gain Food consumption / per animal Relative liver weight
Males
1155 Not applicable Not applicable Not applicable Not applicable
2310 Not applicable Not applicable Not applicable Not applicable
7700 No effect -18% -17% +11%
15400 No effect -25% -17% +20%
Females
1155 Not applicable Not applicable Not applicable Not applicable
2310 Not applicable Not applicable Not applicable Not applicable
7700 No effects -25% -13% +5%
15400 -6% -25% -15% +17%

Extended OECD TG 422
Mg/kg diet Body weight Body weight gain Food consumption / per animal Relative liver weight
Males
800 Not applicable Not applicable Not applicable Not applicable
2500 Not applicable Not applicable Not applicable Not applicable
7500 -7% -15% Not affected +14%
Females
800 Not applicable Not applicable Not applicable Not applicable
2500 Not applicable Not applicable Not applicable Not applicable
7500 No real effects -16% only during premat Not affected No effects

Maternal examinations:

CAGE SIDE 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.

BODY WEIGHT:
Body weights of the parental female animals were recorded on gestation days (GD) 0, 6, 9, 12, 15, 18 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE:
The food consumed for each mated female was measured daily during gestation. The results were expressed in gram per animal per day.

POST-MORTEM EXAMINATIONS:
- The females were sacrificed by decapitation after CO2/O2 anaesthesia on GD 21 and examined for gross abnormalities.
- In addition to the following organs and tissues, all gross lesions were preserved.
- The following organs were weighted: uterus, containing placentas and fetuses, uterus (empty), ovaries, live fetuses (individually) and corresponding placentas.
- Abnormalities of tissues or organs in dams

Ovaries and uterine content:

All females sacrificed on GD 21 were examined for the following parameters: number of corpora lutea, number of implantation sites, number of early and late resorptions, number of live and dead fetuses, sex of the fetuses, number of grossly visible malformed fetuses and fetuses with external abnormalities, gross evaluation of placentas.

Fetal examinations:

Fetuses were examined for external alterations and sacrificed by hypothermia. Subsequently, approximately half of the fetuses of each litter was fixed in Bouin's fixative, examined for soft tissue anomalies according to a method modified after Barrow and Taylor (1969) and then discarded. Abnormal tissues were preserved. The other half of the fetuses were fixed in 70% alcohol, subsequently partly eviscerated, and then cleared in potassium hydroxide and stained with Alizarin Red S modified after Dawson (1926). They were examined for skeletal abnormalities and then retained. During the fetopathological examination, the observer was unaware of the dose group of the fetuses.

Statistics:

The resulting data were analysed using the methods mentioned below. Tests were generally performed as two-sided tests with results taken as significant where the probability of the results is p<0.05 (*) or p<0.01 (**).
Continuous data were tested for normality b the Shapiro-Wilks test. If the data was not normally distributed, log-transformation was done and the log-transformed data were tested for normality. The homogeneity of normally distributed data was tested with the Levene's median adjusted test. Homogeneous data were analysed using Anova and, for significant differences, the Dunnett or Turkey-Kramer test. Not normally distributed data or data with a lack of homogeneity were tested with the Kruskal Wallis test and, in case of significant differences, the Dunn's or Wilcoxon's test.
Dichotomous data were analysed for difference between proportions with the Chi-square test, where significant findings in the Chi-square test were followed by the Fisher's exact test. The Cochran-Armitage test was used for trends in proportions.

Indices:

The female fertility index ((number of pregnant females / number of inseminated females) x 100) and gestation index ((number of females with live fetuses / number of pregnant females) x 100) were calculated.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The clinical signs observed (wounds, encrustations and sparsely haired areas) are considered not related to the treatment. One animal in the high dose group showed hunched posture and piloerection from gestation day 6 to 8. In addition the animal showed minimal food consumption. In order to stimulate food intake, the animal received a single oral gavage administration of approximately 1.5 gram test diet mixed with drinking water. Thereafter the animal recovered.
An overview the clinical signs can be found in tabular form in the attached study report on page 25.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No animals died unscheduled.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight and mean body weight gain were statistically significantly decreased in the high dose group throughout a major part of gestation. This effect is considered to be related to the effect on food intake in the high dose group. In the low and mid dose groups mean body weight and mean body weight gain was comparable to the control group.
An overview of body weight and body weight gain can be found in tabular form in the attached study report on page 26 and 27, respectively.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean daily food consumption was statistically significantly lower in the high dose group on gestation days 0-6, 10-12, gestation day 14-15 and 16-21. This was considered to be related to the palatability of the test substance. In the mid dose group mean food consumption was decreased at the onset of exposure (from day 0-1 of gestation). In the low dose group mean daily food consumption was comparable to the control group. The mean test substance intake was calculated from the daily food consumption. Mean test substance intake over the complete gestation period (day 0-21 of gestation was 0, 55, 166 and 444 mg/kg body weight for the control group, low dose, mid dose and high dose group, respectively.
An overview of food consumption and test substance intake can be found in tabular form in the attached study report on page 28 to 30 and 31, respectively.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No statistically significant effects were observed on the mean uterine weight (including fetuses) and on the mean empty uterus weight. Mean ovary weight was comparable in all groups.
An overview of uterine weight can be found in tabular form in the attached study report on page 35. Placental weight can be found on page 36.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

One animal of the control group showed a yellow stomach deposition. One animal of the low dose and mid dose were sparsely haired. One animal of the mid dose group showed a wound on the skin and another animal from this dose group showed encrustations on the skin. These observations are common in this strain of rats and not considered to be related to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No statistical significant differences were observed in the mean pre-implantation loss between the control group (6.8%) and the animals in the low, mid and high dose groups (11.3%, 11.1% and 10.2%, respectively). Mean post-implantation loss varied from 1.7% (control group) to 6.8% (mid dose group).
An overview of pre- and post-implantation loss can be found in tabular form in the attached study report on page 32 to 34.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

No increase was observed in the mean number of early and late resorptions.
An overview of early and late resoptions can be found in tabular form in the attached study report on page 32 to 34.

Dead fetuses:

no effects observed

Description (incidence and severity):

The mean number of live fetuses was comparable in all groups( 11.9, 11.5, 11.8 and 11.3 for the control group, low dose, mid dose and high dose group, respectively).
An overview of the number of dead fetuses can be found in tabular form in the attached study report on page 32 to 34.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

Two animals in the mid dose group and one animal in the high dose group were not pregnant, resulting in 24 litters in the control and low dose group, 22 litters in the mid dose group and 23 litters in the high dose group. The female fertility index was therefore 100% in the control and low dose group, 92% for the mid dose group and 96% for the high dose group, respectively).

Other effects:

no effects observed

Description (incidence and severity):

All pregnant females had live fetuses, resulting in a gestation index of 100% for all groups. The mean number of implantation sites was comparable in all groups (12.1, 12.2, 12.4 and 11.8 for the control group, low dose, mid dose and high dose, respectively).

Key result

Dose descriptor:

NOAEL

Effect level:

>= 444 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed at highest tested dose

Remarks on result:

other: Corresponding with 7700 mg/ kg diet

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean fetus weight (both sexes together) was statistically significantly lower in the high dose group. When split to males and females, the mean male fetus weight in the high dose group was statistically significantly lower, whereas the mean female fetus weight did not reach statistical significance. The decrease in fetus weight in the high dose group is considered to be related to the decrease in maternal body weight and is only minor (approximately 6% as compared to mean fetus weight in the control group, whereas mean maternal body weight in the high dose group was approximately 20% lower as compared to the control group). In addition no effects were observed on the ossification of the fetuses and therefore this slight effect on fetus weight is not considered adverse. No effect on mean fetus weight were observed in the low dose and mid dose group as compared to the control group.
An overview of fetal body weight changes can be found in tabular form in the attached study report on page 36.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

An overview of the number of live offspring can be found in tabular form in the attached study report on page 32.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The mean percentage male fetuses per litter (sex ratio) was comparable in all groups (52.4%, 46.8%, 44.4% and 52.0% for the control group, low dose, mid dose and high dose group, respectively).
An overview of changes in sex ratio can be found in tabular form in the attached study report on page 34.

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No fetal external malformations were observed. One fetus in the low dose group was small and another one showed subcutaneous hemorrhages.
An overview of external malformations and variations can be found in tabular form in the attached study report on page 38 and 39, respectively.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The following malformations were observed: one fetus in the control group showed a small pubis and another fetus in the control group showed branched ribs and a fused centrum of the thoracic vertebra. One fetus in the mid dose group also showed a fused centrum of the thoracic vertebra. These observations were considered malformations. Variations observed were mainly related to the partly incomplete ossification status of the fetal bones. In addition, wavy ribs, irregular shapes, fusions or supernumerary sternebrae were observed.
An overview of skeletal malformations and variations can be found in tabular form in the attached study report on page 43 and 44 to 56, respectively.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No fetal visceral malformations were observed. Visceral variations included observations in the liver (discolored lobe), eyes (unilateral or bilateral retina fold), variations in the ureters (unilateral or bilateral bent or kinked), kidneys (dilated renal pelvis), urinary bladder (distended) and mouth (dark content). A distended urinary bladder was observed in fetuses of the treatment groups only. However, this is a common variation in this strain of rats. Historical background data show that the incidences observed in this study are considered common for this observation. Therefore the observation “distended urinary bladder” is not considered to be treatment-related.
An overview of visceral malformations and variations can be found in tabular form in the attached study report on page 40 and 41 to 42, respectively.

Other effects:

no effects observed

Description (incidence and severity):

No effects were observed on mean placenta weight in the exposed groups as compared to the control group.
An overview of placental weight can be found in tabular form in the attached study report on page 36.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 444 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at highest tested dose

Remarks on result:

other: Corresponding with 7700 mg/ kg diet

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

For the substance a NOAEL of > 444 mg/kg bw/day (corresponding to > 7700 mg/kg in diet) is derived for maternal and developmental toxicity. The basis for this effect level is the absence of adverse effects at the highest dose tested. The only treatment effects observed on maternal toxicity are decreased food intake and body weight in the high dose group, which both result from reduced palatability of the high-dose diet and thus not adverse. The reduced fetal body weights are linked to the decrease in maternal food intake and body weight, and are not considered adverse.

Executive summary:

A prenatal developmental toxicity study was performed according to OECD TG 414 and GLP. The substance was administered to female Wistar Han IGS rats (Crl:WI(Han)) at dose levels of 800, 2500 and 7700 mg/kg in diet (corresponding to 55, 166 and 444 mg/kg bw total dose).

The dose levels were selected based on the results of a 14-day DRF for a previously performed 70-90-days extended OECD TG 422 study (Triskelion report V20063; A. Wolterbeek, 2012). In the DRF the dose of ca 15400 mg/kg diet (nominal 1000 mg/kg bw) was used. In this DRF at this high dose decrease in food consumption and body weight was seen and these effects were related to the palatability of the test item Verdox. Verdox is a fragrance which can smell awkwardly and is therefore not tasty either. At this 15400 mg/kg diet in the DRF the relative liver weight increased >=17% in males and females, which we considered to be due to metabolic saturation. This liver weight increase is also expected in combination with Verdox’s MW (196) and physico-characteristics, which indicate full and fast oral absorption.

The dietary 80-90-day Repeated dose / Reproscreen study with the highest dose of 7500 mg/kg diet (converted to nominal 500 mg/kg bw) showed decrease in body weight gain (ca. 15%), which was considered test item related but not adverse. Based on this the 7500 mg/kg diet was sufficiently high to present (absence of adverse) effects for the OECD TG 414 study and not muddle the outcome of the study due to potential palatability issues. In this Repeated dose / Reproscreen study the relative liver weights in males were increased around 14%, which we related to increased metabolic capacity and considered this sufficiently high to set the maximum dose levels at 7500 mg/kg diet in the follow up OECD TG 414 study.

 

The 24 females/ dose level were exposed from gestation day 0 (defined by a sperm-positive vaginal smear) to necropsy at gestation day 21. A control group on plain diet was included. Analysis of the test diets showed that the test substance was homogeneously distributed in the test diets and that the concentrations were close to intended. Based on the limited stability in the test feeds, the feed was replaced daily with fresh portions from the freezer.

Clinical observations and feed consumption were measured daily. Body weight was determined on gestation days 0, 6, 9, 12, 15, 18 and 21. At gestation day 21 the animals were sacrificed and cesarean section was performed. On the maternal animals gross pathology was performed and weights of reproductive organs were recorded. In addition, the number and distribution of implantations, fetuses and resorptions was recorded. Fetuses were weighed and examined for external observations and then sacrificed and processed for visceral or skeletal fetal evaluations.

During the study no mortalities or morbidity was observed. Statistically significant decreased mean body weight and body weight gain in the 7700 mg/kg diet group which were related to decreased food intake in the in this dose-group. The test substance is a fragrance and probably reduced palatability of the high-dose diet resulted in a lower food intake. Hence the reduced food intake and the accompanying lower body weights in the high-dose group are considered to be due to reduced palatability rather than to the test substance per se, and are not considered to be adverse. No effects on maternal reproductive organ weights (full and empty uterus, ovary weight) fertility and litter parameters (pre- and post-implantation loss, number of live fetuses and resorptions), placenta weight and fetus sex ratio were observed. No treatment related effects on external, visceral and skeletal observations were observed. In the high dose group, a decrease in mean fetus weight in the 7700 mg/kg diet group was observed. This effect is considered to be related to the decrease of maternal food intake and maternal body weight. In view of the minor effect on fetal body weight as compared to the maternal effects and in absence of a delay in ossification this fetal body weight decrease is not considered to be adverse. As the maternal effects on body weight and food consumption observed in the high dose group are considered related to the palatability of the test substance and are not considered adverse, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity is ≥ 7700 mg/kg diet (corresponding to a dose level of 444 mg/kg body weight/day). In absence of effects on ossification the slight effect on fetus weight in the high dose group was not considered adverse and therefore the NOAEL for developmental toxicity is ≥ 7700 mg/kg diet (corresponding to a dose level of 444 mg/kg body weight/day). The substance is therefore not considered to be a developmental toxicant.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Additional information

Verdox tested in an OECD TG 414

A prenatal developmental toxicity study was performed according to OECD TG 414 and GLP. The substance was administered to female Wistar Han IGS rats (Crl:WI(Han)) at dose levels of 800, 2500 and 7700 mg/kg in diet (corresponding to 55, 166 and 444 mg/kg bw total dose).

The dose levels were selected based on the results of a 14-day DRF for a previously performed 70-90-days extended OECD TG 422 study (Triskelion report V20063; A. Wolterbeek, 2012). In the DRF the dose of ca 15400 mg/kg diet (nominal 1000 mg/kg bw) was used. In this DRF at this high dose decrease in food consumption and body weight was seen and these effects were related to the palatability of the test item Verdox. Verdox is a fragrance which can smell awkwardly and is therefore not tasty either. At this 15400 mg/kg diet in the DRF the relative liver weight increased >=17% in males and females, which we considered to be due to metabolic saturation. This liver weight increase is also expected in combination with Verdox’s MW (196) and physico-characteristics, which indicate full and fast oral absorption.

The dietary 80-90-day Repeated dose / Reproscreen study with the highest dose of 7500 mg/kg diet (converted to nominal 500 mg/kg bw) showed decrease in body weight gain (ca. 15%), which was considered test item related but not adverse. Based on this the 7500 mg/kg diet was sufficiently high to present (absence of adverse) effects for the OECD TG 414 study and not muddle the outcome of the study due to potential palatability issues. In this Repeated dose / Reproscreen study the relative liver weights in males were increased around 14%, which we related to increased metabolic capacity and considered this sufficiently high to set the maximum dose levels at 7500 mg/kg diet in the follow up OECD TG 414 study.

Main test: The 24 females/ dose level were exposed from gestation day 0 (defined by a sperm-positive vaginal smear) to necropsy at gestation day 21. A control group on plain diet was included. Analysis of the test diets showed that the test substance was homogeneously distributed in the test diets and that the concentrations were close to intended. Based on the limited stability in the test feeds, the feed was replaced daily with fresh portions from the freezer.

Clinical observations and feed consumption were measured daily. Body weight was determined on gestation days 0, 6, 9, 12, 15, 18 and 21. At gestation day 21 the animals were sacrificed and cesarean section was performed. On the maternal animals gross pathology was performed and weights of reproductive organs were recorded. In addition, the number and distribution of implantations, fetuses and resorptions was recorded. Fetuses were weighed and examined for external observations and then sacrificed and processed for visceral or skeletal fetal evaluations.

During the study no mortalities or morbidity was observed. Statistically significant decreased mean body weight and body weight gain in the 7700 mg/kg diet group which were related to decreased food intake in the in this dose-group. The test substance is a fragrance and probably reduced palatability of the high-dose diet resulted in a lower food intake. Hence the reduced food intake and the accompanying lower body weights in the high-dose group are considered to be due to reduced palatability rather than to the test substance per se, and are not considered to be adverse. No effects on maternal reproductive organ weights (full and empty uterus, ovary weight) fertility and litter parameters (pre- and post-implantation loss, number of live fetuses and resorptions), placenta weight and fetus sex ratio were observed. No treatment related effects on external, visceral and skeletal observations were observed. In the high dose group, a decrease in mean fetus weight in the 7700 mg/kg diet group was observed. This effect is considered to be related to the decrease of maternal food intake and maternal body weight. In view of the minor effect on fetal body weight as compared to the maternal effects and in absence of a delay in ossification this fetal body weight decrease is not considered to be adverse. As the maternal effects on body weight and food consumption observed in the high dose group are considered related to the palatability of the test substance and are not considered adverse, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity is ≥ 7700 mg/kg diet (corresponding to a dose level of 444 mg/kg body weight/day). In absence of effects on ossification the slight effect on fetus weight in the high dose group was not considered adverse and therefore the NOAEL for developmental toxicity is ≥ 7700 mg/kg diet (corresponding to a dose level of 444 mg/kg body weight/day). The substance is therefore not considered to be a developmental toxicant.

Verdox developmental toxicity as seen in the >=10 week extended OECD TG 422

No effects were seen as is presented in the fertility section above where the study is summarised.

Justification for classification or non-classification

Based on the available data, classification of the test material for effects on fertility or developmental toxicity is not warranted according to EU CLP (EC No. 1272/2008 and its updates).