Tetrahydro-4-methyl-2-(2-methylprop-1-enyl)pyran

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Remarks:

The submitted data refer to a draft study report before QAU check. The study entry will be updated, (if necessary) as soon as the final report is available.

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

Rangefinder study with focus on maternal toxicity only

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:Wl(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: About 10-12 weeks
- Fasting period before study: no
- Housing: 1 animal per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: From GD 0 (day of supply) to the beginning of administration (GD 6).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Concentration in vehicle: 12.5 and 25 g/100 ml in the mid and high dose group respectively
- Amount of vehicle (if gavage): 4 ml/kg bw/d

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of the test substance in corn oil at room temperature over a period of 7 days had been verified prior to the start of the study in a similar batch.

Details on mating procedure:

Animals time-mated by the breeder

Duration of treatment / exposure:

GD6 - 19

Frequency of treatment:

daily

Duration of test:

GD0 - 20

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

7 time-mated females

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
Mortality: A check for moribund and dead animals was made twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays (GD O to 20).
Clinical signs: at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity. During the administration period (GD 6 - 19) all animals were checked daily for any abnormal clinically signs before the administration as well as within 2 hours and within 5 hours after the administration.

BODY WEIGHT: Yes
- Time schedule for examinations: recorded on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: recorded for GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

WATER CONSUMPTION: Yes
- Time schedule for examinations: recorded for GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

CLINICAL PATHOLOGY: Yes
Blood samples were taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia.
Parameters examined from the dams - Hematology
1. Leukocytes
2. Erythrocytes
3. Hemoglobin
4. Hematocrit
5. Mean corpuscular volume (MCV)
6. Mean corpuscular hemoglobin (MCH)
7. Mean corpuscular hemoglobin concentration (MCHC)
8. Platelets
9. Differential blood count
10. Reticulocytes

Parameters examined from the dams -Clinical chemistry
1. Alanine aminotransferase
2. Aspartate aminotransferase
3. Alkaline phosphatase
4. Serum y-glutamyl transferase
5. lnorg. phosphate
6. Calcium
7. Urea
8. Creatinine
9. Glucose
10. Total bilirubin
11. Total protein
12. Albumin
13. Globulins
14. Triglycerides
15. Cholesterol

POST-MORTEM EXAMINATIONS: Yes
On GD 20, the surviving dams were anesthetized with isoflurane, sacrificed by decapitation. Moribund animals and animals that die intercurrently were examined in the same way, if possible (with the exception of the uterus weight and organ weights).
- Necropsy of exsanguinated animals and assessement by gross pathology, special attention being given to the reproductive organs.
- Organ weights:
1. Adrenal glands
2. Kidneys
3. Liver
4. Spleen

Ovaries and uterine content:

On GD 20, the surviving dams were anesthetized with isoflurane, sacrificed by decapitation. Moribund animals and animals that die intercurrently were examined in the same way, if possible (with the exception of the uterus weight and organ weights).
Examinations:
- Weight of the unopened uterus

Fetal examinations:

On GD 20, the surviving dams were anesthetized with isoflurane, sacrificed by decapitation. Moribund animals and animals that die intercurrently were examined in the same way, if possible (with the exception of the uterus weight and organ weights).
Examinations:
- Early resorptions according to SALEWSKI E (1964) in animals that do not appear to be pregnant

Statistics:

KRUSKAL-WALLIS and WILCOXON test: Carcass weights and absolute and relative organ weights; Clinical pathology parameters; Weight of the unopened uterus
DUNNETT's test: Water consumption, food consumption, body weight, body weight change, corrected body weight gain, carcass weight

Details on results:

CLINICAL SIGNS
1000 mg/kg bw/d:
Piloerection (2/7); Salivation (7/7); Plough nose-first into bedding (7/7)
500 mg/kg bw/d: Piloerection (1/7); Salivation (7/7); Plough nose-first into bedding (7/7)

BODY WEIGHTS
1000 mg/kg bw/d: Body weight loss (d 6-8). Statistically significant decrease in body weight change for d 6-10 and overall dosing period d6-19 (-20% vs.ctrl.). Decrease in corrected body weight gain (-30% vs. ctrl.).
500 mg/kg bw/d: Statistically significant decrease in body weight change for d 8-10. Decrease for the overall dosing period d6-19 (-13% vs.ctrl.; not statistically significant). Decrease in corrected body weight gain (-14% vs. ctrl.).

FOOD CONSUMPTION
1000 mg/kg bw/d: Statistically significant decrease (-16% vs ctrl.) for test substance administration period (d 6-19)
500 mg/kg bw/d: Statistically significant decrease (-12% vs ctrl.) for test substance administration period (d 6-19)

WATER CONSUMPTION
1000 mg/kg bw/d: Significant increase (approx. 50% vs ctrl.) for test substance administration period (d 6-19)

ORGAN WEIGHTS:
1000 mg/kg bw/d: Decrease in uterus weight (-20% vs ctrl.)
500 mg/kg bw/d: Decrease in uterus weight (-15% vs ctrl.)

CLINICAL CHEMISTRY:
1000 mg/kg bw/d:
Statistically significant decrease of erythrocytes, eosinophils, mean corpuscular hemoglobin concentration, creatinine, total bilirubin
Statistically significant increase of mean corpuscular volume, mean corpuscular hemoglobin, reticulocytes, alanine aminotransferase, alkaline phosphatase, cholesterol

500 mg/kg bw/d:
Statistically significant decrease of erythrocytes, mean corpuscular hemoglobin concentration, total bilirubin
Statistically significant increase of mean corpuscular volume, reticulocytes, alkaline phosphatase, cholesterol

ORGAN WEIGHTS:
1000 mg/kg bw/d: Statistically significant increase of absolute/relative liver (+32%/39% vs ctrl.) and kidney (+21%/29% vs ctrl.) weights
500 mg/kg bw/d: Statistically significant increase of absolute/relative liver (+27%/33% vs ctrl.) and kidney (+10%/15% vs ctrl.) weights

GROSS PATHOLOGY:
1000 mg/kg bw/d: Enlarged livers in 6 of 7 animals

Based on the results of this range-finding study, dose levels of 50, 250 and 800 mg/kg bw/d have been selected for the main developmental toxicity study acc. to OECD 414 (BASF 2019; 30R0624/07R071).

Executive summary:

In the maternal toxicity dose range-finding study dose levels of 500 and 1000 mg/kg bw/d were tested (BASF 2019; 10R0624/07R067). The test substance was administered to pregnant female Wistar rats on GD 6 through GD 19. In this study clinical signs like transient salivation and animals ploughing their nose into bedding indicated a bad taste and/or a slightly irritating potential of the test item in the upper gastrointestinal tract. Piloerection was noted in 1/2 animal(s) at 500/1000 mg/kg bw/d.

At 1000 mg/kg bw/d an increase of water consumption (48% above control), a decrease of food consumption (16% below control), decrease of gross (20% below control) and corrected (30% below control) body weight gain was noted during treatment (GD 6-19). These animals transiently lost weight after initiation of treatment (GD 6-8). A dose of 500 mg/kg bw/d still caused a decrease of food consumption (12% below control) and a decrease of gross (13% below control) and corrected (14% below control) body weight gain.

In addition, at 500 and 1000 mg/kg bw/d the dams were affected by a regenerative hypochromic, macrocytic anemia, as was shown by decreased red blood cell (RBC) counts and mean corpuscular hemoglobin concentration (MCHC) as well as increased absolute reticulocyte counts and mean corpuscular volume (MCV). The anemia was accompanied by dose-dependently increased absolute/relative liver and kidney weights along with corresponding changes of clinical chemistry parameters. At 1000 mg/kg bw/d livers appeared already macroscopically enlarged.

Thus, dose levels of 50, 250 and 800 mg/kg bw/d were considered appropriate for the present definitive study, as the degree of the adverse effects should neither be potentially lethal nor should it cause severe suffering. Thus, the selected high dose for the present study meets the principles of guidelines OECD 414 (adopted 2001) and OPPTS 870.3700 (US EPA), as well as ECHA practical guide 10 (“how to avoid unnecessary testing on animals”; chapter 4 “animal welfare”; ECHA-10-B-17-EN, 2010) which is in in compliance with EU Di-rective 86/609/EEC on animal protection.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetrahydro-4-methyl-2-(2-methylprop-1-enyl)pyran

EC Number:

240-457-5

EC Name:

Tetrahydro-4-methyl-2-(2-methylprop-1-enyl)pyran

Cas Number:

16409-43-1

Molecular formula:

C10H18O

IUPAC Name:

tetrahydro-4-methyl-2-(2-methylprop-1-enyl)pyran

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-12 weeks
- Weight at study initiation: 166.4 – 224.6 g (pregnant animals on day 0)
- Fasting period before study: No
- Housing: individually in Polycarbonate cages type III
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: GD0 - GD6

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature. For the test substance preparations, the specific amount of test substance was weighed, topped up with corn oil in a graduated flask and intensely mixed with a magnetic stirrer until it was completely dissolved. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 1.25, 6.25 and 20 g/100 ml for low, mid and high dose group, respectively
- 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:

Analytical verifications of the stability of the test substance in corn oil over a period of 7 days at room temperature had been verified prior to the start of the study in a similar batch.
Given that the test substance is completely miscible with corn oil, solutions were considered to be homogenous without further analysis.
Samples of preparations were analyzed twice (at the beginning of administration and during administration ) for verification of the test substance concentrations. The results of the analysis confirmed the correctness of the prepared concentrations. The single values of the measured concentrations corresponded to the expected values within the limits of the analytical method, i.e. were above 90% and below 110% of the nominal concentrations.

Details on mating procedure:

The animals were paired by the breeder (“time-mated”); the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory. The following day was designated as “GD 1”.

Duration of treatment / exposure:

GD 6-19

Frequency of treatment:

daily

Duration of test:

GD 0-20

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

800 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25 time-mated females (1 animal of the low dose and 1 animal of the high dose group were not pregnant)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:

In the maternal toxicity dose range-finding study dose levels of 500 and 1000 mg/kg bw/d were tested (BASF 2019; 10R0624/07R067). The test substance was administered to pregnant female Wistar rats on GD 6 through GD 19. In this study clinical signs like transient salivation and animals ploughing their nose into bedding indicated a bad taste and/or a slightly irritating potential of the test item in the upper gastrointestinal tract. Piloerection was noted in 1/2 animal(s) at 500/1000 mg/kg bw/d.
At 1000 mg/kg bw/d an increase of water consumption (48% above control), a decrease of food consumption (16% below control), decrease of gross (20% below control) and corrected (30% below control) body weight gain was noted during treatment (GD 6-19). These animals transiently lost weight after initiation of treatment (GD 6-8). A dose of 500 mg/kg bw/d still caused a decrease of food consumption (12% below control) and a decrease of gross (13% below control) and corrected (14% below control) body weight gain.
In addition, at 500 and 1000 mg/kg bw/d the dams were affected by a regenerative hypochromic, macrocytic anemia, as was shown by decreased red blood cell (RBC) counts and mean corpuscular hemoglobin concentration (MCHC) as well as increased absolute reticulocyte counts and mean corpuscular volume (MCV). The anemia was accompanied by dose-dependently increased absolute/relative liver and kidney weights along with corresponding changes of clinical chemistry parameters. At 1000 mg/kg bw/d livers appeared already macroscopically enlarged.
Thus, dose levels of 50, 250 and 800 mg/kg bw/d were considered appropriate for the present definitive study, as the degree of the adverse effects should neither be potentially lethal nor should it cause severe suffering. Thus, the selected high dose for the present study meets the principles of guidelines OECD 414 (adopted 2001) and OPPTS 870.3700 (US EPA), as well as ECHA practical guide 10 (“how to avoid unnecessary testing on animals”; chapter 4 “animal welfare”; ECHA-10-B-17-EN, 2010) which is in in compliance with EU Di-rective 86/609/EEC on animal protection.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
A check for mortality was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20). A cage-side examination was conducted at least once daily before and after treatment period (GD 0-5 and 20). During treatment period (GD 6-19) all rats were checked daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity before administration as well as within 2 hours and within 5 hours after administration.

DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. The body weight change of the animals was calculated based on the obtained results. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13 15, 15-17, 17-19 and 19-20.


WATER CONSUMPTION: Yes
- Time schedule for examinations: The consumption of water was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13 15, 15-17, 17-19 and 19-20.

POST-MORTEM EXAMINATIONS: Yes
Sacrifice on gestation day 20;
- Necropsy: sacrificed by decapitation under isoflurane anesthesia in a randomized sequence. The ex-sanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.
- Organ weights: Kidneys, Liver, Spleen, Thyroid glands (with parathyroid glands) (fixed).
- Histopathology: Fixation in 4% neutral buffered formaldehyde solution. Organs evaluated: Liver, Thyroid glands

OTHER: Clinical pathology
- HAEMATOLOGY: Yes
Blood was taken from the retro-bulbar venous plexus from animals anaesthetized using isoflurane.
The following parameters were determined in blood with EDTA K3 as anticoagulant:
- Leukocyte count
- Erythrocyte count
- Hemoglobin
- Hematocrit
- Mean corpuscular volume
- Mean corpuscular hemoglobin
- Mean corpuscular hemoglobin concentration
- Platelet count
- Differential blood count
- Reticulocytes

- CLINICAL CHEMISTRY: Yes
Blood was taken from the retro-bulbar venous plexus from animals anaesthetized using isoflurane.
The following parameters were determined:
- Alanine aminotransferase
- Aspartate aminotransferase
- Alkaline phosphatase
- gamma-Glutamyltransferase
- Inorganic phosphate
- Calcium
- Urea
- Creatinine
- Glucose
- Total bilirubin
- Total protein
- Albumin
- Globulins
- Triglycerides
- Cholesterol
- TSH, T3, T4
Thyroid hormones: The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter. T3 and T4 was determined via an Elisa.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Number of dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)

Fetal examinations:

- External examinations: Yes: [all per litter]
Each fetus was weighed and sexed. The sex was determined by observing the distance between the anus and the base of the genitalia. Viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded. External tissues and all orifices were examined macroscopically. The anogenital distance (defined as the distance from the center of the anal opening to the base of the genital tubercle) measurements were conducted, using a measuring ocular, on all liveborn fetuses.

- Soft tissue examinations: Yes: [half per litter]
The fetuses fixed in Harrison’s fluid were examined for any visceral findings according to the method of BARROW and TAYLOR.

- Skeletal examinations: Yes: [half per litter]
The skeletons of the fetuses fixed in ethanol were stained according to a modified method of KIMMEL and TRAMMELL. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope.

Statistics:

DUNNETT-test: Water consumption, food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetus-es, proportions of preimplanta-tion loss, pro¬portions of postim-plantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight, anogenital dis-tance, anogenital index
FISHER´S EXACT-test: Female mortality, females preg-nant at terminal sacrifice, number of litters with fetal findings
WILCOXON-test: Proportions of fetuses with mal-formations, variations and/or unclassified observations in each litter, Blood parameters (unidirectional)
KRUSKAL-WALLIS test + WILCOXON-test (post-test): Blood parameters (bidirectional), organ weight parameters

Indices:

Conception rate (in %) : number of pregnant animals / number of fertilized animals x 100
Pre-implantation loss (in %): number of corpora lutea – number of implantations / number of corpora lutea x 100
Post-implantation loss (in %): number of implantations – number of live fetuses / number of implantations x 100
Anogenital index: anogenital distance [mm] / cubic root of fetal weight [g]

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test group 800 mg/kg bw/d:
- Salivation (<2h after treatment) in 25/25 animals starting at GD6
- Ploughing nose-first into bedding in 25/25 animals starting at GD6
- Unsteady gait (<2h after treatment) in 19/25 animals
- Piloerection in 2/25 animals (<2h after treatment; GD18-19) and 25/25 animals (>2h-<5h after treatment; GD7-19)

Test group 250 mg/kg bw/d:
- Salivation (<2h after treatment) in 25/25 animals starting at GD6
- Ploughing nose-first into bedding in 25/25 animals starting at GD6

Test group 50 mg/kg bw/d:
- Salivation (<2h after treatment) in 8/25 animals starting at GD8
- Ploughing nose-first into bedding in 12/25 animals starting at GD11

Salivation and ploughing nose-first into bedding are considered to be treatment-related, most likely as a local irritation of the upper digestive tract or as a result of the bad taste of the test substance/vehicle preparation. Unsteady gait and piloerection are considered as treatment-related signs of maternal toxicity.
No clinical signs or changes of general behavior were detected in any female at all dose levels before the daily treatment (GD 6-19).

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related or spontaneous mortalities in any females of all test groups (0, 50, 250 or 800 mg/kg bw/d).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test group 800 mg/kg bw/d:
Body weights: lower from GD 8 until GD 20 (final weight - 5% vs. ctrls), without statistical significance.
Body weight gain: Distinctly and statistically significantly reduced during GD 6-8 (body weight loss). Inconsistent course GD 8-20, i.e. decreases and increases versus controls. Phases of lower weight gain prevailed as the high-dose dams gained overall 16% less weight (statistically significant) than the controls during the treatment period (GD 6-19).
Mean body weight gain Days 6 to 8: -4.6** g vs. 6.1 g in controls.
Mean body weight gain Days 6 to 19: 71.0** g vs. 84.7 g in controls.
**p<=0.01
Corrected body weight gain: distinctly and statistically significantly lower (about -33% vs ctrls.).
Carcass weight: reduced without attaining statistical significance (about -4% vs. ctrls).

Test group 250 mg/kg bw/d:
Body weigths, body weight gain, corrected body weight gain, carcass weight: unaffected by the treatment

Test group 50 mg/kg bw/d:
Body weigths, body weight gain, corrected body weight gain, carcass weight: unaffected by the treatment

Effects in the high dose group are assessed as direct, test substance-related signs of maternal toxicity.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test group 800 mg/kg bw/d:
Statistically significantly reduced during GD 6-13 (up to -44% vs. ctrls) but recovered afterwards and was comparable to controls throughout the remaining study period. During the treatment period (GD 6-19), the food consumption overall 14% less than controls.

Test group 250 mg/kg bw/d:
Generally comparable to the concurrent control group throughout the entire study period.

Test group 50 mg/kg bw/d:
Generally comparable to the concurrent control group throughout the entire study period.

Reduction in food consumption in the high dose group is assessed as treatment-related and adverse.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Test group 800 mg/kg bw/d:
Statistically significantly increased from GD 8 to GD 20 (up to +78% vs ctrl.). During the treatment period (GD 6-19) consumption of water 47% more water than controls.

Test group 250 mg/kg bw/d:
Increased (from GD 10 onwards), statistical signifiicant on GD 17-19 (+20% vs control). During the treatment period (GD 6-19), consumption of water about 13% more than controls.

Test group 50 mg/kg bw/d:
Comparable to the concurrent control group throughout the entire study period.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Test group 800 mg/kg bw/d:
- Red blood cell (RBC) counts significantly decreased (-11.7% vs. ctrl.)
- Mean corpuscular hemoglobin concentration (MCHC) significantly decreased (-4.4% vs ctrl.)
- Mean corpuscular volume (MCV) significantly increased (+15.5% vs. ctrl.)
- Mean corpuscular hemoglobin content (MCH) significantly increased (+10.4% vs. ctrl.)
- Absolute reticulocyte counts significantly increased (+64.7% vs. ctrl.)
These changes were regarded as treatment-related and adverse.

- relative eosinophil cell counts significantly decreased (-36.4% vs. ctrl.)
- absolute eosinophil cell counts decreased (-20.4% vs. ctrl.)
This was the only relevant differential cell fraction alteration. Therefore, this change was regarded as treatment-related but non-adverse (ECETOC Technical Report No. 85, 2002).

Test group 250 mg/kg bw/d:
- Red blood cell (RBC) counts significantly decreased (-3.5% vs. ctrl.)
- Mean corpuscular hemoglobin concentration (MCHC) significantly decreased (-0.8% vs. ctrl.)
- Mean corpuscular volume (MCV) significantly increased (+3.0% vs. ctrl.)
- Mean corpuscular hemoglobin content (MCH) significantly increased (+2.3% vs. ctrl.)
Values were within historical control ranges. Thus, these alterations are likely precursory to a more distinct anemia, but still adaptive changes which are not yet adverse.

Test group 50 mg/kg bw/d:
No treatment related, adverse effects observed.


Statistically significant WBC and differential blood cell count changes were observed but regarded as incidental and not treatment-related. These were either within historical control ranges or the corresponding absolute cell fractions were within these ranges:
- Increase in total white blood cell (WBC) counts, absolute lymphocyte and monocyte counts (mid, high dose group)
- Increase in absolute basophil cell counts as well as absolute and relative large unstained cell (LUC) counts, relative lymphocyte counts (high dose group)
- Decrease in relative neutrophil cell counts (high dose group)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical chemistry
Test group 800 mg/kg bw/d:
Inorganic phosphate significantly decreased (-35.9% vs. ctrl.)
Calcium significantly decreased (-4.1% vs. ctrl.)
These changes were regarded as treatment-related and adverse.

Test group 250 mg/kg bw/d:
Inorganic phosphate significantly decreased (-17.2% vs. ctrl.)
This was the only relevantly changed clinical chemistry parameter. Therefore, this change was regarded as treatment-related but non-adverse (ECETOC Technical Report No 85, 2002).

Test group 50 mg/kg bw/d:
No treatment related, adverse effects observed.

The following statistically significant clinical chemistry parameter changes were regarded as incidental and not treatment-related. The were not dose-dependent, were within historical control ranges or corresponding parameters (i.e. , albumin and globulins for total protein) were within these ranges.
- Increase in alanine aminotransferase (ALT) activities (mid and high dose group)
- Decrease in total bilirubin (mid and high dose group)
- Increase in cholesterol (high dose group)
- Decrease in creatinine, total protein, albumin and globulin levels (high dose group)
- Increase in triglycerides (mid dose group)


Thyroid hormones

Test group 800 mg/kg bw/d:
T3 significantly decreased (-14.3% vs. ctrl.)
T4 significantly decreased (-8.4% vs. ctrl.), within the historical control range
TSH significantly increased (+27.6% vs. ctrl.)

Test group 250 mg/kg bw/d:
T3 significantly decreased (-10.3% vs. ctrl.)
T4 significantly decreased (-8.5% vs. ctrl.), within the historical control range
TSH significantly increased (+22.6% vs. ctrl.)

Test group 50 mg/kg bw/d:
T3 significantly decreased (-9.1% vs. ctrl.), within the historical control range

The significantly changed hormone levels in mid and high dose groups (250 and 800 mg/kg bw/d) were regarded as treatment-related and adverse, whereas the only decreased T3 level in dams of the low dose group (50 mg/kg bw/d) was regarded as non-adverse (ECETOC Technical Report No. 85, 2002).

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Description (incidence and severity):

UTERUS WEIGHTS
Gravid uterus [mean g]: 60.9, 60.2, 61.0, 56.7 in controls, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.

Mean gravid uterus weight of high dose animals (800 mg/kg bw/d) was slightly reduced (about - 7% vs. ctrls.; not statistically significant). This was likely to be a subsequent effect of reduced mean fetal weights in this test group and not an independent effect.
No test substance related effects in of the low- and mid-dose animals (50 and 250 mg/kg bw/d).

PLACENTAL WEIGHTS
The mean placental weights of the low-, mid- and high-dose groups were comparable to the corresponding control group.

OTHER ORGAN WEIGHTS
- Liver: In the mid and high dose group, the significant absolute and relative weight increase was considered treatment-related.
- Kidneys: In the high dose group, significant absolute and relative weight increases were above the historical control ranges and therefore regarded as possibly treatment-related. In the mid dose group, the significant absolute weight increase were also above the historical control ranges, whereas the significant relative weight increase was within the historical control values. Therefore, the absolute weight increase of kidneys in mid dose animals was regarded as incidental.
- Spleen: In the high dose group, significant absolute and relative weight increases were above the historical control ranges and therefore regarded as possibly treatment-related.

All other mean absolute and relative weight parameters did not show significant differences when compared to the control group.

Absolute organ weights:
Kidneys [% vs. ctrls.]: 105, 108**, 119** in low, mid and high dose group (50, 250, 800 mg/kg bw/d), respectively.
Liver [% vs. ctrls.]: 100, 120**, 133** in low, mid and high dose group (50, 250, 800 mg/kg bw/d), respectively.
Spleen [% vs. ctrls.]: 104, 103, 117** in low, mid and high dose group (50, 250, 800 mg/kg bw/d), respectively.
**p<=0.01

Relative organ weights:
Kidneys [% vs. ctrls.]: 103, 109**, 124** in low, mid and high dose group (50, 250, 800 mg/kg bw/d), respectively.
Liver [% vs. ctrls.]: 104, 120**, 138** in low, mid and high dose group (50, 250, 800 mg/kg bw/d), respectively.
Spleen [% vs. ctrls.]: 103, 104, 122** in low, mid and high dose group (50, 250, 800 mg/kg bw/d), respectively.
**p<=0.01

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic changes occurred individually and were considered to be incidental in nature and not related to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in the liver and thyroid gland:

- Thyroid: Minimal (grade 1) thyroid hypertrophy/hyperplasia in 0/25, 0/24, 0/25 and 2/24 animals of controls, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively. These findings were considered treatment related.

- Liver: Moderate (grade 3) increase of cytoplasmic vacuolation of hepatocytes in 1/25, 1/24, 17/25 and 17/24 animals of controls, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively. The finding is characterized by coarse and irregular defined vacuoles of different sizes with a cloudy content, which is a typical pattern of glycogen accumulation.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

No test substance related and/or biologically relevant differences between the different test groups in the number aborted
Females Aborted [n]: 0, 0, 0, 0 in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance related and/or biologically relevant differences between the different test groups in implantation sites and the values calculated for the pre- and post-implantation losses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.
The only notable finding was a 100% post-implantation loss in one high-dose female (800 mg/kg bw/d), where 2 early resorptions were recorded after staining the empty uterus at C-section (i.e. pregnant by stain). In the other high-dose dams post-implantation losses and number of early resorptions were comparable to the control. Thus, this isolated finding was regarded as incidental and not treatment-related.

Preimplantation loss [mean %+/- SD]: 5.9 (+/-14.5), 3.2 (+/-4.9), 3.1 (+/-5.9), 7.3 (+/-18.3) in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.
Postimplantation loss [mean %+/- SD]: 5.8 (+/-6.2), 9.2 (+/-7.9), 5.6 (+/-6.8), 10.3 (+/-22.2) in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

The only notable finding was a 100% post-implantation loss in one high-dose female (800 mg/kg bw/d), where 2 early resorptions were recorded after staining the empty uterus at C-section (i.e. pregnant by stain). In the other high-dose dams post-implantation losses and number of early resorptions were comparable to the control. Thus, this isolated finding was regarded as incidental and not treatment-related.

Dams with all resorptions [n]: 0, 0, 0, 1 in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance related and/or biologically relevant differences between the different test groups in the number of resorptions. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.
The only notable finding was a 100% post-implantation loss in one high-dose female (800 mg/kg bw/d), where 2 early resorptions were recorded after staining the empty uterus at C-section (i.e. pregnant by stain). In the other high-dose dams post-implantation losses and number of early resorptions were comparable to the control. Thus, this isolated finding was regarded as incidental and not treatment-related.

Early resorptions [mean % +/- SD]: 5.8 (+/-6.2), 8.8 (+/-7.8), 5.3 (+/-6.6), 9.0 (+/-21.3) in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.
Late resorptions [mean % +/- SD]: 0.0 (+/-0.0), 0.3 (+/-1.6), 0.3 (+/-1.3), 1.3 (+/-6.3) in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.

Dead fetuses:

no effects observed

Description (incidence and severity):

No test substance related and/or biologically relevant differences between the different test groups in viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.

Dead fetuses [n]: 0, 0, 0, 0 in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

No test substance related and/or biologically relevant differences between the different test groups in conception rate. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.

Females pregnant [n]: 25, 24, 25, 24 in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.

Other effects:

no effects observed

Description (incidence and severity):

No test substance related and/or biologically relevant differences between the different test groups in the mean number of corpora lutea. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.

Corpora lutea [mean]: 12.2, 12.3, 12.2, 12.5 in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

haematology

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean fetal weights of mid and high dose group animals (250 and 800 mg/kg bw/d) were statistically significantly reduced (about 5% and 11% below control - both sexes combined). No biologically relevant differences in the low dose group in comparison to the control group.

Fetal weights of all viable fetuses (on a litter basis) [g]: 3.7, 3.6, 3.5**, 3.3** in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.
**p<=0.01

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses low, mid and high dose groups (50, 250 and 800 mg/kg bw/d) was comparable to the control fetuses.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

MALFORMATIONS
The overall incidences of external malformations were comparable to those found in the historical control data. One fetus of the low dose group had an external malformation (anasarca) associated with multiple skeletal malformations. This is considered to be an incidental finding.
Affected fetuses/litter [mean%]: 0.0, 0.4, 0.0, 0.0 in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.

VARIATIONS
No external variations recorded.

UNCLASSIFIED OBSERVATIONS
Two unclassified external observations were recorded (blood coagulum around placenta in one control litter; placentae fused in one high-dose litter. These findings were not considered biologically relevant, since they were single events.
Affected fetuses/litter [mean%]: 0.4, 0.0, 0.0, 0.3 in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

MALFORMATIONS
The total incidences of skeletal malformations in treated animals did not differ significantly from the control group and were comparable to the historical control data.
Skeletal malformations were detected in single fetuses of all treated groups. One female low-dose fetushad multiple skeletal malformations affecting the sternum, skull, thoracic vertebral column and ribs (plus an additional external malformation), all of which are considered to be spontaneous in origin and not treatment-related.
The malformations in the mid- and high-dose fetuses are not assessed as treatment-related since they were single events in single fetuses. All of them can be found in the historical control data at comparable incidences.
Affected fetuses/litter [mean%]: 0.0, 0.7, 1.7, 2.0 in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.

VARIATIONS
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared in the majority of cases without a relation to dosing. The overall affected fetuses/litter incidences of skeletal variations were statistically significantly increased in the mid- and high-dose groups. However, the incidences in mid and high dose animales were well within the historical control range, whereas the incidences of the control and the low-dose groups were below (HCD: mean% 97.8 [94.0 - 100.0]).
Affected fetuses/litter [mean%]: 92.0, 93.8, 98.7**, 100.0** in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.

Concerning specific skeletal variations, the affected fetuses/litter rates of ‘incomplete ossification of skull; unchanged cartilage’ and ‘unossified sternebra; unchanged cartilage’ were statistically significantly increased and outside the historical control range in mid dose and and high dose animals, whereas the rate in the low dose group was well within the historical control range, respectively. Furthermore, the rate of ‘incomplete ossification of sacral arch; cartilage present’ was increased and outside the historical control range in high dose animals. These findings are most likely associated with the treatment. Other findings were either not related to the dose or were covered by the historical control range. They are in any case not considered as adverse events.
In addition to these findings, the litter incidence for ‘supernumerary rib (14th); cartilage not present’ was statistically significantly increased in low dose animals (100%). However, the increase was not dose-related and the value was within the range of the historical control data (mean 90.9%, range of 80.0 - 100.0%). Therefore, it is not considered to be treatment-related.

Findings outside the historical control range:
Incomplete ossification of skull; unchanged cartilage [mean% affected fetuses/litter]: 5.5, 10.2*, 24.6**, 35.8** in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.
Unossified sternebra; unchanged cartilage [mean% affected fetuses/litter]: 1.2, 5.3*, 12.0**, 23.0** in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.
Incomplete ossification of sacral arch; cartilage present [mean% affected fetuses/litter]: 0.5, 0.7, 2.7, 8.8** in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.

Findings within the historical control range:
Incomplete ossification of parietal; unchanged cartilage [mean% affected fetuses/litter]: 5.3, 6.4, 12.2*, 4.5 in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.
Incomplete ossification of supraoccipital; unchanged cartilage [mean% affected fetuses/litter]: 7.8,20.9*, 21.3**, 33.0** in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.
Incomplete ossification of temporal [mean% affected fetuses/litter]: 0.0, 0.0, 0.0, 2.0* in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.
Incomplete ossification of sternebra; unchanged cartilage [mean% affected fetuses/litter]: 58.3, 72.4**, 87.1**, 89.2** in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.
Misshapen sternebra; unchanged cartilage [mean% affected fetuses/litter]: 26.2, 25.6, 27.5, 43.6* in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.
Wavy rib [mean% affected fetuses/litter]: 1.8, 2.7, 7.4*, 12.8** in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.
Incomplete ossification of pubis; cartilage present [mean% affected fetuses/litter]: 0.0, 0.0, 0.0, 2.3* in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.
**p <=0.05; **p<=0.01

UNCLASSIFIED OBSERVATIONS
Some isolated cartilage findings without impact on the respective bony structures occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the ribs and the sternum and did not show any relation to the dose.
Affected fetuses/litter [mean%]: 64.4, 66.4, 57.5, 51.6 in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

MALFORMATIONS
Two soft tissue malformations were recorded (abnormal lung lobation in one control fetus; hydronephrosis in one high-dose fetus). These findings can be found in the historical control data at comparable incidences and were single events in single fetuses. No cluster of any of these individual malformations were seen in the other offspring of this or the other treated groups. Thus, no association to the treatment was assumed.
Affected fetuses/litter [mean%]: 0.8, 0.0, 0.0, 0.6 in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.

VARIATIONS
Four soft tissue variations were detected( supernumerary branch of subclavian artery and short innominate in high dose group; dilated renal pelvis and dilated ureter in all test groups).
The incidences of these variations were neither statistically significantly nor dose-dependently increased in the treated groups. Except for ‘supernumerary branch of subclavian artery’, all of them can be found in the historical control data at comparable incidences. Therefore, they were not assessed as treatment-related.
Affected fetuses/litter [mean%]: 4.6, 3.5, 1.5, 9.7 in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.

UNCLASSIFIED OBSERVATIONS
One unclassified soft tissue observation (yellowish discolored liver) was recorded in 5/12 fetuses of 1 mid dose litter and 6/12 and 3/12 fetuses of 2 high dose litters without attaining statistical significance. The anatomy of the affected livers was otherwise normal. The cause and toxicological significance of this discoloration is not known.
Affected fetuses/litter [mean%]: 0.0, 0.0, 3.3, 7.0 in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Anogenital distance/anogenital index
Male fetuses: The anogenital distance and anogenital index of all treated groups was comparable to the concurrent control values.
Female fetuses: The anogenital index of the high-dose female fetuses was marginally, but statistically significantly above the concurrent control value , while the anogenital distance was not affected. This is solely due to the lower body weight of these fetuses and neither is it an independent effect on its own, nor does it constitute a sign of female virilization.
The anogenital distance of all female fetuses of all treated groups and the anogenital index of all female fetuses in low and mid dose groups was comparable to the concurrent control values.

AG distance - males [mean, mm]: 3.0, 3.0, 3.0, 2.9 in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.
AG distance - females [mean, mm]: 1.5, 1.5, 1.5, 1.5 in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.
AG index - males [mean]: 1.93, 1.92, 1.95, 1.96 in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.
AG index - females [mean]: 1.00, 0.99, 1.01, 1.04** in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.
**<=0.01

Details on embryotoxic / teratogenic effects:

Overall assessment of malformations
There were external, soft tissue and skeletal malformations in 1 fetus, each, of the control and the low-dose groups and in 3 fetuses, each, of the mid- and high-dose groups.
Fetuses, carrying more than one malformation:
- 1 high-dose male fetus: severely malformed skull bones (some skull bones misshapen, small or with a hole).
- 2 mid-dose male fetuses (same litter): skeletal malformations concerning the upper limbs (i.e. shortened scapula, shortened humerus).
- 1 low dose female fetus: anasarca combined with multiple skeletal malformations (for example malpositioned and bipartite sternebra, misshapen basisphenoid, absent thoracic vertebra, branched rib).
Further malformations, i.e. abnormal lung lobation, hydronephrosis, misshapen basisphenoid, and bent rib, were observed in individual fetuses, unrelated to dose and all of them can be found in the historical control data.
All these findings were single cases, no ontogenetic pattern is recognizable for all these individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of these test groups. They also do neither form a pattern or syndrome with other minor anomalies which may raise toxicological concern nor do they influence the overall rate of malformations in this study. There is no evidence for any association of these scattered findings with the treatment.

Overall assessment of variations
External variations did not occur in any fetus in this study. Four soft tissue variations and a range of skeletal variations were noted in all test groups including the controls.
If all different types of variations were summarized, the incidence in the high dose group was statistically significantly increased but within the historical control range. This slight increase was mainly driven by skeletal variations.
The affected fetuses/litter rates of ‘incomplete ossification of skull; unchanged cartilage’ and ‘unossified sternebra; unchanged cartilage’ were statistically significantly increased and outside the historical control range in mid and high dose animals (250 and 800 mg/kg bw/d), whereas the rate in the low dose group was well within the historical control range, respectively. Furthermore, the rate of ‘incomplete ossification of sacral arch; cartilage present’ was increased and outside the historical control range in the high dose group. These findings are most likely associated with the treatment.
Incomplete ossifications represent slight delays of ossification which do not constitute structural alterations of the skeleton, as the underlying cartilage template was completely intact in all these cases. Further, the delayed ossification noted in the mid- and high-dose groups was associated with maternal toxicity and decreased fetal weights. As can be seen from the historical background data, increased incidences of such incomplete or non-ossifications of the affected skeletal elements are routinely quantified and are among the most frequently noted skeletal variants in control populations of this Crl:WI(Han) rat strain. This indicates that these findings reflect species-specific anatomic variation at the time around birth without any detrimental effects on further development. Thus, their toxicological relevance is considered to be rather low.
Concerning all other statistically significant findings, no dose dependency was observed and/or all values were clearly inside the historical control range, thus, an association to the test substance and a toxicological relevance is not assumed.

Overall assessment of unclassified observations
A spontaneous origin is assumed for the unclassified external and skeletal cartilage observations, which were observed in several fetuses of all test groups. The distribution and type of these findings do not suggest any relation to treatment.
One unclassified soft tissue observation, i.e. yellowish discolored liver, was recorded in 5/12 fetuses of 1 litter in mid dose and 6/12 and 3/12 fetuses of 2 litters in the high dose group (250 and 800 mg/kg bw/d), without attaining statistical significance. The anatomy of the affected livers was otherwise normal. The cause and toxicological significance of this discoloration is not known.


Total fetal malformations:
Affected fetuses/litter [mean%]: 0.4, 0.4, 0.9, 1.3 in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.

Total fetal variations:
Affected fetuses/litter [mean%]: 50.3, 51.0, 52.6, 57.0** in control, low, mid and high dose group (0, 50, 250, 800 mg/kg bw/d), respectively.
**p<=0.01

In conclustion, the findings observed suggest that the test item caused a slight delay of embryofetal development at dose levels of 250 and 800 mg/kg bw/d. These dose levels also caused maternal toxicity.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Overall developmental toxicity

Developmental effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Any other information on results incl. tables

The conception rate was 96% in the low- and high-dose groups (50 and 800 mg/kg bw/d) and 100% in the control and the mid-dose groups (0 and 250 mg/kg bw/d).With these rates, a sufficient number of pregnant females were available for the purpose of this study according to the respective test guidelines).

Applicant's summary and conclusion

Executive summary:

The test substance, tested for its prenatal developmental toxicity, was administered as an oily preparation to groups of 25 time-mated female Wistar rats by gavage at doses of 50, 250 and 800 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (corn oil) in parallel. A standard dose volume of 4 mL/kg body weight was used for each test group. At terminal sacrifice on GD 20, 24-25 females per group had implantation sites.

Water consumption, food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On GD 20, blood samples were obtained from all females by retrobulbar venous puncture following isoflurane anesthesia. After blood sampling all females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including sampling of thyroid glands (with parathyroid glands) and weight determinations of the kidneys, liver, spleen, thyroid glands (with parathyroid glands), unopened uterus and placentas). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Anogenital distance measurements were conducted on all liveborn fetuses. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings.

 

All females of the high- and mid-dose groups (800 and 250 mg/kg bw/d) and 8 females of the low-dose group (50 mg/kg bw/d) showed transient salivation during the treatment period. Furthermore, all females of the high- and mid-dose groups and 12 females of the low-dose group ploughed nose-first into bedding. Both findings occurred in most of the respective animals only within the 2-hour examination interval immediately after treatment, however in some mid- and high-dose dams it continued beyond 2 hours for a maximum of 5-hours. They are considered to be treatment-related, likely as a result of the bad taste of the test substance/vehicle preparation or due to local irritation of the upper digestive tract. They are not considered to be signs of systemic toxicity.

For 19/25 high-dose females (800 mg/kg bw/d) unsteady gait shortly after treatment (i.e. 0-2h) was recorded on GD 7-14, the highest number of animals being affected on GD 9 and 10. Furthermore, all females of the high-dose group had piloerection during the treatment period. Both findings are considered as treatment-related signs of systemic maternal toxicity.

Generally, clinical observations revealed no effects of treatment-related systemic maternal toxicity in the animals receiving 50 mg/kg bw/d of the test substance.

The mean food consumption of the high-dose dams (800 mg/kg bw/d) was decreased statistically significantly on GD 6-13 (: up to -44% vs. ctrls.). Afterwards it recovered, but during the treatment period (GD 6-19) the high-dose dams consumed 14% less food in comparison to the concurrent control group. The high-dose of the test substance consistently affected the gross and corrected (net) body weight gain (body weight loss GD6-8) of the dams, gaining overall about 16% (gross) or 33% (net) less weight than the concurrent control during the treatment period (GD 6-19). The carcass weight was about 4% below the concurrent control group. These effects were regarded to be treatment-related and adverse.

Regarding clinical pathology, in high dose dams (800 mg/kg bw/d) a regenerative hypochromic, macrocytic anemia was observed because of decreased red blood cell (RBC) counts and mean corpuscular hemoglobin concentration (MCHC) as well as increased absolute reticulocyte counts, mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH). Lower inorganic phosphate and calcium levels in these individuals was found, but the reason for these findings cannot be elucidated. In mid dose dams (250 mg/kg bw/d), RBC counts and MCHC were lower and MCV and MCH higher compared to controls. However, all values in this test group were within historical control ranges. Thus, these alterations are likely precursory to a more distinct anemia, but still adaptive changes which are not yet adverse.

In mid and high dose dams (250 and 800 mg/kg bw/d) decreased T3 and T4 values and increased TSH values indicated a hypothyroidism.

Regarding pathology, the significant absolute and relative weight increase of the liver in the mid and high dose animals (250 and 800 mg/kg bw/d) was consistent with an increased hepatocytic accumulation of, most likely, glycogen. Histopathologically, this accumulation was similarly present in both test groups and was regarded as treatment-related but not as adverse since neither signs of cytotoxicity nor changes in clinical chemistry parameters indicative of hepatic alteration were noted.

In the thyroid glands, histopathology revealed a minimal hypertrophy/hyperplasia of the follicular cells in 2/24 high-dose dams (800 mg/kg bw/d). The minimal incidence and grading of these findings were assumed as possibly treatment-related and in combination with the results of hormonal measurements (increased TSH and decreased T3 and T4) as potentially adverse.

In high dose dams, the absolute and relative kidneys and spleen weights were significantly increased above the historical control ranges. In a previous study with a 28-day exposure to the same test substance, significant absolute and relative kidneys weight increases occurred in females at 300 and 1000 mg/kg bw/d without a histopathological correlate, whereas significant relative spleen weight increases at 1000 mg/kg bw/d were consistent with extramedullary hematopoiesis and hematological signs of regenerative anemia. The data of the 28-day study and the absence of altered clinical chemical markers of kidney toxicity in the present study indicate that the kidney weight increases were treatment-related but not adverse. Although no histopathological investigation of the spleen was performed in the present study, the significant weight increases of the organ in the high dose group were assumed to be a treatment-related adaptive response, relating to the evident adverse hematological signs of regenerative anemia.

No differences of toxicological relevance between the control and the treated groups (50, 250 or 800 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and post-implantation loss. Similarly, no toxicologically relevant influence of the test substance on sex distribution and anogenital distance/index of the fetuses was noted at any dose.

The mean fetal weights of the mid- and high-dose groups (250 and 800 mg/kg bw/d) were statistically significantly reduced (about 5% and 11% below control). This correlated with mild delays in ossification of a few skeletal elements (skull, sternebrae, sacral arch). These delays of ossification do not constitute structural alterations of the skeleton, as the underlying cartilage template was completely intact in all these cases. Further, the delayed ossification noted in the mid- and high-dose group was associated with beginning (250 mg/kg bw/d) or distinct (800 mg/kg bw/d) maternal toxicity. As can be seen from the historical background data, increased incidences of such incomplete or non-ossifications of skeletal elements are routinely quantified and are among the most frequently noted skeletal variants in control populations of this Crl:WI(Han) rat strain. This indicates that these findings reflect species-specific anatomic variation at the time around birth without any detrimental effects on further development. Thus, their toxicological relevance is considered to be rather low.

Under the conditions of this study the test item is not teratogenic.

 

In conclusion, the oral administration of the test substance to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused evidence of distinct maternal toxicity (such as adverse clinical findings, reductions of food consumption and body weight/body weight gain, anemia, thyroid perturbation at 800 mg/kg bw/d) under the conditions of this prenatal developmental toxicity study, Beginning maternal toxicity was detectable already at 250 mg/kg bw/d where clinical findings, precursory signs of anemia and thyroid perturbation were noted. Each of these effects was not strong enough to constitute adverse events when looked at them individually, but as a whole provide evidence for disturbance of maternal homeostasis. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 50 mg/kg bw/d.

Further, the test item caused a slight delay of embryofetal development (lower fetal weights, mild delays in ossification) at dose levels of 250 and 800 mg/kg bw/d. These dose levels also caused maternal toxicity. In conclusion, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 50 mg/kg bw/d. However, test substance did not show any teratogenic potential under the conditions of this study.