Indeno[4,3a-b]furan, decahydro-2,2,7,7,8,9,9-heptamethyl-

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Dec 2011 to 03 Feb 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

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:

14 December 2011 to 3 February 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Source: Harlan, Horst, the Netherlands
- Age at study initiation: (P) 10 - 11 weeks
- Weight at study initiation: Males: 319.1 g (control), 324.1 g (low-dose), 321.8 g (mid-dose), 317.0 g (high-dose); Females: 196.8 g (control), 199.7g (low-dose), 197.4g (mid-dose), 198.9 g (high-dose).
- Fasting period before study: No
- Housing: Macrolon cages with a bedding of wood shavings (Lignocel, Type 3/4) and strips of paper (Enviro-dri) and a wooden block as environmental enrichment. Pre-mating in groups of 4/sex, during mating 1 male/1 female, after mating individually.
- Diet: Cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3). Ad libitum.
- Water: Domestic mains tap-water suitable for human consumption given in polypropylene bottles. Ad libitum.
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS:
- Temperature (°C): 22 ± 2
- Humidity (%): 45 - 65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 21 Dec 2011 To: 03 Feb 2012

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The feed was provided as a powder in stainless steel cans, covered by a perforated stainless steel plate that served to prevent spillage. The feed in the feeders was replaced daily. The test substance was incorporated in the basal diet by mixing in a mechanical blender and divided into daily amounts of diets to be stored in plastic bags in the freezer.

DIET PREPARATION:
- Rate of preparation of diet: Short before start of the study and after 2 weeks
- Mixing appropriate amounts with: Cereal-based rodent diet
- Storage temperature of food: <-18 °C

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test substance in the diet was determined by extraction of diet samples with acetonitrile. Duplicate portions of approximately 10 grams of (accurately weighed) diet were mixed with 50 ml acetonitrile. After ultra sonic treatment for 15 minutes, the samples were centrifuged.A clear supernatant aliquot was analysed using HPLC with UV detection at 220 nm. The analytical method was validated by analysing 3 spiked samples per low dose level and 3 samples per high dose level, to conform to linearity (correlation coefficient >=0.996), recovery (70 % - 110 %) and repeatability (relative standard deviation in the percentage recovery of 3 spiked diet samples per concentration level should be < 10 %).
The homogeneous distribution, stability (after one day in the animal room and after storage for 5 weeks in a freezer at <-18 °C) and achieved concentration of the test substance in the diet were analysed in the first batch of diets prepared or this study.

Duration of treatment / exposure:

Males: 14 days pre-mating, during mating and up to the day of sacrifice (total exposure up to 33 days).
Females: 14 days pre-mating, during mating, during gestation and lactation and up to the day of sacrifice (total exposure up to 44 days).

Frequency of treatment:

Continously

Dose / conc.:

225 mg/kg diet

Remarks:

15 mg/kg bw nominal

Dose / conc.:

750 mg/kg diet

Remarks:

50 mg/kg bw nominal

Dose / conc.:

1 500 mg/kg diet

Remarks:

100 mg/kg bw nominal

No. of animals per sex per dose:

12

Control animals:

yes, plain diet

Details on study design:

Details on mating procedure
- M/F ratio per cage: 1/1
- Length of cohabitation: Till mating occurred or 1 week
- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: Individually
- Any other deviations from standard protocol: No additional attempts after unsuccessful pairing

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: Twice daily
- Cage side observations checked: Signs of toxicity, mortality

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Daily

BODY WEIGHT:
- Time schedule for examinations: 1 day before start of study and on day 0. Males weighed weekly. Females weighed weekly during premating and mating. Pregnant females weighed on days 0, 7, 14 and 21 during presumed gestation and on days 1 and 4 of lactation. Non-mated females weighed weekly after mating period. Animals were weighed on scheduled necropsy date to be able to calculate the correct organ to body weight ratios.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

HAEMATOLOGY:
- Time schedule for blood collection: Upon sacrifice
- How many animals: 3-5 animals/sex/group
- Parameters checked: Total white blood cell count
 

Sacrifice and pathology:

SACRIFICE:
- Male animals: All surviving animals after mating period.
- Maternal animals: All surviving animals at day 4 of lactation.

GROSS NECROPSY:
- Gross necropsy consisted of external examinations. Corpora lutea in the ovaries and implantation sites in the uterus were counted.

HISTOPATHOLOGY / ORGAN WEIGHTS:
- Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate-buffered 4 % solution of formaldehyde; except for the testes which was preserved in Bouin's fixative: ovaries, uterus, testes*, epididymides*, seminal vesicles, prostate, liver*, kidneys*, all gross lesions. The *marked organs were weighed (paired organs together) as soon as possible after dissection to avoid drying.Tissues for microscopic examination were embedded in paraffin wax, sectioned at 5 µm, 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. Furthermore, organs showing gross lesions of animals of all groups were microscopically examined.

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were observed.

For more details see Table 1 (Page 27 – 28), Table 2 (Page 29), and Table 3 (Page 30) of the attached study report under IUCLID Chapter 7.8.1.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistically significant differences were observed in body weight and body weight gain for the males and females during the study.

For more details see Table 4 (Page 31 – 32), Table 5 (Page 33 - 34), Table 6 (Page 35), Table 7 (Page 36), Table 8 (Page 37) and Table 9 (Page 38) of the attached study report under IUCLID Chapter 7.8.1.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment-related effects on food consumption were observed in males and females during the study. The statistically significantly increased food consumption (expressed in g/animal/day) in males of the mid-dose group between day 21 to 28 was considered to be an incidental finding and therefore not of toxicological relevance.
The actual test substance intake was lower than initially foreseen for animals in the low-, mid-, and high-dose groups.

For more details see Table 10 (Page 39 – 40), Table 11 (Page 41 - 42), Table 12 (Page 43 - 44), Table 13 (Page 45), Table 14 (Page 46), Table 15 (Page 47), Table 16 (Page 48), Table 17 (Page 49), and Table 18 (Page 50) of the attached study report under IUCLID Chapter 7.8.1.

Haematological findings:

no effects observed

Description (incidence and severity):

No statistically significant effects on mean white blood cell count were observed in males and females.

For more details see Table 23 (Page 58) and Table 24 (Page 59) of the attached study report under IUCLID Chapter 7.8.1.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A statistically significantly increased relative liver weight was observed in the males of the mid- (10.6%), and high-dose groups (12.6%) which is considered an adaptive response (<20%) and therefore not of toxicological relevance. No other treatment related effects were observed.
- Male: Absolute liver weight: 3.5, 9.7, and 8.2% increased at low, mid and high dose, respectively
- Female: Absolute liver weight: 4.8, 6.0, and 9.1% increased at low, mid and high dose, respectively
- Male: Relative liver weight: 3.8, 10.6, and 12.6% increased at low, mid and high dose, respectively
- Female: Relative liver weight: 0, 1.8, and 5.7% increased at low, mid and high dose, respectively

For more details see Table 24 (Page 60 - 61) and Table 25 (Page 62 - 63) of the attached study report under IUCLID Chapter 7.8.1.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic observations at necropsy revealed no treatment-related abnormalities.

For more details see Table 26 (Page 64) of the attached study report under IUCLID Chapter 7.8.1.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic examination revealed no treatment-related abnormalities.

For more details see Table 27 (Page 65 - 66) of the attached study report under IUCLID Chapter 7.8.1.

Other effects:

no effects observed

Dose descriptor:

NOAEL

Effect level:

>= 64 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No systemic toxicity observed up to the highest dose tested 100 mg/kg bw (1500 mg/kg nominal in the diet).

Dose descriptor:

NOAEL

Effect level:

>= 83 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No systemic toxicity observed up to the highest dose tested 100 mg/kg bw (1500 mg/kg nominal in the diet).

Key result

Critical effects observed:

no

STABILITY, HOMOGENEITY AND CONTENT OF THE TEST SUBSTANCE:

The concentration of the test substance was lower than intended in all test diets. Relative differences of -28 %, -26 % and -14 % were observed for the measured concentrations in the low-, the mid- and the high dose group, respectively. The test substance was considered to be stable in diet after storage at ambient temperature in an open container for 24 hours for the low- and mid-dose level. However, for the high dose level feed should be stored in a closed container in the freezer (< -18 °C), as the concentration decreased with 11% at ambient temperature (24 hours). The test substance was considered to be homogeneously distributed in all batches of diet.

Table 1. Mean (range) test substance intake (mg/kg bw/day), based on the nominal concentration of the test substance in the diet. The actual test substance intake was lower because the actual dietary concentration was lower than intended.

	Mean (range) test substance intake (mg/kg bw/day)
	Low dose	Mid dose	High dose
Males during study	12.49 (11.54-13.25)	43.04 (41.87-43.97)	85.26 (81.39-89.70)
Pre-mating females	15.55 (14.99-16.11)	47.99 (46.65-49.33)	110.84 (105.99-115.69)
Gestation females	16.93 (15.86-19.15)	55.06 (50.91-63.18)	108.28 (99.52-123.57)
Lactation females	19.01	62.95	135.24

Conclusions:

Under the conditions of the test (OECD TG 421, GLP), the systemic NOAEL of the test substance was determined to be at least 64 and 83 mg/kg bw/day for males and females, respectively.

Executive summary:

For the Reproscreen study a 14-day dose range finder was performed at 225, 1125, 2250 and 6750 mg/kg diet which are nominal 15, 75, 150 and 450 mg/kg bw. The dietary route was selected to prevent effects due to bolus dosing. At the mid high and high dose increased relative liver weights were seen in males +26 and 38% and females +19 and +41%, respectively. These adverse relative liver weight were used to set a maximum dose level of nominal 100 mg/kg bw to prevent repeated dose effects that could possibly obscure reproductive effects. 

The the reproduction/developmental toxicity screening test was performed according to OECD TG 421 and under GLP conditions. Twelve Wistar rats/sex/dose received diets containing Amber Xtreme at concentrations of 0, 225, 750 or 1500 mg Amber Xtreme/kg diet during a premating period of 2 weeks and during mating (1 week), gestation and lactation until postnatal day 4. The nominal concentrations were 15, 50 and 100 mg/kg bw. Amber Xtreme was homogeneously distributed in the diets and stable in diet stored in an open container for 24 hours, except for the high-dose level (-11%). When stored in a closed container in the freezer (< -18°C), the high dose of Amber Xtreme in diet was stable for 5 weeks. The concentration of Amber Xtreme was lower than intended in all diets. Relative differences of -28 %, -26 % and -14 % were observed for the measured concentrations in the low-, the mid- and the high dose group, respectively. As a results the maxiumum intake of the highest dose group was 64 and 83 mg/kg bw, for males and females, respectively.

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. Furthermore, 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. Relative liver weight was increased with 12.6% in males but not in females. Macroscopic and microscopic examination of organs did not reveal any treatment-related changes.

In conclusion, the results of the reproduction/developmental toxicity screening test in rats indicate no toxicity up to and including the highest dose tested (1500 mg/kg diet, nominal concentration (100 mg/kg bw)). The NOAEL for systemic toxicity was at least 64 mg/kg bw/day for males and at least 83 mg/kg bw/day for females based on the actual dose of test material ingested.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

Jul 1995

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

449-360-4

EC Name:

-

Cas Number:

647828-16-8

Molecular formula:

C18H32O

IUPAC Name:

decahydro-2,2,6,6,7,8,8-heptamethyl-2H-Indeno[4,5-b] furan

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Source: Harlan, Horst, the Netherlands
- Age at study initiation: (P) 10 - 11 weeks
- Weight at study initiation: Males: 319.1 g (control), 324.1 g (low-dose), 321.8 g (mid-dose), 317.0 g (high-dose); Females: 196.8 g (control), 199.7g (low-dose), 197.4g (mid-dose), 198.9 g (high-dose).
- Fasting period before study: No
- Housing: Macrolon cages with a bedding of wood shavings (Lignocel, Type 3/4) and strips of paper (Enviro-dri) and a wooden block as environmental enrichment. Pre-mating in groups of 4/sex, during mating 1 male/1 female, after mating individually.
- Diet: Cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3). Ad libitum.
- Water: Domestic mains tap-water suitable for human consumption given in polypropylene bottles. Ad libitum.
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS:
- Temperature (°C): 22 ± 2
- Humidity (%): 45 - 65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 21 Dec 2011 To: 03 Feb 2012

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The feed was provided as a powder in stainless steel cans, covered by a perforated stainless steel plate that served to prevent spillage. The feed in the feeders was replaced daily. The test substance was incorporated in the basal diet by mixing in a mechanical blender and divided into daily amounts of diets to be stored in plastic bags in the freezer.

DIET PREPARATION:
- Rate of preparation of diet: Short before start of the study and after 2 weeks
- Mixing appropriate amounts with: Cereal-based rodent diet
- Storage temperature of food: <-18 °C

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: Till mating occurred or 1 week
- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: Individually
- Any other deviations from standard protocol: No additional attempts after unsuccessful pairing

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test substance in the diet was determined by extraction of diet samples with acetonitrile. Duplicate portions of approximately 10 grams of (accurately weighed) diet were mixed with 50 ml acetonitrile. After ultra sonic treatment for 15 minutes, the samples were centrifuged.A clear supernatant aliquot was analysed using HPLC with UV detection at 220 nm. The analytical method was validated by analysing 3 spiked samples per low dose level and 3 samples per high dose level, to conform to linearity (correlation coefficient >=0.996), recovery (70 % - 110 %) and repeatability (relative standard deviation in the percentage recovery of 3 spiked diet samples per concentration level should be < 10 %).
The homogeneous distribution, stability (after one day in the animal room and after storage for 5 weeks in a freezer at <-18 °C) and achieved concentration of the test substance in the diet were analysed in the first batch of diets prepared or this study.

Duration of treatment / exposure:

Males: 14 days premating, during mating and up to the day of sacrifice (total exposure up to 33 days).
Females: 14 days premating, during mating, during gestation and lactation and up to the day of sacrifice (total exposure up to 44 days).

Frequency of treatment:

Continuously

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

225 mg/kg diet

Remarks:

15 mg/kg nominal

Doses / concentrations 2

Dose / conc.:

750 mg/kg diet

Remarks:

50 mg/kg nominal

Doses / concentrations 3

Dose / conc.:

1 500 mg/kg diet

Remarks:

100 mg/kg nominal

No. of animals per sex per dose:

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.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: Twice daily
- Cage side observations checked: Signs of toxicity, mortality

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Daily

BODY WEIGHT:
- Time schedule for examinations: 1 day before start of study and on day 0. Males were weighed weekly. Females were weighed weekly premating and mating. Pregnant females weighed on days 0, 7, 14 and 21 during presumed gestation and on days 1 and 4 of lactation. Non-mated females weighed weekly after mating period. In addition, animals were weighed on scheduled necropsy date to be able to calculate the correct organ to body weight ratios.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg bw/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

HAEMATOLOGY:
- Time schedule for blood collection: Upon sacrifice
- How many animals: 3 - 5 animals/sex/group
- Parameters checked: Total white blood cell count

Litter observations:

PARAMETERS EXAMINED:
The following parameters were examined in F1 offspring: Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, mean body weight.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities. Necrospy was performed for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE:
- Male animals: All surviving animals after mating period by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia.
- Maternal animals: All surviving animals at day 4 of lactation by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia.

GROSS NECROPSY:
- Gross necropsy consisted of external examinations. Corpora lutea in the ovaries and implantation sites in the uterus were counted.

HISTOPATHOLOGY / ORGAN WEIGHTS:
- Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate-buffered 4 % solution of formaldehyde; except for the testes which was preserved in Bouin's fixative: ovaries, uterus, testes*, epididymides*, seminal vesicles, prostate, liver*, kidneys*, all gross lesions. The *marked organs were weighed (paired organs together) as soon as possible after dissection to avoid drying.Tissues for microscopic examination were embedded in paraffin wax, sectioned at 5 µm, 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. 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 and pups were discarded.

Statistics:

- Clinical findings, mortality data and data of the pathology of parent animals were evaluated by Fisher's exact probability test. This test was also 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.
- Body weight, body weight gain, food consumption, organ weight and total white blood cell count data were subjected to one way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests.
- Pre-coital time (mean number of days), duration of gestation, number of corpora lutea and implantation sites, 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 the Mann-Whitney U test.

Reproductive indices:

- 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 corpora lutea, number of implantation sites, number of lost implantations.
- The following parameters are calculated: Pre-coital time, duration of gestation, mating index, male fertility index, female fertility index, female fecundity index, gestation index, pre-implantation loss, number of lost implantations, post-implantation loss, litter size.

Offspring viability indices:

- The following parameters are determined: Live birth index, viability index day 1-4, pup mortality day 1 or 4, sex ratio day 1 or 4, 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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were observed.

For more details see Table 1 (Page 27 – 28), Table 2 (Page 29), and Table 3 (Page 30) of the attached study report.

Mortality:

no mortality observed

Description (incidence):

No mortality observed.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistically significant differences were observed in body weight and body weight change between the males of the control and the test substance exposed groups during the study. For the females no effects were observed on body weights or body weight changes during premating, gestation and lactation.

For more details see Table 4 (Page 31 – 32), Table 5 (Page 33 - 34), Table 6 (Page 35), Table 7 (Page 36), Table 8 (Page 37) and Table 9 (Page 38) of the attached study report.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects on food consumption were observed in males during the study. The statistically significantly increased food consumption (expressed in g/animal/day) in males of the mid-dose group between day 21 to 28 was considered to be an incidental finding. The effect was not dose-related. Food consumption of the females was comparable between the control and the test substance exposed groups during premating, gestation and lactation.
The actual test substance intake was lower than initially foreseen for animals in the low-, mid-, and high-dose groups.

For more details see Table 10 (Page 39 – 40), Table 11 (Page 41 - 42), Table 12 (Page 43 - 44), Table 13 (Page 45), Table 14 (Page 46), Table 15 (Page 47), Table 16 (Page 48), Table 17 (Page 49), and Table 18 (Page 50) of the attached study report.

Haematological findings:

no effects observed

Description (incidence and severity):

No statistically significant effects on mean white blood cell count were observed in males and females.

For more details see Table 23 (Page 58) and Table 24 (Page 59) of the attached study report.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic examination revealed no treatment-related abnormalities.

For more details see Table 27 (Page 65 - 66) of the attached study report.

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

In each group 12 females were placed with a male from the same dose group. All females were mated as confirmed by a vaginal plug and/or sperm-positive vaginal smear. The mean pre-coital time was comparable for all groups. The number of pregnant females and the number of females with liveborn pups amounted 12 in all groups.

No differences were observed on male and female fertility indices, female fecundity index, gestation index or duration of gestation. All females survived delivery. Six females in the control group, and 2, 3, and 1 females in the low-, mid-, and high-dose groups, respectively, 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 treated groups and the control group.

For more details see Table 19 (Page 51 - 53) of the attached study report.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

>= 64 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

>= 83 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

3

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

>= 64 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

4

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

>= 81 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

At postnatal day 1, all pups of dam 17 of the control group were pale. At postnatal day 4, clinical signs were observed in 6 pups from dam 71 of the mid-dose group (5 pups were pale and 1 pup with a swollen abdomen). These signs were not considered as treatment-related.

For more details see Table 20 (Page 54) of the attached study report.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The mean number of live pups per litter was comparable in all groups on day 1 and on day 4 of lactation, exept for an incidental decrease in mean number of live pups per litter on day 4 in the mid-dose group. A statistically significantly increased number of male pups in the high-dose group at postnatal day 4 was observed. Based on a normal sex ratio in all groups, this was considered not to be toxicologically relevant.

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 exposed groups. Additionally, the number of pups that were lost between postnatal days 1-4 was statistically significantly lower in all exposed groups compared to the control group (52 in the control group compared to 34, 24 and 3 in the low-, mid-, and high dose groups, respectively). The number of litters lost entirely between postnatal days 1 and 4 was relatively higher in the control group compared to the exposed groups (5 in the control group compared to 2, 1 and 0 in the low-, mid-, and high dose groups, respectively). The high mortality of pups due to cannibalism by the dams in all groups is comparable with the results obtained in other studies with this rat strain and is not considered to be affected by the test item. There were no adverse treatment-related effects on litter size and pup survival.

For more details see Table 19 (Page 51 - 53) of the attached study report.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean pup body weight on postnatal day 1 and 4 and pup body weigh changes between postnatal day 1 - 4 were comparable in all groups.

For more details see Table 21 (Page 55 - 56) of the attached study report.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Most pups showed no milk in the stomach, didn’t have distended lungs or were partly cannibalised. A dilated urinary bladder was observed in 2 female pups that died on day 2 (one dam; control group) and in 1 male pup that died on day 4 (one; mid-dose group). Based on the incidence and frequency of these observations, this was not considered to be treatment-related.

For more details see Table 22 (Page 57) of the attached study report.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 81 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

STABILITY, HOMOGENEITY AND CONTENT OF THE TEST SUBSTANCE:

The concentration of the test substance was lower than intended in all test diets. Relative differences of -28 %, -26 % and -14 % were observed for the measured concentrations in the low-, the mid- and the high dose group, respectively. The test substance was considered to be stable in diet after storage at ambient temperature in an open container for 24 hours for the low- and mid-dose level. However, for the high dose level feed should be stored in a closed container in the freezer (< -18 °C), as the concentration decreased with 11 % at ambient temperature (24 hours). The test substance was considered to be homogeneously distributed in all batches of diet.

Table 1. MEASURED TEST SUBSTANCE INTAKE.

	Mean (range) substance intake (mg/kg bw/day), based on the nominal concentration of the test substance in the diet. The actual substance intake was lower because the actual dietary concentration was lower than intended.
	Low-dose	Mid-dose	High-dose
Males during study	12.49 (11.54-13.25)	43.04 (41.87-43.97)	85.26 (81.39-89.70)
Premating females	15.55 (14.99-16.11)	47.99 (46.65-49.33)	110.84 (105.99-115.69)
Gestation females	16.93 (15.86-19.15)	55.06 (50.91-63.18)	108.28 (99.52-123.57)
Lactation females	19.01	62.95	135.24

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test (OECD TG 421, GLP), the systemic NOAEL of the test substance was determined to be at least 64 and 83 mg/kg bw/day for males and females, respectively. The fertility NOAEL and the developmental NOAEL were considered to be at least 64 and 81 mg/kg bw/day for males and females, respectively.

Executive summary:

In a reproduction/developmental toxicity screening test that was performed according to OECD TG 421 and under GLP conditions, 12 Wistar rats/sex/dose received diets containing the test substance at concentrations of 0, 15, 50 and 150 mg/kg bw (0, 225, 750 or 1500 mg/kg diet) during a premating period of 2 weeks and during mating (1 week), gestation and lactation until postnatal day 4. The test substance was homogeneously distributed in the diets and stable in diet stored in an open container for 24 hours, except for the high-dose level (-11 %). When stored in a closed container in the freezer (< -18°C), the high dose of the test substance in diet was stable for 5 weeks. The concentration of the test substance was lower than intended in all diets. Relative differences of -28 %, -26 % and -14 % were observed for the measured concentrations in the low-, the mid- and the high dose group, respectively.

Results: 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. Relative liver weight was statistically significant increased with 10.6 and 12.7% in mid dose and high dose males, respectively, but not in females. The toxicological relevance of this finding is doubtful, because there was no clear dose-response and no histopathological changes in the liver were observed. Macroscopic and microscopic examination of organs did not reveal any treatment-related changes. 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. No effects were observed on litter size, pup sex and weight and pup survival. Macroscopic observations of stillborn pups or pups that died during lactation did not reveal any treatment-related changes.

Conclusion: The results of the reproduction/developmental toxicity screening test in rats indicate no systemic, fertility, and developmental toxicity up to and including the highest dose tested 100 mg/kg bw (1500 mg/kg diet, nominal concentration). In male rats, the NOAEL for systemic toxicity and fertility was established to be at least 64 mg/kg bw/day based on the actual dose of test substance ingested. In female rats, the NOAEL for systemic toxicity was established to be at least 83 mg/kg bw/day and the NOAEL for fertility and development was at least 81 mg/kg bw/day.