Indeno[4,5-d]-1,3-dioxin, 4,4a,5,6,7,8,9,9b-octahydro-7,7,8,9,9-pentamethyl-

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

xxx

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability one is derived because study is carried out according to the respective OECD Guideline and under GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

DOSE RANGE FINDING STUDY:
Test animals:
- Source: Harlan, Horst, the Netherlands
- Weight on the day before initiation of treatment: Males: 300.6g (control), 288.7g (low-dose), 292.0g (mid-dose), 294.3g (high-dose), 293.0 (top dose); Females: 195.5g (control), 196.3g (low-dose), 190.6g (mid-dose), 191.5g (high-dose), 192.4 (top-dose)
- Housing: Pairwise (2 animals of the same sex and dosing group per cage) in macrolon cages with a bedding of wood shavings (Lignocel, Type ¾), strips of paper (Enviro-dri) and a wooden block as environmental enrichment under conventional conditions.
- Diet: ad libitum
- Water: ad libitum

MAIN STUDY
Test animals:
- Source: Harlan, Horst, the Netherlands
- Age at study initiation: 8-10 weeks
- Weight on the day before initiation of treatment: Males: 264.1g (control), 266.5g (low-dose), 260.4g (mid-dose), 266.3g (high-dose); Females: 187.9g (control), 192.8g (low-dose), 193.1g (mid-dose), 191.4g (high-dose).
- Housing: In macrolon cages with a bedding of wood shavings (Lignocel, Type ¾) and strips of paper (Enviro-dri) and a wooden block as environmental enrichment. During the premating period, the animals were housed in groups of 4/sex. For mating, one male and one female were housed together. Mated females were housed individually in macrolon cages.
- Diet: A cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, Witham, England), available ad libitum
- Water: Domestic mains tap-water suitable for human consumption available ad libitum
- Acclimation period: 7 days
Environmental conditions:
- Temperature (°C): 22 ± 2
- Humidity (%): 45-65
- 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 feed was provided as a powder (in order to mix the test item into the diet), in stainless steel cans, covered by a perforated stainless steel plate that served to prevent spillage. In the reproduction/developmental toxicity screening test, the feed in the feeders was replaced with fresh portions every 3 to 4 days. The test item was incorporated in the basal diet by mixing in a mechanical blender. Just before preparation of the experimental diets, the test item was warmed (up to 45 ºC), to facilitate incorporation into the basal diet. The experimental diets were prepared shortly before the start of the study and subsequently every 2-3 weeks. After preparation, the experimental diets were divided into amounts of diets sufficient for 4 days and were stored in plastic bags in a freezer (<-18°C). When the feed needed to be refreshed, one bag per group was removed from the freezer to feed the animals.
The report on the dose range finding study does not give information on diet storage conditions or preparation/refreshing frequency.

Details on mating procedure:

At the end of the premating period, each female was caged with one male from the same group. Animals were caged together until mating occured or 1 week had elapsed. Mating pairs were clearly identified. 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 were killed not earlier than 21 days after copulation. All 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:

METHODS
- During the dose-range finding study (non-GLP), an analytical method to measure the concentration of the test item Nebulone in the experimental diets was developed. Although the measured concentration of the test item in the experimental diets of the dose-range finding study were lower than intended, probably due to evaporation during preparation, the test item appeared to be stable when the diets were directly stored in a freezer (<-18°C) and when diets were stored at room temperature in the animal room for up to 4 days. For that reason, the feed was replaced every 3 to 4 days. In the first batch of diets prepared for this study, the analytical method was validated.
- Principle of the analytical methods: The concentration of Nebulone in RM3 diet (Rat & Mouse No.3 Breeding Diet, RM3) was determined by extraction of diet samples with acetonitrile. Duplicate portions of approximately 10 grams of (accurately weighed) diet was mixed with 50 mL acetonitrile. After ultrasonic treatment for 15 minutes, the samples was centrifuged at 1800 x g for 5 minutes. A supernatant aliquot was transferred to an HPLC vial. The samples were analyzed with HPLC (High Performance Liquid Chromatography) with UV detection at 220nm. A Phenomenex Luna 5 µm C18 150 mm/ 4.6 mm column was used with an isocratic method and acetonitrile only for the mobile phase. Quantification was obtained by comparison of the peak area of Nebulone in the sample extracts with those in calibration solutions containing known amounts of the test item.
- Analyses: The homogeneous distribution, stability (after 4 days in the animal room and after storage for 5 weeks in a freezer at <-18°C) and achieved concentration of the test item in the RM3 rat feed were analysed in the first batch of diets prepared for this study. Directly after mixing of each diet, samples for the homogeneity and stability experiments were taken from the mixer. Firstly, five homogeneity samples were taken at different locations in the feed container. Secondly, two samples per group for examination of the stability were taken.
RESULTS
- Analytical results of the dose range finding study: The samples that were stored in the freezer directly after preparation showed a lower measured content than intended: The measured concentration was only 91% of the intended concentration in the low-dose group (225 mg/kg). The measured concentration was only 73% of the intended concentration in the top-dose group (6750 mg/kg). Nebulone seems to be quite stable in the diet, when stored at room temerature for 7 days in an open container in the animal room. The concentration decreased about 7% in the low-dose group. The concentration decreased about 2% in the top-dose group. Therefore, it was proposed to give fresh diet with Nebulone every 3 to 4 days
- Analytical results of the main study: Based on the results of the stability analysis Nebulone was considered to be stable in the low-dose diet when stored at ambient temperature in an open container in the animal room for 4 days. However, for the mid- and the high-dose level, Nebulone was not quite stable when stored in an open container in the animal room for 4 days (relative decrease of 13% and 14% for the mid- and high-dose level, respectively). When stored in a closed container in the freezer (< -18°C) Nebulone was considered to be stable for 5 weeks. The concentration of Nebulone was “close to intended” (relative difference < 10%) for all dose levels. Nebulone was considered to be homogeneously distributed in all batches of diet.

Duration of treatment / exposure:

Dose range finding study: 14 days
Main study: Males: 14 days premating, 1-6 days mating, up till sacrifice (total exposure up to 33 days); Females: 14 days premating 1-6 days mating, 21/22 days gestation, 4 days lactation (total exposure up to 44 days)

Frequency of treatment:

Continuously in feed

Remarks:

Doses / Concentrations:
15, 75, 150 and 450 mg/kg bw (225, 1125, 2250, 6750 ppm in feed)
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
15, 50 and 125 mg/kg bw (225, 750, 1875 ppm)
Basis:
nominal in diet
main study

No. of animals per sex per dose:

Dose range finding study: 4
Main study: 12

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were determined in consultation with the sponsor and were based on the results of a 2-week dose-range finding study and a 28-day repeated-dose toxicity study.

Parental animals: Observations and examinations:

DOSE RANGE FINDING STUDY:
- Clinical observations: Each animal was observed daily in the morning hours. On working days, all cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. All abnormalities, signs of ill health or reactions to treatment were recorded.
- Body weight: The body weight of each animal was recorded twice weekly.
- Feed consumption: Feed consumption was recorded twice weekly.
- Section: At autopsy all animals were examined macroscopically. Testes, kidney, liver and spleen were weighed.

MAIN STUDY:
- General clinical observations: Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. All cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study.
- Body weight: Body weights of male and female rats were recorded one day before the start of administration of the test item at randomization and at the start of the study (Day 0). Males were weighed weekly until sacrifice. Females were 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. Non-mated females were weighed once per week after the mating period. 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, the food consumption was measured per cage over the same periods as the body weight was measured.
- Intake of test item: The intake of the test item per kg body weight per day was calculated from the nominal dietary concentration of the test item in the diet, the food consumption and the mean body weight measured at the beginning and the end of the pertaining period.
- Parturition: 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 observations:

- 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 surviving 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. Based on the results of a 28-day repeated-dose toxicity study with Nebulone, potential sensitive organs (liver and kidneys) and total white blood cell count were included.
- Total white blood cell count was conducted on 4-5 animals/sex/group. Males were selected from each of 3 cages/group (2, 2 and 1 animals with the lowest animal number of each cage/group, respectively). Only pregnant females were selected. Erroneously, no blood was taken from female 21 for white blood cell analysis. At necropsy, blood samples were taken from the abdominal aorta of the rats whilst under CO2/O2 anaesthesia. K3 EDTA was used as anticoagulant. In each sample total white blood cell count was analyzed using the Advia 120 haematology analyzer, Bayer HealthCare, Ireland.
- The following organs were weighed (paired organs together) as soon as possible after dissection to avoid drying: testes, epididymides, liver, kidneys.
- 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 the corpora lutea), uterus (after counting of the implantation sites), epididymides, seminal vesicles, prostate, liver, kidneys, all gross lesions. Tissues for microscopic examination were embedded in paraffin wax, sectioned at 5µm, and stained with haematoxylin and eosin, except for sections of the testes which were stained with PAS haematoxylin.
Microscopic examination was performed on the collected organs, except for liver and kidneys, of all animals of the control and high-dose group. In addition, reproductive organs of males that failed to sire (did not mate or mated females were not pregnant) and females that were 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.

Postmortem examinations (offspring):

- Pathology of pups: Grossly malformed pups were sacrificed and macroscopically examined. A necropsy was performed on stillborn pups and on pups that died during the study. At necropsy of the dams, at day 4 of lactation, all other pups were examined externally for gross abnormalities and killed by CO2/O2. After sacrifice, pups were stored in a freezer. No further skeletal and visceral examination was necessary (after consulation with the sponsor) and pups were discarded.

Statistics:

DOSE RANGE FINDING STUDY:
Clinical findings and parental necropsy observations were evaluated by Fisher's exact probability test. Body weight, body weight gain, organ weights and feed consumption data were subjected to one way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests. All analyses were two-sided. Group mean differences with an associated probability of less than 0.05 were considered to be statistically significant.

MAIN STUDY:
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 be 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, number of pups lost, number of male pups on day 1 and 4, 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. Mortality data and data of the pathology of parent animals were evaluated by the Fisher’s exact probability test. Total white blood cell count was subjected to one way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests.

Reproductive indices:

MAIN STUDY
- Fertility and reproductive performance: number of females placed with males, number of males mated with females, number of successful copulations, 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 at day 1 and 4, number of pups lost, number of litters lost entirely, number of male pups at day 1 and 4, number of corpora lutea, number of implantation sites, number of lost implantations, litter size
- The following parameters are calculated: pre-coital time, duration of gestation, mating index, male fertility index, female fertility index, female fecundity index, gestation index, live birth index, viability index day 1-4, pup mortality day 1 or 4, sex ratio day 1 or 4, pre-implantation loss, number of lost implantations, post-implantation loss.

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

DOSE RANGE FINDING STUDY:
- Clinical signs and survival: No treatment-related clinical signs were observed.
- Body weights: Mean body weights were statistically significantly decreased in females from the mid-, high, and top-dose groups on day 10 and day 14. Mean body weight change was statistically significantly reduced in females of the top-dose group from day 0 to day 14. In males, no effect was observed on body weights. However, body weight change was statistically significantly reduced in the top-dose males from day 7 to day 10.
- Feed consumption: No effects were observed in food consumption.
- Necropsy observations: Relative kidney and liver weights were statistically significantly increased in males form the top-dose group. Relative liver weight was also statistically significantly increased in females of the top-dose group. One female in the high-dose group had a swollen uterus (bilateral) with hypogenesis of the right uterine horn.

MAIN STUDY:
- Mortalities and clinical signs: No mortality or treatment-related clinical signs were observed.
- Body weight and body weight change: In males, no treatment-related effects on body weight were observed during the study. Body weight change was statistically significantly lower in the Nebulone-treated groups on several intervals during the study. However, based on the apparent incidental distribution in time and in absence of a dose-respons relationship and/or an effect on mean body weight, this was considered not to be toxicologically relevant. In females, no treatment-related effects on body weight were observed during premating, gestation and lactation. A statistically significantly reduced body weight change was observed in the high-dose group during the first week of dosing (in the premating phase).
- Food consumption: No effects on food consumption were observed for all dose levels in males during the enire study period and in females during premating, gestation and lactation.
- Test substance intake: Test substance intake (mg Nebulone/kg body weight/day) was calculated over the study period (males), premating period (females), gestation period (females) and lactation period from day 1-4 (females) and is summarized in the "other information on results". The intake of Nebulone 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 Nebulone was not stable in the mid- and high-dose diets, when stored at ambient temperature in an open container for 4 days (relative decrease 13% and 14%, respectively), the actual test substance intake was lower than indicated in the table for animals in the mid-, and high-dose groups.
- Fertility and reproductive performance: In each group 12 females were placed with a male from the same dose group. All 12 females in each group were mated as confirmed by a vaginal plug and/or sperm-positive vaginal smear of which 8, 10, 8, and 8 females were pregnant in the control, low-, mid-, and high-dose groups, respectively. The mean pre-coital time was comparable for all groups. The number of females with liveborn pups amounted to 7, 10, 8 and 8 for the control group and the low-, mid-, and high-dose group, respectively. One pregnant female (no. 23) of the control group showed implantation sites but no pups. No effects were observed on mating index, fecundity index, fertility index or gestation index. The duration of gestation was comparable in all groups. All females survived delivery. Two females in the control group, 2 females in the low-dose group and 1 female in the high-dose group delivered both live and stillborn pups. None of the females delivered stillborn pups only. The number of corpora lutea, implantation sites, lost implantations and the calculated indices, pre- and post implantation loss were comparable between the Nebulone-treated groups and the control group.
- Total white blood cell analysis: A slight decrease in mean white blood cell count was observed in males (14.4%) and females (13.3%) of the high-dose group, although not statistically significant and not dose-related.
- Organ weights of parental animals: A statistically significant increase in relative liver weight was observed in the males of the high-dose group. However, no clear dose-respons relationship was observed, the increase was less than 10% and not accompanied by macroscopic changes. Therefore, this finding was considered not to be toxicologically relevant.
- Macroscopic examination of parental animals: Macroscopic observations at necropsy revealed no treatment-related abnormalities. The uterus was swollen in some females, but this is considered to be part of the normal reproduction cycle. The remaining macroscopic findings were unremarkable and considered to be part of the background pathology of rats of this strain and age.
- Microscopic examination of parental animals: Microscopic examination revealed no treatment-related abnormalities. In the uterus, luminal dilatation was found in some animals which explained the macroscopic observation of the swollen uteri. This is considered to be part of the normal reproduction cycle. The histopathological findings in the other organs were unremarkable and considered to be part of the background pathology of rats of this strain and age.

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

>= 107 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No systemic toxicity seen up to the highest dose tested (1875 mg/kg diet, providing at least 107 mg/kg bw/day).

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

>= 110 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No systemic toxicity seen up to the highest dose tested (1875 mg/kg diet, providing at least 110 mg/kg bw/day).

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

>= 107 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects on male fertility seen up to the highest dose tested (1875 mg/kg diet, providing at least 107 mg/kg bw/day).

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

>= 110 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects on female fertility seen up to the highest dose tested (1875 mg/kg diet, providing at least 121 mg/kg bw/day).

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

- Litter size and sex: The total number of pups delivered was comparable for all groups. The control group showed a relatively high incidence of stillborn pups compared to the mid- and high-dose groups. The number of pups lost between postnatal days 1 and 4 was low, resulting in high viability indices for all groups (99%, 95%, 100% and 99% for the control, low dose, mid dose and high dose groups, respectively). The mean number of live pups per litter was comparable in all groups on day 1 and on day 4 of lactation. Sex ratio was comparable in all groups on day 1 and 4 of lactation. There were no adverse treatment-related effects on litter size and pup survival.
- Pup observations: On postnatal day 1, a few pups from two females of the low-dose group (no. 25 and 37) were cold or had no milk in the stomach. These findings were not related to treatment. No findings were observed on postnatal day 4.
- Pup body weight: Mean pup body weight on postnatal day 1 and 4 and pup body weight changes between postnatal day 1-4 were comparable in all groups.
- Macroscopic observations of pups that died: Most pups showed no milk in the stomach, not distended lungs or were partly cannibalized. Based on the incidence and frequency of these observations, this was not considered to be treatment-related.

Reproductive effects observed:

not specified

Conclusions:

In an oral (diet) reproduction/developmental toxicity screening test in rats (according to OECD 421, GLP compliant), no toxicity was observed up to and including the highest dose tested (1875 mg/kg diet, nominal concentration). In male rats, the NOAEL for systemic toxicity and fertility was at least 107 mg/kg bw/day. In female rats, the NOAEL for systemic toxicity was at least 110 mg/kg bw/day and the NOAEL for fertility and development was at least 121 mg/kg bw/day. These NOAELs were calculated on the basis of the nominal concentration of the substance in the diet corrected for the lower actual concentration due to instability as observed at the dose confirmation analysis.

Executive summary:

A developmental / reproscreen study was performed according to OECD TG 421. The doses were established after a dose-range finding study consisting of one control group and four test groups, each comprising 4 males and 4 females, which were administered different dose levels of the test substance by diet for two consecutive weeks. The animals received diets containing 15, 50, 125 and 450 mg/kg bw (0, 225, 1125, 2250 and 6750 mg/kg diet). Based on increased liver and kidney weights at the high dose lower dosing was done in the main study.

In the main study 12 Wistar rats/sex/dose received Nebulone in the diet at concentrations of 0, 225, 750 or 1875 mg/kg diet during a premating period of 2 weeks and during mating (1 week), gestation and lactation until postnatal day 4. Nebulone was homogeneously distributed in the diets. Nebulone was stable in the diet for the low-dose level but not for the mid- and high-dose level (relative decrease of 13% and 14%, respectively) when stored in an open container for 4 days. After storage in a closed container in the freezer (<-18°C) Nebulone was stable for 5 weeks. The concentration of Nebulone was close to the intended’ in all diets. Daily clinical observations did not reveal any treatment-related changes in the animal’s appearance, general condition or behaviour. No treatment-related effects were observed in mean body weight, body weight changes and food consumption throughout the study. No treatment-related effects were observed on pre-coital time, mating index, female fecundity index, male and female fertility indices, gestation index, duration of gestation, number of corpora lutea, implantation sites, lost implantations and the calculated indices, pre- and post-implantation loss. The effects on pups are described at the developmental toxicity section. No treatment-related effects of toxicological relevance were observed on organ weights or after macroscopic and microscopic examination of organs.

In conclusion, in male rats the highest dose of at least 107 mg/kg bw/day) was a No-Observed-Adverse-Effect Level (NOAEL) because no effects were seen on systemic toxicity parameters and male fertility. In female rats, the highest dose of at least 110 mg/kg bw/day (intake before gestation) was derived in absence of systemic and fertility effects.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

The quality of the database is high because in two different tests fertility has been investigated: the OECD TG 421 and in the 28-repeated dose toxicity study both conducted according OECD guidelines and under GLP.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Information on fertility has been derived from a reproscreen study. In addition, in the 28 -day repeated dose study no effects on fertility were seen in male and female reproductive organs, further confirming the absence of effects on fertility.

Short description of key information:
In a GLP-compliant reproduction/developmental toxicity screening test, performed according to OECD Guideline 421, 12 Wistar rats/sex/dose received Nebulone in the diet at concentrations of 0, 15, 50 and 150 mg/kg bw (0, 225, 750 or 1875 mg/kg diet) during a premating period of 2 weeks and during mating (1 week), gestation and lactation until postnatal day 4. Nebulone was homogeneously distributed in the diets. Feed with Nebulone was freshly prepared every day because in the dose range finding it was shown not to be stable in open container for 4 days. The concentration of Nebulone was close to the intended concentrations in all diets. Daily clinical observations did not reveal any treatment-related changes in the animal’s appearance, general condition or behaviour. No treatment-related effects were observed in mean body weight, body weight changes and food consumption throughout the study. No treatment-related effects were observed on pre-coital time, mating index, female fecundity index, male and female fertility indices, gestation index, duration of gestation, number of corpora lutea, implantation sites, lost implantations and the calculated indices, pre- and post-implantation loss. In conclusion, in male rats the highest dose of at least 107 mg/kg bw/day) was a No-Observed-Adverse-Effect Level (NOAEL) because no effects were seen on systemic toxicity parameters and male fertility. In female rats, a NOAEL of at least 110 mg/kg bw/day was derived for systemic and fertility effects in absence of any effects at this dose.

Justification for selection of Effect on fertility via oral route:
A reproscreening study is available being conducted according to OECD-guideline and under GLP conditions, which is adequate for covering this endpoint.

Effects on developmental toxicity

Description of key information

In a GLP-compliant reproduction/developmental toxicity screening test, performed according to OECD Guideline 421, 12 Wistar rats/sex/dose received Nebulone in the diet at concentrations of 0, 15, 50 and 150 mg/kg bw (0, 225, 750 or 1875 mg/kg diet) during a premating period of 2 weeks and during mating (1 week), gestation and lactation until postnatal day 4. Nebulone was homogeneously distributed in the diets. Feed with Nebulone was freshly prepared every day because in the dose range finding it was shown not to be stable in open container for 4 days. The concentration of Nebulone was close to the intended concentrations in all diets. For estimating the dose in mg/kg bw  for dams and offspring the intake of the substance during gestation and lactation were used. No treatment related findings were seen in the dams up to the highest dose considering appearance, general condition or behaviour. No treatment-related effects were observed in mean body weight, body weight changes and food consumption throughout the study. No effects were observed on litter size, pup sex and weight and pup survival. No treatment-related effects of toxicological relevance were observed on macroscopic and microscopic examination. Therefore in dams the highest dose of at least 121 mg/kg bw/day (based on actual intake) was a No-Observed-Adverse-Effect Level (NOAEL) for maternal and developmental toxicity.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 is assigned because the study is conducted according to the respecive OECD guidelines and GLP.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD TG 421

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

see 7.8.1

Route of administration:

oral: feed

Vehicle:

other: Substance was provided in feed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

see 7.8.1

Details on mating procedure:

see 7.8.1

Duration of treatment / exposure:

see 7.8.1

Frequency of treatment:

see 7.8.1

Duration of test:

see 7.8.1

Remarks:

Doses / Concentrations:

Basis:
actual ingested

No. of animals per sex per dose:

see 7.8.1

Details on study design:

see 7.8.1

Maternal examinations:

see 7.8.1

Fetal examinations:

to be filled

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
see 7.8.1

Dose descriptor:

NOAEL

Effect level:

>= 121 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

>= 121 mg/kg bw (total dose)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

In a GLP-compliant reproduction/developmental toxicity screening test, performed according to OECD Guideline 421 no effects were seen up to the highest dose tested (121 mg/kg bw) resulting in the NOAEL.

Executive summary:

In a GLP-compliant reproduction/developmental toxicity screening test, performed according to OECD Guideline 421, 12

Wistar rats/sex/dose received Nebulone in the diet at concentrations of 0, 15, 50 and 150 mg/kg bw (0, 225, 750 or 1875 mg/kg diet) during a premating period of 2 weeks and during mating (1 week), gestation and lactation until postnatal day 4. Nebulone was h omogeneously distributed in the diets. Feed with Nebulone was freshly prepared every day because in the dose range finding it was shown not to be stable in open container for 4 days. The concentration of Nebulone was close to the intended’ in all diets. No treatment related findings were seen in the dams up to the highest dose considering appearance, general condition or behaviour. No treatment-related effects were observed in mean body weight, body weight changes and food consumption throughout the study. No effects were observed on litter size, pup sex and weight and pup survival. No treatment-related effects of toxicological relevance were observed on macroscopic and microscopic examination. Therefore in dams the highest dose of at least 121 mg/kg bw/day (based on actual intake during gestation and lactation) was a No-Observed-Adverse-Effect Level (NOAEL) for systemic and development toxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The quality of the database is high because the reproscreen study combined with the additional information on developmental toxicity the endpoint is adequately covered.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Justification for selection of Effect on developmental toxicity: via oral route:
A reproscreening / developmental toxicity study is conducted according to OECD-guideline 421 and under GLP conditions and is the one experimental study available. The study is adequate to cover the developmental toxicity endpoint.

Justification for classification or non-classification

Based on the absence of adverse effects on fertility and of developmental toxicity in the reproduction / developmental toxicity screening test in rats and the absence of effects in the gonads of the repeated dose toxicity classification of the test material for effects on fertility or developmental toxicity is not warranted in accordance with EU Directive 67/548/EEC and EU Classification (DSD), Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 (CLP).