Dimethylcyclohex-3-ene-1-carbaldehyde

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12-08-2016 to 24-10-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12-08-2016 to 24-10-2016

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 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

28 July 2015

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

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: females 12 weeks and males 11 weeks at the start of pre-treatment period
- Weight at study initiation: mean males between 332.96 - 335.90 g, mean females between 222.60 - 225.63 g
- Housing: in makrolon cages with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. During the pre-treatment and pre-mating period the animals were housed four rats to a cage (separated by sex). For mating, one male and one female were housed together. Mated females were housed individually in cages, which were placed in another cage rack.
- Diet: ad libitum, cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England). The diets were given as a powder in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The food in the cans was replaced twice per week with fresh portions from the freezer.
- 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). The water was given in polypropylene bottles ad libitum, which were cleaned weekly and filled as needed.
- Acclimation period: 8 days, followed by a two-week pre-treatment period during which the animals were not dosed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): relative humidity of at least 45% and not exceeding 65% other than during short periods due to room cleaning.
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 3-8-2016 To: 24-10-2016

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Weekly, one bottle of test formulation per dose level was prepared. Preparation of the test formulation was performed one day before or on the first day of the dosing period and at weekly intervals therein until the completion of the dosing phase of the study. The solutions were stirred for a minimum time of 15 minutes until confirmation of dissolution. Subsequently, under continuous stirring, this was divided into 8 aliquots (7 days plus 1 extra) according to the volume required for each dosing day. Aliquots that were not used on the day of preparation were stored in a refrigerator. Aliquots from the refrigerator were stirred for a minimum of 30 minutes before use in order to homogenize and equilibrate to ambient temperature. All aliquots were continuously stirred on a magnetic stirrer during the entire daily administration period, in order to maintain the homogeneity of the test substance in the vehicle.

VEHICLE
- Concentration in vehicle: Dose levels: 0, 50, 150 and 500 mg/kg bw/day correspond to 0, 12.5, 37.5 and 125 mg/mL test substance in dose formulation.
- Amount of vehicle (if gavage): 4 mL/kg body weight

Details on mating procedure:

- M/F ratio per cage: At the end of the pre-mating period, each female was caged with one male from the same dose group (1:1)
- Length of cohabitation: maximally one week until mating occurred
- Proof of pregnancy: Every consecutive 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At three different weeks during the study (first week of the study, first week of the gestation period and first week of the lactation period), and just after preparation of the test formulations (whilst stirring), 3 aliquots from each of the dosing formulations (bottom, middle and top) and one aliquot of the control vehicle were taken, placed into separate vials and frozen (≤ -18°C) until analysis.

The following analyses were conducted. Homogeneity: The homogeneity (and content) of the test substance in the dosing solutions were demonstrated in the first batch, by analysing the three samples (taken at different locations in the bottle) in the dose formulation of each group, in duplicate. One sample of the control group was analysed. Concentration: The concentration of test substance at each dose level was determined in the two other batches of dosing formulations prepared in the study (one prepared during the gestation period and one prepared during the lactation period). Hereto, only the middle sample was used. Stability: The stability of the test substance under experimental conditions was demonstrated in the first batch of dosing formulations prepared during the study. For this purpose, samples of one batch of test formulations (low-dose, mid-dose and high-dose) and of the vehicle was analyzed at t=0 and reanalyzed after storage in the refrigerators for 7 days.

Duration of treatment / exposure:

Administration of the test substance during the different study phases:
- Pre-exposure period: male and females were not dosed with the test substance.
- Premating period: male and female animals were dosed with the test substance during 2 weeks prior to mating.
- Mating period: male and female rats were dosed until mating occurs. Subsequently, male rats and non-mated female rats were dosed until sacrifice.
- Gestation period: Female animals were dosed from the start of the gestation period (the finding of a sperm positive vaginal smear was considered gestation day 0) up to delivery of the pups. Dosing volume was not adjusted after gestation day 14.
- Lactation period: Females were dosed up to sacrifice on lactation day 13 or shortly thereafter.

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS
Each animal was observed daily in the morning hours by cage-side observations. All cages were checked again in the afternoon for dead or moribund animals. All abnormalities, signs of ill health or reactions to treatment were recorded

BODY WEIGHT
Body weights of male and female animals were recorded just before the start of the treatment (to enable randomization) and at the start of the study (day 0). Subsequently males were weighed weekly until sacrifice. Females were weighed once per week during the pre-mating and mating period. Mated females were weighed on days 0, 7, 14 and 20 during presumed gestation and on days 0, 4, 7 and 13 of lactation. The animals were weighed on their scheduled necropsy date in order to calculate the organ to body weight ratios.

FOOD CONSUMPTION
Food consumption was measured per cage for the same periods as the body weights were measured, except during the mating period when food intake was not registered. The results are expressed in g per animal per day.

HORMONE DETERMINATIONS
During necropsy blood was taken from the aorta under CO2/O2 anaesthesia from all male and female adult animals for determination of TSH and T4 hormone levels in serum samples. This blood was collected to determine T4 and TSH hormone levels in serum samples. Analysis were performed with commercially available ELISA kits of Cloud-Clone Corp (kit CEA463Ra for TSH and kit CEA452Ge for T4, respectively).

Oestrous cyclicity (parental animals):

Vaginal smears to evaluate the oestrus cycle length and normality were made daily from the start of the pre-treatment period, during the pre-mating period and until confirmation of mating. An additional vaginal smear was made at the day of sacrifice. Smears were stained and examined in all females.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment was applied. Preference was to retain four male pups in order to have sufficient male pups for nipple retention determinations on lactation day 13); excess pups were killed and discarded.

PARTURITION AND LITTER EVALUATION
At the end of the gestation period (GD 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, PUP SURVIVAL, SEX 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 0, 4, 7 and 13 of lactation. The pups were individually weighed on days 0, 4, 7 and 13 of lactation. Mean pup weight was calculated per sex and for both sexes combined per dose group.

OTHER PARAMETERS EXAMINED
- At lactation day 4 the anogenital distance (AGD) was measured of each pup before culling of the litter. The AGD is reported as mean per litter and corrected by the cube root of body weight.
- On postnatal day 13 all surviving male pups were examined for the presence of nipples and/or areolas.
- Any abnormal behaviour of pups was recorded on day 0, 4, 7 and 13 of lactation. Grossly malformed pups were sacrificed and examined.
- Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.

Postmortem examinations (parental animals):

SACRIFICE
Prior to sacrifice, all adult male and female animals were fasted overnight (water was freely available).
- Male animals: All males were sacrificed after the mating period on day 30 of the study.
- Maternal animals: Dams were sacrificed, after overnight fasting, on day 14 of lactation

ORGAN WEIGHTS
At scheduled necropsy, the following organs of the parent animals were weighed (paired organs together) as soon as possible after dissection to avoid drying:
- Male reproductive organs (all males): Epididymides (paired), Prostate (dorsolateral and ventral), Seminal vesicles and coagulation glands (paired), Testes, Levator ani plus bulbocavernosus muscle complex, Cowper’s glands and glans penis.
- Female reproductive organs (all females): Ovaries (paired), Uterus, including cervix
- Other organs (all males and females): Kidney (paired), Liver, Gastro-intestinal tract, Thyroid gland

HISTOPATHOLOGY
Samples of the following tissues and organs of the parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes, epididymides and ovaries, which were preserved in Bouin’s fixative:
- Male reproductive organs (all males): Epididymides (paired), Prostate (dorsolateral and ventral), Seminal vesicles and coagulation glands (paired), Testes, Levator ani plus bulbocavernosus muscle complex, Cowper’s glands and glans penis.
- Female reproductive organs (all females): Ovaries (paired), Uterus, including cervix
- Other organs (all males and females): Kidney (paired), Liver, Gastro-intestinal tract, Thyroid gland, all gross lesions

Microscopic examinations were performed on the preserved organs (including gross lesions) of all animals of the control and high-dose group.
In addition, reproductive organs (ovaries, uterus, testes, epididymides, seminal vesicles and prostate) of males that failed to sire (did not mate or mated females were not pregnant) and females that were non-mated or non-pregnant, of the low- and mid-dose groups, were microscopically examined. Furthermore, organs showing gross lesions of animals of all groups were microscopically examined. To assess possible alpha hydrocarbon nephropathy, the kidneys of all males of all groups were immunohistochemically stained for α2-microglobulin and microscopically evaluated.

Postmortem examinations (offspring):

GROSS NECROSPY
A necropsy was also performed on stillborn pups and pups that died during the study and macroscopic observations of these pups were recorded. At necropsy of the dams and litters, at or shortly after day 13 of lactation, pups were examined externally for gross abnormalities and killed by decapitation. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.

HORMONE DETERMINATIONS
Blood samples were collected from the surplus pups per litter at culling on lactation day 4 (blood of all culled pups of each litter was pooled, PN 4 pups were sacrificed by decapitation for blood collection) and from two pups per litter at sacrifice on or shortly after lactation day 13 (individual, blood was collected from the heart whilst under CO2/O2 anaesthesia). This blood was collected to determine T4 and TSH hormone levels in serum samples. Analysis were performed with commercially available ELISA kits of Cloud-Clone Corp (kit CEA463Ra for TSH and kit CEA452Ge for T4, respectively).

Statistics:

Tests were generally performed as two-sided tests with results taken as significant where the probability of the results was p<0.05 (*) or p<0.01 (**):
- Continuous data were subjected to a decision tree for continuous data.
- Dichotomous data were evaluated using a decision tree for dichotomous data.
- Functional observational battery: one-way analysis of variance followed by Dunnett’s multiple comparison tests (continuous data), Kruskal-Wallis non-parametric analysis of variance followed by multiple comparison tests (rank order data) or Pearson chi-square analysis (categorical data).
- Motor activity data: total distance moved: one-way analysis of variance followed by Dunnett’s multiple comparison tests; habituation of activity: repeated measures analysis of variance on time blocks (each session consists of 5 time blocks of 6 minutes each).
- Clinical pathology data (hematology, clinical chemistry): ‘Generalized Anova/Ancova Test’ (abbreviation GEN AN) with ‘Automatic’ as data transformation method. This test is an decision tree consisting of:
(1) Data preprocessing tests. First, normality of data distribution (Shapiro-Wilks test) and homogeneity of variances (Levene test) are checked (initial transformation ‘None’ [Identity]). If any of these checks fail (p<0.05) they are repeated using Log transformation. If checks on log-transformed data fail, data are rank-transformed
(2) A group test assessing whether or not group means are all equal (parametric for untransformed or log-transformed data: one-way analysis of variance [Anova]; nonparametric for rank transformed data: Kruskal-Wallis test).
(3) Post-hoc analysis. If the group test shows significant (p<0.05) non-homogeneity of group means, pairwise comparisons with the control group are conducted by Dunnett’s multiple comparison test (parametric after Anova, non-parametric after Kruskal-Wallis; significance
levels 0.01 and 0.05).
- Incidences of histopathological changes: Fisher’s exact probability test.

Reproductive indices:

- mating days until Day 0 pc = time between the start of mating and successful copulation
- duration of gestation = time between gestation day 0 and day of delivery
- female mating index = (number of females inseminated / number of females placed with males) x 100
- male mating index = (number of males placed with females / number of females inseminated) x 100
- female fertility index = number of pregnant females x 100 / number of inseminated females
- male fertility index = number of males with pregnant females x 100 / number of males placed with females
- gestation index = (number of females with live pups / number of females pregnant) x 100
- prenatal loss = (Total number of Implantations - Total number of pups delivered) x 100 / Total number of Implantation Sites

Offspring viability indices:

- perinatal loss = (Total number of pups delivered - Total number of alive pups delivered) x 100 / Total number of pups delivered
- live birth index = (number of pups born alive/number of pups born) x 100
- viability index day 0 - 4 = (number of pup surviving 4 days/number of liveborn on day 0) x 100
- viability index day 4-13 = (number of pup surviving 13 days/number of liveborn on day 4) x 100
- sex ratio day 0 and 13 = (number of live male or female pups on day 0 or 13/ number of live pups on day 0 or 13) x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The only animals that showed remarkable observations was one female of the control group that was pale the day before delivery, one female of the mid-dose group showing a subcutaneous nodule in the neck region for 16 days and one male of the high dose group that salivated during part of the mating and post-mating period. These findings were considered to be not related to treatment. Overall, there were no treatment-related clinical signs during the pre-mating period, mating period, post-mating period, gestation or the lactation period.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortalities were observed.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on body weights and body weight changes of male and/or female animals during the pre-mating period, post-mating period, gestation period and the lactation period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- There were no treatment-related effects on food consumption of male and female animals during the pre-mating period.
- During the post-mating period food consumption of the male animals of the mid- and high-dose groups was statistically significantly higher than of the males of the control group.
- During the gestation period food consumption of the pregnant females of the high-dose group was statistically significantly higher than of the control group.
- No statistically significant differences were observed during the lactation period.
- The observed increases in food consumption were not considered as an adverse effect of the test substance.

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Microscopic evaluation revealed a treatment-related increase of accumulation of hyaline droplets in tubular epithelial cells in the outer cortex of the kidneys, accompanied by degenerative changes of these cells in 6/12 high dose males. In most cases the hyaline droplets were also present in the lumen of the tubules. This finding, along with the fact that it was only present in males, was suggestive of accumulation of alpha 2 urinary microglobulins (alpha 2u). However, other typical findings, such as an increase of basophilic tubules and dilated tubules containing eosinophilic granular debris, were not observed. To further specify these changes the kidneys were immunohistochemically stained with a monoclonal antibody against alpha 2u. The result was that the hyaline droplets did not stain positively. Based on the above results it was concluded that there was insufficient evidence to identify the hyaline droplets as alpha 2 urinary microglobulins.
- The other organs and tissues did not reveal treatment related histopathological changes. The 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.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

No statistically significant effects were observed on T4 hormone and, except for a statistically significant increase of concentration of TSH in the blood of male parental animals of the high-dose group, on TSH hormone. The increased concentration of TSH as observed in the male parental animals of the high-dose group was considered a chance finding and of no toxicological significance since in male parental animals no effects were observed on any related parameter (e.g. body weight/growth, thyroid organ weight, microscopy of the thyroid) and no effects were observed in pups.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No statistically significant effects were observed on the estrus cycle during the 2 weeks pretreatment and during the pre-mating phase, up to and including the day the animals were mated. In line with the microscopic examination of vagina and uterus no remarkable differences were observed among the groups.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The results of mating was not affected by treatment. In each group 12 females were placed with males for mating and all females were mated (one female of the control group was misjudged to be not mated). Consequently, the male and female mating indices were 100% for all groups. The mean time to mating (days until day 0 pc) was statistically significantly increased in the high-dose group but all animals were mated within 5 days (approximately one estrus cycle) and, therefore, the extended time to mating as was observed in the high-dose group was considered a chance-finding. Pregnancy was not affected by treatment, except for one female of the mid-dose group, all mated females were pregnant. In addition, one female of the mid-dose group appeared to be pregnant but delivered no pups and had only implantations. At a first preliminary check, one female of the control group had 3 male and 5 female pups of which 3 female pups were already partly cannibalized. At the scheduled check of the litter, only 3 dead pups were left (two stillborn and one found dead, the other pups were cannibalized (since these pups were missing before the scheduled examination of the litters, the cannibalized pups were considered as lost implantations). One female of the mid-dose group delivered only one stillborn pup. The number of implantation sites and number of pups delivered were comparable among the groups. Consequently, no statistically significant differences were observed on prenatal loss between the control and treatment groups. The incidences of live- and stillborn pups and perinatal loss indices were also comparable among the groups.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: Absence of adverse systemic effects at the highest dose tested

Key result

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive performance

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment-related signs in pups during the lactation period.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

- One dam of the control group delivered 8 pups of which 5 were considered as post-implantation loss.
- The number of stillborn or pups found dead at first scheduled litter inspection after birth accounted 7, 1, 1 and 2 for the control, low-, mid- and high-dose groups, respectively, whereas the number of number of pups that died or were missing between days 0-4 accounted 0, 1, 0 and 0 for the control, low-, mid- and high dose, respectively.
- After culling on day 4, no pups were lost.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment-related differences in mean pup weights between the test groups and the controls on day 0, 4, 7 and 13 of lactation.

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 on day 0 and day 13 was comparable among the groups.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no effects of the weight of the thyroid of male and female pups on post-natal day 13.

Gross pathological findings:

no effects observed

Description (incidence and severity):

- Macroscopic examinations of stillborn pups and pups that died did not reveal treatment-related effects.
- At necropsy of the pups no treatment related macroscopic changes were observed.

Histopathological findings:

no effects observed

Description (incidence and severity):

Microscopical evaluation of the thyroid gland of the pups did not reveal treatment related histopathological changes.

Other effects:

no effects observed

Description (incidence and severity):

- There were no effects on nipple retention of male pups on post-natal day 13.
- There were no treatment related effects on the absolute (expressed in mm) and corrected (expressed in mm/g) anogenital distance of male and female pups
- No toxicological adverse effects on T4 and TSH hormones were observed in culled pups on postnatal day 4 and in male and female pups on postnatal day 13.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Observational study: there were no treatment-related signs in pups during the lactation period.

Developmental immunotoxicity:

not examined

Key result

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

The test substance was considered to be homogeneously distributed and to be stable in the gavage liquids under the experimental conditions. Although the concentration of the test substance of the mid-dose groups of the gavage liquids prepared on 3 October 2016 was 14% lower than intended, in general, the concentration of the test substance was close to intended (90-110% of the intended concentration).

Conclusions:

For the substance a parental NOAEL for systemic effects of 150 mg/kg bw/day was derived, based on the effects on the organs weights of liver (male and female) and kidneys (male) and microscopic effects observed in the kidneys (male) of the high-dose group. No adverse effect were observed on fertility parameters, reproductive performance and developmental parameters up to and including 500 mg/kg bw/ day, the highest dose level tested. Based on the absence of adverse effects on reproduction parameters, the NOAEL reproduction in this study is equal or greater than 500 mg/kg bw/day.

Executive summary:

A reproduction/ developmental toxicity screening was performed according to OECD TG 421 and GLP. The substance was administered orally (gavage) to male and female Wistar Han IGS rats at dose levels of 50, 150 and 500 mg/kg bw/day (12 rats/sex/dose level). Concurrent controls (12 rats/sex) were performed with the vehicle (corn oil). Parental animals were exposed 2 weeks prior mating and during mating. Male rats and non-mated female rats were exposed until sacrifice (study day 30). Mated females were dosed from the start of the gestation period (the finding of a sperm positive vaginal smear was considered gestation day 0) up to delivery of the pups. During the lactation period, females were dosed up to sacrifice, after overnight fasting, on day 14 of lactation. The test substance was considered to be homogeneously distributed and to be stable in the gavage liquids under the experimental conditions. Although the concentration of the test substance of the mid-dose groups of the gavage liquids prepared was 14% lower than intended, in general, the concentration of the test substance was close to intended (90-110% of the intended concentration).

Repeated dose effects, clinical signs: For the male and female animals, there were no mortalities observed and no treatment related effects on macroscopic parameters and body weight were observed. In addition, no treatment related changes in estrus cyclicity, fertility and reproductive performance in P0 was observed. Although food consumption was affected in the post-mating period of the male animals in mid- and high dose groups and in pregnant females in the high dose group, this was not considered as an adverse effect of the treatment. In male animals of the high-dose group, the absolute- (+13.6%) and relative (+16.8%) liver weights and the relative kidney weight (+8.0%) were statistically significantly increased as compared to the control animals. Microscopic evaluation revealed a treatment-related increase of accumulation of hyaline droplets in tubular epithelial cells in the outer cortex of the kidneys, accompanied by degenerative changes of these cells in 6/12 high dose males in most cases the hyaline droplets were also present in the lumen of the tubules. Immunohistochemically staining with a monoclonal antibody against alpha 2u revealed that there was insufficient evidence to identify the hyaline droplets as alpha 2 urinary microglobulins. In female animals of the mid- and high-dose groups, the absolute (+9.9% and +24.7%, respectively) and relative (+9.0% and +24.2%, respectively) weights of the liver were statistically significantly increased as compared to the control group. No toxicological adverse effects on T4 and TSH hormones were observed in male and female parental animals.

Fertility: No treatment related changes in estrus cyclicity, fertility and reproductive performance in P0 was observed. Minor food consumption effects were observed in the post-mating period of the male animals in mid- and high dose groups and in pregnant females in the high dose group, but this was not considered as an adverse effect of the treatment.

Developmental toxicity: In the pups, no treatment related effects were observed in observational studies, body weight, anogenital distance, nipple retention and thyroid weight. No effects were observed during macroscopic observations in both still born/ found-dead pups and after sacrifice (post-natal day 13). Microscopic evaluation of the thyroid gland did not reveal treatment related histopathologicalchanges. No toxicological adverse effects on T4 and TSH hormones were in culled pups on postnatal day 4 and in male and female pups on postnatal day 13.

 

In conclusion, systemic effects were observed in males in the high dose group (500 mg/kg bw/day). A NOAEL of 150 mg/kg bw/ day was therefore derived based on effects on microscopic effects observed in the kidneys (male), which upon staining were not confirmed to be alpha hydrocarbon nephropathy*. No adverse effects were observed on fertility parameters, reproductive performance and developmental parameters up to the highest dose tested. Therefore, the NOAEL for reproduction is ≥500 mg/kg bw/day.

*In this OECD TG 421 study the kidney effects in males were seen. In an additional OECD TG 407 study kidney effects were seen both in the control and dose groups and alpha-2u globulin was identified with immune staining. As the effects were not considered adverse in the OECD TG 407, they were also not considered adverse in this OECD TG 421. Therefore the male kidney effects were not taken into account for assessing the parental NOAEL. 

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

12-08-2016 to 24-10-2016

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:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

28 July 2015

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

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: females 12 weeks and males 11 weeks at the start of pre-treatment period
- Weight at study initiation: mean males between 332.96 - 335.90 g, mean females between 222.60 - 225.63 g
- Housing: in makrolon cages with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. During the pre-treatment and pre-mating period the animals were housed four rats to a cage (separated by sex). For mating, one male and one female were housed together. Mated females were housed individually in cages, which were placed in another cage rack.
- Diet: ad libitum, cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England). The diets were given as a powder in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The food in the cans was replaced twice per week with fresh portions from the freezer.
- 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). The water was given in polypropylene bottles ad libitum, which were cleaned weekly and filled as needed.
- Acclimation period: 8 days, followed by a two-week pre-treatment period during which the animals were not dosed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): relative humidity of at least 45% and not exceeding 65% other than during short periods due to room cleaning.
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 3-8-2016 To: 24-10-2016

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Weekly, one bottle of test formulation per dose level was prepared. Preparation of the test formulation was performed one day before or on the first day of the dosing period and at weekly intervals therein until the completion of the dosing phase of the study. The solutions were stirred for a minimum time of 15 minutes until confirmation of dissolution. Subsequently, under continuous stirring, this was divided into 8 aliquots (7 days plus 1 extra) according to the volume required for each dosing day. Aliquots that were not used on the day of preparation were stored in a refrigerator. Aliquots from the refrigerator were stirred for a minimum of 30 minutes before use in order to homogenize and equilibrate to ambient temperature. All aliquots were continuously stirred on a magnetic stirrer during the entire daily administration period, in order to maintain the homogeneity of the test substance in the vehicle.

VEHICLE
- Concentration in vehicle: Dose levels: 0, 50, 150 and 500 mg/kg bw/day correspond to 0, 12.5, 37.5 and 125 mg/mL test substance in dose formulation.
- Amount of vehicle (if gavage): 4 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At three different weeks during the study (first week of the study, first week of the gestation period and first week of the lactation period), and just after preparation of the test formulations (whilst stirring), 3 aliquots from each of the dosing formulations (bottom, middle and top) and one aliquot of the control vehicle were taken, placed into separate vials and frozen (≤ -18°C) until analysis.

The following analyses were conducted. Homogeneity: The homogeneity (and content) of the test substance in the dosing solutions were demonstrated in the first batch, by analysing the three samples (taken at different locations in the bottle) in the dose formulation of each group, in duplicate. One sample of the control group was analysed. Concentration: The concentration of test substance at each dose level was determined in the two other batches of dosing formulations prepared in the study (one prepared during the gestation period and one prepared during the lactation period). Hereto, only the middle sample was used. Stability: The stability of the test substance under experimental conditions was demonstrated in the first batch of dosing formulations prepared during the study. For this purpose, samples of one batch of test formulations (low-dose, mid-dose and high-dose) and of the vehicle was analyzed at t=0 and reanalyzed after storage in the refrigerators for 7 days.

Duration of treatment / exposure:

Administration of the test substance during the different study phases:
- Pre-exposure period: male and females were not dosed with the test substance.
- Premating period: male and female animals were dosed with the test substance during 2 weeks prior to mating.
- Mating period: male and female rats were dosed until mating occurs. Subsequently, male rats and non-mated female rats were dosed until sacrifice.
- Gestation period: Female animals were dosed from the start of the gestation period (the finding of a sperm positive vaginal smear was considered gestation day 0) up to delivery of the pups. Dosing volume was not adjusted after gestation day 14.
- Lactation period: Females were dosed up to sacrifice on lactation day 13 or shortly thereafter.

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS
Each animal was observed daily in the morning hours by cage-side observations. All cages were checked again in the afternoon for dead or moribund animals. All abnormalities, signs of ill health or reactions to treatment were recorded

BODY WEIGHT
Body weights of male and female animals were recorded just before the start of the treatment (to enable randomization) and at the start of the study (day 0). Subsequently males were weighed weekly until sacrifice. Females were weighed once per week during the pre-mating and mating period. Mated females were weighed on days 0, 7, 14 and 20 during presumed gestation and on days 0, 4, 7 and 13 of lactation. The animals were weighed on their scheduled necropsy date in order to calculate the organ to body weight ratios.

FOOD CONSUMPTION
Food consumption was measured per cage for the same periods as the body weights were measured, except during the mating period when food intake was not registered. The results are expressed in g per animal per day.

Sacrifice and pathology:

SACRIFICE
Prior to sacrifice, all adult male and female animals were fasted overnight (water was freely available).
- Male animals: All males were sacrificed after the mating period on day 30 of the study.
- Maternal animals: Dams were sacrificed, after overnight fasting, on day 14 of lactation

ORGAN WEIGHTS
At scheduled necropsy, the following organs of the parent animals were weighed (paired organs together) as soon as possible after dissection to avoid drying:
- Male reproductive organs (all males): Epididymides (paired), Prostate (dorsolateral and ventral), Seminal vesicles and coagulation glands (paired), Testes, Levator ani plus bulbocavernosus muscle complex, Cowper’s glands and glans penis.
- Female reproductive organs (all females): Ovaries (paired), Uterus, including cervix
- Other organs (all males and females): Kidney (paired), Liver, Gastro-intestinal tract, Thyroid gland

HISTOPATHOLOGY
Samples of the following tissues and organs of the parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes, epididymides and ovaries, which were preserved in Bouin’s fixative:
- Male reproductive organs (all males): Epididymides (paired), Prostate (dorsolateral and ventral), Seminal vesicles and coagulation glands (paired), Testes, Levator ani plus bulbocavernosus muscle complex, Cowper’s glands and glans penis.
- Female reproductive organs (all females): Ovaries (paired), Uterus, including cervix
- Other organs (all males and females): Kidney (paired), Liver, Gastro-intestinal tract, Thyroid gland, all gross lesions

Microscopic examinations were performed on the preserved organs (including gross lesions) of all animals of the control and high-dose group.
In addition, reproductive organs (ovaries, uterus, testes, epididymides, seminal vesicles and prostate) of males that failed to sire (did not mate or mated females were not pregnant) and females that were non-mated or non-pregnant, of the low- and mid-dose groups, were microscopically examined. Furthermore, organs showing gross lesions of animals of all groups were microscopically examined. To assess possible alpha hydrocarbon nephropathy, the kidneys of all males of all groups were immunohistochemically stained for α2-microglobulin and microscopically evaluated.

Other examinations:

HORMONE DETERMINATIONS
During necropsy blood was taken from the aorta under CO2/O2 anaesthesia from all male and female adult animals for determination of TSH and T4 hormone levels in serum samples. This blood was collected to determine T4 and TSH hormone levels in serum samples. Analysis were performed with commercially available ELISA kits of Cloud-Clone Corp (kit CEA463Ra for TSH and kit CEA452Ge for T4, respectively).

Statistics:

Tests were generally performed as two-sided tests with results taken as significant where the probability of the results was p<0.05 (*) or p<0.01 (**):
- Continuous data were subjected to a decision tree for continuous data.
- Dichotomous data were evaluated using a decision tree for dichotomous data.
- Functional observational battery: one-way analysis of variance followed by Dunnett’s multiple comparison tests (continuous data), Kruskal-Wallis non-parametric analysis of variance followed by multiple comparison tests (rank order data) or Pearson chi-square analysis (categorical data).
- Motor activity data: total distance moved: one-way analysis of variance followed by Dunnett’s multiple comparison tests; habituation of activity: repeated measures analysis of variance on time blocks (each session consists of 5 time blocks of 6 minutes each).
- Clinical pathology data (hematology, clinical chemistry): ‘Generalized Anova/Ancova Test’ (abbreviation GEN AN) with ‘Automatic’ as data transformation method. This test is an decision tree consisting of:
(1) Data preprocessing tests. First, normality of data distribution (Shapiro-Wilks test) and homogeneity of variances (Levene test) are checked (initial transformation ‘None’ [Identity]). If any of these checks fail (p<0.05) they are repeated using Log transformation. If checks on log-transformed data fail, data are rank-transformed
(2) A group test assessing whether or not group means are all equal (parametric for untransformed or log-transformed data: one-way analysis of variance [Anova]; nonparametric for rank transformed data: Kruskal-Wallis test).
(3) Post-hoc analysis. If the group test shows significant (p<0.05) non-homogeneity of group means, pairwise comparisons with the control group are conducted by Dunnett’s multiple comparison test (parametric after Anova, non-parametric after Kruskal-Wallis; significance
levels 0.01 and 0.05).
- Incidences of histopathological changes: Fisher’s exact probability test.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The only animals that showed remarkable observations was one female of the control group that was pale the day before delivery, one female of the mid-dose group showing a subcutaneous nodule in the neck region for 16 days and one male of the high dose group that salivated during part of the mating and post-mating period. These findings were considered to be not related to treatment. Overall, there were no treatment-related clinical signs during the pre-mating period, mating period, post-mating period, gestation or the lactation period.

Mortality:

no mortality observed

Description (incidence):

No mortalities were observed.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on body weights and body weight changes of male and/or female animals during the pre-mating period, post-mating period, gestation period and the lactation period

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- There were no treatment-related effects on food consumption of male and female animals during the pre-mating period.
- During the post-mating period food consumption of the male animals of the mid- and high-dose groups was statistically significantly higher than of the males of the control group.
- During the gestation period food consumption of the pregnant females of the high-dose group was statistically significantly higher than of the control group.
- No statistically significant differences were observed during the lactation period.
- The observed increases in food consumption were not considered as an adverse effect of the test substance.

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:

effects observed, treatment-related

Description (incidence and severity):

- In male animals of the high-dose group, the absolute- (+13.6%) and relative (+16.8%) liver weights and the relative kidney weight (+8.0%) were statistically significantly increased as compared to the control animals.
- In male animals of the low-dose group (and not in the mid-and high-dose groups), the relative weight of the LABC (-8.7%) and the absolute- (-20.7%) and relative (-21.2%) weights of the Cowper’s glands were statistically significantly decreased as compared to the control animals. Since these differences were only observed in the low-dose group, they were considered chance findings and not related to treatment.
- In female animals of the mid- and high-dose groups, the absolute (+9.9% and +24.7%, respectively) and relative (+9.0% and +24.2%, respectively) weights of the liver were statistically significantly increased as compared to the control group.
- The statistically significant effects on liver weights of >10% in male and female animals of the high-dose group, and the statistically significant increased relative kidney weight (in relation to the microscopic effects) in male animals of the high-dose group are considered to be related to treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

At necropsy no treatment related macroscopic changes were observed.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Microscopic evaluation revealed a treatment-related increase of accumulation of hyaline droplets in tubular epithelial cells in the outer cortex of the kidneys, accompanied by degenerative changes of these cells in 6/12 high dose males. In most cases the hyaline droplets were also present in the lumen of the tubules. This finding, along with the fact that it was only present in males, was suggestive of accumulation of alpha 2 urinary microglobulins (alpha 2u). However, other typical findings, such as an increase of basophilic tubules and dilated tubules containing eosinophilic granular debris, were not observed. To further specify these changes the kidneys were immunohistochemically stained with a monoclonal antibody against alpha 2u. The result was that the hyaline droplets did not stain positively. Based on the above results it was concluded that there was insufficient evidence to identify the hyaline droplets as alpha 2 urinary microglobulins.
- The other organs and tissues did not reveal treatment related histopathological changes. The 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.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

No statistically significant effects were observed on T4 hormone and, except for a statistically significant increase of concentration of TSH in the blood of male parental animals of the high-dose group, on TSH hormone. The increased concentration of TSH as observed in the male parental animals of the high-dose group was considered a chance finding and of no toxicological significance since in male parental animals no effects were observed on any related parameter (e.g. body weight/growth, thyroid organ weight, microscopy of the thyroid).

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

The test substance was considered to be homogeneously distributed and to be stable in the gavage liquids under the experimental conditions. Although the concentration of the test substance of the mid-dose groups of the gavage liquids prepared on 3 October 2016 was 14% lower than intended, in general, the concentration of the test substance was close to intended (90-110% of the intended concentration).

Conclusions:

For the substance a parental NOAEL for systemic effects of 150 mg/kg bw/day was derived, based on the effects on the organs weights of liver (male and female) and kidneys (male) and microscopic effects observed in the kidneys (male) of the high-dose group.

Executive summary:

A reproduction/ developmental toxicity screening was performed according to OECD TG 421 and GLP. The substance was administered orally (gavage) to male and female Wistar Han IGS rats at dose levels of 50, 150 and 500 mg/kg bw/day (12 rats/sex/dose level). Concurrent controls (12 rats/sex) were performed with the vehicle (corn oil). Parental animals were exposed 2 weeks prior mating and during mating. Male rats and non-mated female rats were exposed until sacrifice (study day 30). Mated females were dosed from the start of the gestation period (the finding of a sperm positive vaginal smear was considered gestation day 0) up to delivery of the pups. During the lactation period, females were dosed up to sacrifice, after overnight fasting, on day 14 of lactation. The test substance was considered to be homogeneously distributed and to be stable in the gavage liquids under the experimental conditions. Although the concentration of the test substance of the mid-dose groups of the gavage liquids prepared was 14% lower than intended, in general, the concentration of the test substance was close to intended (90-110% of the intended concentration). For the male and female animals, there were no mortalities observed and no treatment related effects on macroscopic parameters and body weight were observed. Although food consumption was affected in the post-mating period of the male animals in mid- and high dose groups and in pregnant females in the high dose group, this was not considered as an adverse effect of the treatment. In male animals of the high-dose group, the absolute- (+13.6%) and relative (+16.8%) liver weights and the relative kidney weight (+8.0%) were statistically significantly increased as compared to the control animals. Microscopic evaluation revealed a treatment-related increase of accumulation of hyaline droplets in tubular epithelial cells in the outer cortex of the kidneys, accompanied by degenerative changes of these cells in 6/12 high dose males in most cases the hyaline droplets were also present in the lumen of the tubules. Immunohistochemically staining with a monoclonal antibody against alpha 2u revealed that there was insufficient evidence to identify the hyaline droplets as alpha 2 urinary microglobulins. In female animals of the mid- and high-dose groups, the absolute (+9.9% and +24.7%, respectively) and relative (+9.0% and +24.2%, respectively) weights of the liver were statistically significantly increased as compared to the control group. The increased liver weighs are somewhat high (>20%) but in abscence of affected bioachemistry parameters and histopathology these are considered non-adverse. No toxicological adverse effects on T4 and TSH hormones were observed in male and female parental animals.

In conclusion, systemic effects were observed in the high dose group (500 mg/kg bw/day). A NOAEL of 150 mg/kg bw/ day was therefore derived based on effects on kidney (male) and microscopic effects observed in the kidneys (male).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

28 July 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dimethylcyclohex-3-ene-1-carbaldehyde

EC Number:

248-742-6

EC Name:

Dimethylcyclohex-3-ene-1-carbaldehyde

Cas Number:

27939-60-2

Molecular formula:

C9H14O

IUPAC Name:

(1R,6R)-3,6-dimethylcyclohex-3-ene-1-carbaldehyde; (1R,6R)-4,6-dimethylcyclohex-3-ene-1-carbaldehyde

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Han IGS

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: females 12 weeks and males 11 weeks at the start of pre-treatment period
- Weight at study initiation: mean males between 332.96 - 335.90 g, mean females between 222.60 - 225.63 g
- Housing: in makrolon cages with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. During the pre-treatment and pre-mating period the animals were housed four rats to a cage (separated by sex). For mating, one male and one female were housed together. Mated females were housed individually in cages, which were placed in another cage rack.
- Diet: ad libitum, cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England). The diets were given as a powder in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The food in the cans was replaced twice per week with fresh portions from the freezer.
- 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). The water was given in polypropylene bottles ad libitum, which were cleaned weekly and filled as needed.
- Acclimation period: 8 days, followed by a two-week pre-treatment period during which the animals were not dosed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): relative humidity of at least 45% and not exceeding 65% other than during short periods due to room cleaning.
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 3-8-2016 To: 24-10-2016

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Weekly, one bottle of test formulation per dose level was prepared. Preparation of the test formulation was performed one day before or on the first day of the dosing period and at weekly intervals therein until the completion of the dosing phase of the study. The solutions were stirred for a minimum time of 15 minutes until confirmation of dissolution. Subsequently, under continuous stirring, this was divided into 8 aliquots (7 days plus 1 extra) according to the volume required for each dosing day. Aliquots that were not used on the day of preparation were stored in a refrigerator. Aliquots from the refrigerator were stirred for a minimum of 30 minutes before use in order to homogenize and equilibrate to ambient temperature. All aliquots were continuously stirred on a magnetic stirrer during the entire daily administration period, in order to maintain the homogeneity of the test substance in the vehicle.

VEHICLE
- Concentration in vehicle: Dose levels: 0, 50, 150 and 500 mg/kg bw/day correspond to 0, 12.5, 37.5 and 125 mg/mL test substance in dose formulation.
- Amount of vehicle (if gavage): 4 ml/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At three different weeks during the study (first week of the study, first week of the gestation period and first week of the lactation period), and just after preparation of the test formulations (whilst stirring), 3 aliquots from each of the dosing formulations (bottom, middle and top) and one aliquot of the control vehicle were taken, placed into separate vials and frozen (≤ -18°C) until analysis.

The following analyses were conducted. Homogeneity: The homogeneity (and content) of the test substance in the dosing solutions were demonstrated in the first batch, by analysing the three samples (taken at different locations in the bottle) in the dose formulation of each group, in duplicate. One sample of the control group was analysed. Concentration: The concentration of test substance at each dose level was determined in the two other batches of dosing formulations prepared in the study (one prepared during the gestation period and one prepared during the lactation period). Hereto, only the middle sample was used. Stability: The stability of the test substance under experimental conditions was demonstrated in the first batch of dosing formulations prepared during the study. For this purpose, samples of one batch of test formulations (low-dose, mid-dose and high-dose) and of the vehicle was analyzed at t=0 and reanalyzed after storage in the refrigerators for 7 days.

Details on mating procedure:

- M/F ratio per cage: At the end of the pre-mating period, each female was caged with one male from the same dose group (1:1)
- Length of cohabitation: maximally one week until mating occurred
- Proof of pregnancy: Every consecutive 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 (GD) 0.
- Upon evidence of copulation the females were caged individually for the birth and rearing of their pups.

Duration of treatment / exposure:

Administration of the test substance during the different study phases:
- Pre-exposure period: male and females were not dosed with the test substance.
- Premating period: male and female animals were dosed with the test substance during 2 weeks prior to mating.
- Mating period: male and female rats were dosed until mating occurs. Subsequently, male rats and non-mated female rats were dosed until sacrifice.
- Gestation period: Female animals were dosed from the start of the gestation period (the finding of a sperm positive vaginal smear was considered gestation day 0) up to delivery of the pups. Dosing volume was not adjusted after gestation day 14.
- Lactation period: Females were dosed up to sacrifice on lactation day 13 or shortly thereafter.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS
Each animal was observed daily in the morning hours by cage-side observations. All cages were checked again in the afternoon for dead or moribund animals. All abnormalities, signs of ill health or reactions to treatment were recorded

BODY WEIGHT
Body weights of female animals were recorded just before the start of the treatment (to enable randomization) and at the start of the study (day 0). Subsequently females were weighed once per week during the pre-mating and mating period. Mated females were weighed on days 0, 7, 14 and 20 during presumed gestation and on days 0, 4, 7 and 13 of lactation. The animals were weighed on their scheduled necropsy date in order to calculate the organ to body weight ratios.

FOOD CONSUMPTION
Food consumption was measured per cage for the same periods as the body weights were measured, except during the mating period when food intake was not registered. The results are expressed in g per animal per day.

HORMONE DETERMINATIONS
During necropsy blood was taken from the aorta under CO2/O2 anaesthesia from all female adult animals for determination of TSH and T4 hormone levels in serum samples. This blood was collected to determine T4 and TSH hormone levels in serum samples. Analysis were performed with commercially available ELISA kits of Cloud-Clone Corp (kit CEA463Ra for TSH and kit CEA452Ge for T4, respectively).

SACRIFICE
Prior to sacrifice, all female animals were fasted overnight (water was freely available). Dams were sacrificed, after overnight fasting, on day 14 of lactation

ORGAN WEIGHTS
At scheduled necropsy, the following organs of the parent animals were weighed (paired organs together) as soon as possible after dissection to avoid drying:
- Female reproductive organs (all females): Ovaries (paired), Uterus, including cervix
- Other organs (all males and females): Kidney (paired), Liver, Gastro-intestinal tract, Thyroid gland

HISTOPATHOLOGY
Samples of the following tissues and organs of the parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for ovaries, which were preserved in Bouin’s fixative:
- Female reproductive organs (all females): Ovaries (paired), Uterus, including cervix
- Other organs (all females): Kidney (paired), Liver, Gastro-intestinal tract, Thyroid gland, all gross lesions

Microscopic examinations were performed on the preserved organs (including gross lesions) of all animals of the control and high-dose group.
In addition, reproductive organs (ovaries, uterus, testes, epididymides, seminal vesicles and prostate) of males that failed to sire (did not mate or mated females were not pregnant) and females that were non-mated or non-pregnant, of the low- and mid-dose groups, were microscopically examined. Furthermore, organs showing gross lesions of animals of all groups were microscopically examined. To assess possible alpha hydrocarbon nephropathy, the kidneys of all males of all groups were immunohistochemically stained for α2-microglobulin and microscopically evaluated.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of resorptions: Yes

Fetal examinations:

PARTURITION AND LITTER EVALUATION
At the end of the gestation period (GD 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, PUP SURVIVAL, SEX 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 0, 4, 7 and 13 of lactation. The pups were individually weighed on days 0, 4, 7 and 13 of lactation. Mean pup weight was calculated per sex and for both sexes combined per dose group.

OTHER PARAMETERS EXAMINED
- At lactation day 4 the anogenital distance (AGD) was measured of each pup before culling of the litter. The AGD is reported as mean per litter and corrected by the cube root of body weight.
- On postnatal day 13 all surviving male pups were examined for the presence of nipples and/or areolas.
- Any abnormal behaviour of pups was recorded on day 0, 4, 7 and 13 of lactation. Grossly malformed pups were sacrificed and examined.
- Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.

GROSS EXAMINATION OF DEAD PUPS
A necropsy was also performed on stillborn pups and pups that died during the study and macroscopic observations of these pups were recorded. At necropsy of the dams and litters, at or shortly after day 13 of lactation, pups were examined externally for gross abnormalities and killed by decapitation.

HORMONE DETERMINATIONS
Blood samples were collected from the surplus pups per litter at culling on lactation day 4 (blood of all culled pups of each litter was pooled, PN 4 pups were sacrificed by decapitation for blood collection) and from two pups per litter at sacrifice on or shortly after lactation day 13 (individual, blood was collected from the heart whilst under CO2/O2 anaesthesia). This blood was collected to determine T4 and TSH hormone levels in serum samples. Analysis were performed with commercially available ELISA kits of Cloud-Clone Corp (kit CEA463Ra for TSH and kit CEA452Ge for T4, respectively).

Statistics:

Tests were generally performed as two-sided tests with results taken as significant where the probability of the results was p<0.05 (*) or p<0.01 (**):
- Continuous data were subjected to a decision tree for continuous data.
- Dichotomous data were evaluated using a decision tree for dichotomous data.
- Functional observational battery: one-way analysis of variance followed by Dunnett’s multiple comparison tests (continuous data), Kruskal-Wallis non-parametric analysis of variance followed by multiple comparison tests (rank order data) or Pearson chi-square analysis (categorical data).
- Motor activity data: total distance moved: one-way analysis of variance followed by Dunnett’s multiple comparison tests; habituation of activity: repeated measures analysis of variance on time blocks (each session consists of 5 time blocks of 6 minutes each).
- Clinical pathology data (hematology, clinical chemistry): ‘Generalized Anova/Ancova Test’ (abbreviation GEN AN) with ‘Automatic’ as data transformation method. This test is an decision tree consisting of:
(1) Data preprocessing tests. First, normality of data distribution (Shapiro-Wilks test) and homogeneity of variances (Levene test) are checked (initial transformation ‘None’ [Identity]). If any of these checks fail (p<0.05) they are repeated using Log transformation. If checks on log-transformed data fail, data are rank-transformed
(2) A group test assessing whether or not group means are all equal (parametric for untransformed or log-transformed data: one-way analysis of variance [Anova]; nonparametric for rank transformed data: Kruskal-Wallis test).
(3) Post-hoc analysis. If the group test shows significant (p<0.05) non-homogeneity of group means, pairwise comparisons with the control group are conducted by Dunnett’s multiple comparison test (parametric after Anova, non-parametric after Kruskal-Wallis; significance
levels 0.01 and 0.05).
- Incidences of histopathological changes: Fisher’s exact probability test

Indices:

- mating days until Day 0 pc = time between the start of mating and successful copulation
- duration of gestation = time between gestation day 0 and day of delivery
- female mating index = (number of females inseminated / number of females placed with males) x 100
- male mating index = (number of males placed with females / number of females inseminated) x 100
- female fertility index = number of pregnant females x 100 / number of inseminated females
- male fertility index = number of males with pregnant females x 100 / number of males placed with females
- gestation index = (number of females with live pups / number of females pregnant) x 100
- prenatal loss = (Total number of Implantations - Total number of pups delivered) x 100 / Total number of Implantation Sites
- perinatal loss= (Total number of pups delivered - Total number of alive pups delivered) x 100 / Total number of pups delivered
- live birth index= (number of pups born alive/number of pups born) x 100
- viability index day 0 - 4 = (number of pup surviving 4 days/number of liveborn on day 0) x 100
- viability index day 4-13 = (number of pup surviving 13 days/number of liveborn on day 4) x 100
- sex ratio day 0 and 13 = (number of live male or female pups on day 0 or 13/ number of live pups on day 0 or 13) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The only animals that showed remarkable observations was one female of the control group that was pale the day before delivery and one female of the mid-dose group showing a subcutaneous nodule in the neck region for 16 days. These findings were considered to be not related to treatment. Overall, there were no treatment-related clinical signs during the pre-mating period, mating period, post-mating period, gestation or the lactation period.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortalities were observed.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on body weights and body weight changes of female animals during the pre-mating period, post-mating period gestation period and the lactation period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- There were no treatment-related effects on food consumption of female animals during the pre-mating period.
- During the gestation period food consumption of the pregnant females of the high-dose group was statistically significantly higher than of the control group
- No statistically significant differences were observed during the lactation period.
- The observed increases in food consumption were not considered as an adverse effect of the test substance.

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:

effects observed, treatment-related

Description (incidence and severity):

- In female animals of the mid- and high-dose groups, the absolute (+9.9% and +24.7%, respectively) and relative (+9.0% and +24.2%, respectively) weights of the liver were statistically significantly increased as compared to the control group.
- The statistically significant effects on liver weights of >10% in female animals of the high-dose group are considered to be related to treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

At necropsy no treatment related macroscopic changes were observed.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The organs and tissues did not reveal treatment related histopathological changes. The 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.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No statistically significant effects were observed on T4 hormone and on TSH hormone.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

- One female of the mid-dose group appeared to be pregnant but delivered no pups and had only implantations
- One dam of the control group delivered 8 pups of which 5 were considered as post-implantation loss.

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

- At a first preliminary check, one female of the control group had 3 male and 5 female pups of which 3 female pups were already partly cannibalized. At scheduled check of the litter, only 3 dead pups were left (two stillbore and one found dead). the other pups were cannibalized (since these pups were missing before the scheduled examination of the litters, the cannibalized pups were considered as lost implantations).
- One female of the mid-dose group delivered only one stillborn pup.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Pregnancy was not affected by treatment, except for one female of the mid-dose group, all mated females were pregnant

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The mean time to mating (days until day 0 pc) was statistically significantly increased in the high-dose group but all animals were mated within 5 days (approximately one estrus cycle) and, therefore, the extended time to mating as was observed in the high-dose group was considered a chance-finding.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There were no treatment-related differences in mean pup weights between the test groups and the controls on day 0, 4, 7 and 13 of lactation.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

The number of stillborn or pups found dead at first scheduled litter inspection after birth accounted 7, 1, 1 and 2 for the control, low-, mid- and high-dose groups, respectively.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio on day 0 and day 13 was comparable among the groups.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

- The number of number of pups that died or were missing between days 0-4 accounted 0, 1, 0 and 0 for the control, low-, mid- and high dose, respectively.
- After culling on day 4, no pups were lost.

External malformations:

no effects observed

Description (incidence and severity):

- There were no treatment related effects on the absolute (expressed in mm) and corrected (expressed in mm/g) anogenital distance of male and female pups.
- There were no effects on nipple retention of male pups on post-natal day 13.
- Macroscopic examinations of stillborn pups and pups that died did not reveal treatment-related effects.
- At necropsy of the pups no treatment related macroscopic changes were observed.

Skeletal malformations:

not examined

Visceral malformations:

no effects observed

Description (incidence and severity):

- Microscopical evaluation of the thyroid gland of the pups did not reveal treatment related histopathological changes.

Other effects:

no effects observed

Description (incidence and severity):

No effects on T4 and TSH hormones were observed in culled pups on postnatal day 4 and in male and female pups on postnatal day 13.

Effect levels (fetuses)

Key result

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

No developmental toxicity was observed up to the highest dose level tested (500 mg/kg bw/day).

Executive summary:

A reproduction/ developmental toxicity screening was performed according to OECD TG 421 and GLP. The substance was administered orally (gavage) to male and female Wistar Han IGS rats at dose levels of 50, 150 and 500 mg/kg bw/day (12 rats/sex/dose level). Concurrent controls (12 rats/sex) were performed with the vehicle (corn oil). Parental animals were exposed 2 weeks prior mating and during mating. Male rats and non-mated female rats were exposed until sacrifice (study day 30). Mated females were dosed from the start of the gestation period (the finding of a sperm positive vaginal smear was considered gestation day 0) up to delivery of the pups. During the lactation period, females were dosed up to sacrifice, after overnight fasting, on day 14 of lactation. The test substance was considered to be homogeneously distributed and to be stable in the gavage liquids under the experimental conditions. Although the concentration of the test substance of the mid-dose groups of the gavage liquids prepared was 14% lower than intended, in general, the concentration of the test substance was close to intended (90-110% of the intended concentration).

Repeated dose effects, clinical signs: For the male and female animals, there were no mortalities observed and no treatment related effects on macroscopic parameters and body weight were observed. In addition, no treatment related changes in estrus cyclicity, fertility and reproductive performance in P0 was observed. Although food consumption was affected in the post-mating period of the male animals in mid- and high dose groups and in pregnant females in the high dose group, this was not considered as an adverse effect of the treatment. In male animals of the high-dose group, the absolute- (+13.6%) and relative (+16.8%) liver weights and the relative kidney weight (+8.0%) were statistically significantly increased as compared to the control animals. Microscopic evaluation revealed a treatment-related increase of accumulation of hyaline droplets in tubular epithelial cells in the outer cortex of the kidneys, accompanied by degenerative changes of these cells in 6/12 high dose males in most cases the hyaline droplets were also present in the lumen of the tubules. Immunohistochemically staining with a monoclonal antibody against alpha 2u revealed that there was insufficient evidence to identify the hyaline droplets as alpha 2 urinary microglobulins. In female animals of the mid- and high-dose groups, the absolute (+9.9% and +24.7%, respectively) and relative (+9.0% and +24.2%, respectively) weights of the liver were statistically significantly increased as compared to the control group. No toxicological adverse effects on T4 and TSH hormones were observed in male and female parental animals.

Fertility: No treatment related changes in estrus cyclicity, fertility and reproductive performance in P0 was observed. Minor food consumption effects were observed in the post-mating period of the male animals in mid- and high dose groups and in pregnant females in the high dose group, but this was not considered as an adverse effect of the treatment.

Developmental toxicity: In the pups, no treatment related effects were observed in observational studies, body weight, anogenital distance, nipple retention and thyroid weight. No effects were observed during macroscopic observations in both still born/ found-dead pups and after sacrifice (post-natal day 13). Microscopic evaluation of the thyroid gland did not reveal treatment related histopathologicalchanges. No toxicological adverse effects on T4 and TSH hormones were in culled pups on postnatal day 4 and in male and female pups on postnatal day 13.

 

In conclusion, systemic effects were observed in the high dose group (500 mg/kg bw/day). A NOAEL of 150 mg/kg bw/ day was therefore derived based on microscopic effects observed in the kidneys (male), which upon staining were not confirmed to be alpha hydrocarbon nephropathy. No adverse effects were observed on fertility parameters, reproductive performance and developmental parameters up to the highest dose tested. Therefore, the NOAEL for reproduction and developmental toxic effects is ≥ 500 mg/kg bw/day.