Cyclopropanecarboxylic acid, [(3S,4R,4aR,6S,6aS,12R,12aS,12bS)-3-[(cyclopropylcarbonyl)oxy]-1,3,4,4a,5,6,6a,12,12a,12b-decahydro-6,12-dihydroxy-4,6a,12b-trimethyl-11-oxo-9-(3-pyridinyl)-2H,11H-naphtho[2,1-b]pyrano[3,4-e]pyran-4-yl]methyl ester

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Multiple studies were conducted. In the key two-generation rat study (2013/1347949) dose levels of 0, 100, 500 and 2000 ppm were tested.

The NOAEL for general, systemic toxicity is 500 ppm for the F0 and F1 parental rats, based on decreased food consumption and body weight/body weight gain observed at 2000 ppm in all F0 and F1 parental animals, as well as effects on hematology and clinical chemistry.

The NOAEL for fertility and reproductive performance for the parental rats is 500 ppm due to the reduction in implantation sites and pups delivered in the F1 parents of the 2000 ppm dose group.

The NOAEL for developmental toxicity in the F1 and F2 progeny is 100 ppm, due to the decrease in the pre-weaning pup body weights/pup weight gains observed at the 500 ppm dose. The NOAEL at 100 ppm was based on an effect only observed during the lactation portion of the study when the effective maternal dose is doubled. In addition, later pharmacokinetic studies in the rat indicated that doses exceeding 15 mg/kg bw/d follow non-linear kinetics, resulting in an even higher effective dose. In the context of risk assessment, the NOAEL of 100 ppm does not represent meaningful evidence of increased pup sensitivity.

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 22, 2012 to January 29, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Batch no.: COD-001545
Expiry: December 31, 2013
Appearance: Solid, yellowish

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The rat is the preferred animal species for reproduction studies according to test guidelines.
This strain was selected since extensive historical control data were available for Wistar rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (F0) 5 wks (36 ± 1 d)
- Weight at study initiation: (F0) Males: 112.5 g - 147.8 g; Females: 103.5 g - 125.2 g
- Housing: Individually except for overnight matings, pregnant animals and their litters were housed together
- Diet: Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: About 8 days
ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 30-70%
- Air changes: ≥15 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours per day, lights on at 06:00 and off at 18:00
IN-LIFE DATES: From: 22-May-2012 To: 12-Feb-2013

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Remarks:

Rodent diet

Details on exposure:

DIET PREPARATION
The required quantity of test substance for each dose group was weighed in a beaker and thoroughly mixed with a small amount of food. Then further amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations. Mixing was carried out for about 1 minute in a laboratory mixer.

Details on mating procedure:

In general, each of the male and female animals was mated overnight at a 1 : 1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group.
The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 7.00-9.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted "gestation day (GD) 0" and the following day "gestation day (GD) 1".

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 the diet for a period of 34 days at room temperature were carried out prior to the start of the study.
Homogeneity and concentration control analyses were carried out at the beginning of the premating phase. Concentration control analyses were carried out towards the end of the premating phase. Duplicate samples were kept in reserve and will be discarded after report finalization.
The stability of the substance in rat diet was demonstrated for a period of 34 days at room temperature.
The homogeneity of the mixture was verified at various time points during the study period. Concentration control analysis demonstrated that all values for the test material were in the expected range of the target concentration (90-110%) demonstrating the correctness of the diet preparations.

Duration of treatment / exposure:

After the acclimatization period, the test substance was administered to the parental animals as addition to the diet continuously throughout the entire study.

Frequency of treatment:

Daily

Details on study schedule:

Seventy-five males and 75 females selected after the quarantine/acclimatization period were assigned to two groups in such a way to equalize group means and standard deviations of body weights as closely as possible. These animals were designated as parental animals. After the acclimatization period, the test substance was administered to the parental animals as addition to the diet. The animals of the control group were treated in the same way, with the vehicle (diet only). Treatment ended about 16 hours before sacrifice. At least 75 days after the beginning of treatment, males and females from the same dose group were mated. The females were allowed to deliver and rear their pups (F1 generation pups) until PND 21. Pups were weaned on day 21 of lactation.

F0 generation parental animals and their progeny:
One hundred males and 100 females selected after the quarantine/acclimatizsation period were assigned to the different test groups in such a way to equalize group means and standard deviations of body weights as closely as possible. These animals were designated as parental animals. After the acclimatization period, the test substance was administered to the parental animals as addition to the diet. The animals of the control group were treated in the same way, with the vehicle (diet only). Treatment ended about 16 hours before sacrifice. At least 75 days after the beginning of treatment, males and females from the same dose group were mated. The females were allowed to deliver and rear their pups (F1 generation pups) until PND 4 (standardization) or PND 21.

F1 generation parental animals and their progeny:
After weaning, 25 male and 25 female F1 pups of each test group became F1 generation parental animals. These animals were chosen by lot and each litter was represented as far as technically feasible. If fewer than 25 litters were available in a group or if one sex was missing in a litter, more animals were taken from the other litters of the respective test group to obtain the required number of animals for pairing. All selected animals were treated with the test substance at the same dose level as their parents, from post-weaning through adulthood. At least 74 days after assignment of the F1 generation parental animals, the males and females were mated. The partners were randomly assigned; mating between siblings was avoided. The females were allowed to deliver and rear their pups (F2 generation pups) until PND 4 (standardization) or PND 21.

Dose / conc.:

100 ppm (nominal)

Dose / conc.:

500 ppm (nominal)

Dose / conc.:

2 000 ppm (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, plain diet

Details on study design:

Dose selection rationale: Dose selection was based on existing reproductive and repeated dose toxicity data.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied.
A cage side examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each animal and reported on a weekly basis.
The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams.
On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
In general, body weights of F0 and F1 parents were determined once weekly. However, during gestation and lactation F0/F1 females were weighed on gestation days (GD) 0, 7, 14 and 20 and on postnatal days (PND) 1, 4, 7, 14 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg bw/d: Yes
Food consumption of the F0 and F1 parents was determined regularly during the gestation (days 0 - 7, 7 - 14, 14 - 20) and lactation periods (days 1 - 4, 4 - 7, 7 - 14 and 14 - 21).

Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
The intake of test substance was calculated from the amount of food consumed and expressed in mg/kg body weight per day (mg/kg bw/d).
The calculation of the group values/day was carried out according to the following formula:
Test substance intake (mg/kg bw/d) = [mean food consumption (g/rat/d) x dietary level (ppm)] / mean body weight (g)

CLINICAL PATHOLOGY
Blood was withdrawn from fasted animals from the orbital sinus or (if applicable) after decapitation from the vena cava cranialis following isoflurane anesthesia. The following hematological and clinical chemistry parameters were determined in 12 animals/group/sex at the end of the study period:
Hematology: Leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular Hb concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes.
Clinical chemistry: Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (gamma-GT), inorganic phosphate (INP), calcium (CA), urea (UREA), creatinine (CREA), glucose (GLUC), total bilirubin (TBIL), total protein (TPROT), albumin (ALB), globulins (GLOB), triglycerides (TRIG), cholesterol (CHOL), troponin.

Oestrous cyclicity (parental animals):

Estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 and F1 female parental rats for a minimum of 3 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 and F1 female with scheduled sacrifice.

Sperm parameters (parental animals):

Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from all male animals.
The following sperm parameter were determined: Motility, morphology, head count (cauda epididymis/testis). Sperm head count were evaluated in the control and high dose group of the F0 generation and in all groups of the F1 generation.

Litter observations:

STANDARDISATION OF LITTERS
On PND 4, the individual litters were standardized in such a way that, where possible, each litter contained 4 male and 4 female pups (always the first 4 pups/sex and litter were taken for further rearing). If individual litters did not have 4 pups/sex, the litters were processed in such a way that the most evenly distributed 8 pups per litter were present for further rearing (e.g., 5 male and 3 female pups). Standardization of litters was not performed in litters with < 8 pups.

PUP DELIVERY STATUS
All pups delivered from the F0 parents (F1 litter) and the F1 parents (F2 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

SEX RATIO
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy. The sex ratio was calculated at PND 0 and PND 21 according to the following formula:
Sex ratio = (number of live male or female pups on PND 0 and 21 / number of live male and female pups on PND 0 and 21) x 100

CLINICAL OBSERVATIONS
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams and documented for each pup.

BODY WEIGHT
The pups were weighed on the day after birth (PND 1) and on PND 4 (before standardization), 7, 14 and 21. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning) and on PND 4 immediately before standardization of the litters.

SEXUAL MATURATION
Vaginal opening: All female F1 pups selected to become the F1 parental generation females (25/group) were evaluated daily for vaginal patency beginning on PND 27. On the day of vaginal opening the body weights of the respective animals were determined.
Preputial separation: All male F1 pups selected to become the F1 parental generation males (25/group) were evaluated daily for preputial separation beginning on PND 38. On the day of preputial separation the body weights of the respective animals were determined.
Comparison of sexual maturation with body weight development: While treatment-related delays may be indicative of specifically slowed sexual development, impaired general growth can also alter the onset of puberty. To differentiate between these specific and non-specific effects, an analysis was performed to graphically compare the ages and weights at puberty of the individual animals which were selected for further breeding with the average growth progression of all F1 and F2 control animals, using the change in body weight as a marker for general animal development.

Postmortem examinations (parental animals):

SACRIFICE
After weaning of F1 pups the F0 generation parental animals were sacrificed. The F1 generation parental animals were sacrificed, shortly after the F2 generation pups had been weaned. All F0 and F1 parental animals were sacrificed by decapitation under isoflurane anesthesia.

GROSS NECROPSY
Any gross pathological findings were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights: Adrenal glands, anesthetized animals, brain, cauda epididymis, epididymides, heart, kidneys, liver, ovaries, pituitary gland, prostate, testes, seminal vesicles including coagulation glands, spleen, thyroid glands (with parathyroid glands), uterus.
Preservation of organs: All gross lesions, adrenal glands, brain, cervix uteri, coagulating glands, heart, kidneys, left epididymis, left testis, liver, ovaries, oviducts, pituitary gland, prostate, seminal vesicles, spleen, thyroid glands (with parathyroid glands), uterus, vagina.
Histopathology: All gross lesions, adrenal glands, cervix uteri, coagulating glands, left testis, left epididymis, ovaries, oviducts, pituitary gland, prostate, seminal vesicles, uterus, vagina.
Differential Ovarian Follicle Count (DOFC) was performed in F1 generation.

Postmortem examinations (offspring):

SACRIFICE
Pups after standardization/weaning:
With the exception of those F1 generation pups, which were chosen as F1 rearing animals, all pups were sacrificed under isoflurane anesthesia with carbon dioxide after standardization or weaning.

GROSS NECROPSY
All pups with scheduled sacrifice (i.e. pups culled on PND 4 or sacrificed on PND 21), all stillborn pups and all pups that died before weaning were examined externally and eviscerated; their organs were assessed macroscopically.

ORGAN WEIGTHS
After the scheduled sacrifice the brain, spleen, heart and thymus of 1 pup/sex and litter from the pups were weighed. For the calculation of the relative organ weights, the in-life pup weights determined on PND 21 were used.

Statistics:

Food consumption, body weight and body weight change (for the pup weights, the litter means were used), gestation days, duration of sexual maturation (days to vaginal opening, days to preputial separation): Simultaneous comparison of all dose groups with the control group using the DUNNETT test (two-sided) for the hypothesis of equal means (* for p ≤ 0.05, ** for p ≤ 0.01).
Male and female mating indices, male and female fertility indices, gestation index, females mated, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups: Pair-wise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions (* for p ≤ 0.05, ** for p ≤ 0.01).
Mating days until day 0 post coitum, %postimplantation loss, pups stillborn, %perinatal Loss: Pair-wise comparison of the dose group with the control group using the WILCOXON test (one-sided+) with BONFERRONI-HOLM adjustment for the hypothesis of equal medians (* for p ≤0.05, ** for p ≤0.01).
Implantation sites, pups delivered, pups liveborn, live pups day x, viability index, lactation index: Pair-wise comparison of the dose group with the control group using the WILCOXON test (one-sided-) with BONFERRONI-HOLM adjustment for the hypothesis of equal medians (* for p ≤0.05, ** for p ≤0.01).
live male day x, %live female day x: Comparison of the dose group with the control group was performed using the WILCOXON test (two-sided) for the hypothesis of equal medians (* for p ≤0.05, ** for p ≤0.01).
Number of cycles and Cycle Length (days 54 -74), pup organ weights (absolute and relative): Non-parametric one-way analysis using the KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pair-wise comparison of the dose groups with the control group was performed using the WILCOXON-test (two-sided) for the hypothesis of equal medians (* for p < 0.05, ** for p < 0.01).

Reproductive indices:

Male mating index (%) = (number of males with confirmed mating / number of males placed with females) x 100
Male fertility index (%) = (number of males proving their fertility / number of males placed with females) x 100
Female mating index (%) = (number of females mated / number of females placed with males) x 100
Female fertility index (%) = (number of females pregnant / number of females mated) x 100
Gestation index (%) = (number of females with live pups on the day of birth / number of females pregnant) x 100
Live birth index (%) = (number of liveborn pups at birth / total number of pups born) x 100
Postimplantation loss (%) = [(number of implantations - number of pups delivered) / number of implantations] x 100

Offspring viability indices:

Pups were checked for death or moribundity twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7, 8-14 and 15-21 (lactation period) were determined.
Viability index (%) = (number of live pups on day 4 after birth / number of live pups on the day of birth) x 100
Lactation index (%) = (number of live pups on day 21 after birth / number of live pups on day 4 after birth) x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

see "Details on results"

Mortality:

no mortality observed

Description (incidence):

see "Details on results"

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see "Details on results"

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see "Details on results"

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

see "Details on results"

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see "Details on results"

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see "Details on results"

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

see "Details on results"

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

see "Details on results"

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

see "Details on results"

CLINICAL SIGNS/MORTALITY
There were no treatment-related clinical observations and mortality at any of the tested doses in either sex.
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the groups.
There were no test substance related clinical findings in any females of all dose groups during the gestation period for F1 litter.
Three sperm positive females of high dose animals, three sperm positive females of mid dose and one sperm positive female of low dose did not deliver F1 pups. These observations were not considered to be associated with the test compound.
There were no test substance-related clinical findings in all F0 females of the low and mid dose groups during the lactation period.
Two dams of of the high dose did not properly nurse their pups. One high dose dam did not properly nurse the pups and had a complete litter loss on PND 5. These observations were considered to be associated with the test compound.

BODY WEIGHT AND WEIGHT CHANGES
High dose parental F0 males (2000 ppm) had statistically significantly lower body weights during major parts of the premating period (up to 7% below the concurrent control values), during the whole mating period (up to 6% below the concurrent control values) and during the whole post mating period (up to 7% below the concurrent control values). The body weights of the mid and low dose parental males (500 ppm and 100 ppm) were comparable to the concurrent control group throughout the entire study period.
The body weight change of the high-dose parental males was statistically significantly below the concurrent control values during premating weeks 0 - 1 about 14%, weeks 5 - 6 about 15%, weeks 7 - 8 about 30% and weeks 0 - 10 about 9%. The body weight change of the high dose males during mating and post mating and the body weight change of the mid and low dose males during the whole study period were comparable to the concurrent control group.
The statistically significantly increased body weight change in the low dose males during premating weeks 9 - 10 and the statistically significantly decreased body weight change during the mating period were considered to be spontaneous in nature.
The body weights/body weight change of the high dose females during premating and the body weights/body weight change of the mid and low dose females during the whole study period were comparable to the concurrent control group. The statistically significantly higher body weights in the mid dose females on PND 14, the statistically significantly increased body weight change in the high dose females during premating weeks 2 - 3, during several parts of the lactation period and the statistically significantly increased body weight change in the mid dose females during several parts of the gestation period were considered to be spontaneous in nature.
High dose parental females had statistically significantly lower body weights on GD 20 (about 5%). The body weights remained to be lower during PND 1 - 14 (up to 10% below the concurrent control values) but recovered to control levels until PND 21. Consequently, in these females the body weight change was lower than control during gestation (10% below control) and higher than control during lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE
Food consumption of the high dose (2000 ppm) F0 males was statistically significantly below the concurrent control values during major parts of the premating period (up to 10%). Food consumption of the male F0 rats in the mid and low dose groups (500 and 100 ppm) was comparable to the concurrent control throughout the entire study.
Food consumption of the high dose F0 females was statistically significantly below control during the entire study period. For the premating period a decrease of up to 13%, for the gestation period up to 15% and for the lactation period up to 24% below the concurrent control values was observed. Food consumption of the female F0 rats in the mid and low dose groups was comparable to the concurrent control throughout the entire study.
Mean test substance intake (dose group 100/500/2000 ppm) in mg/kg bw/d:
F0 males: 7.4 / 37.0 / 143.3
F0 females premating: 8.4 / 41.3 / 155.1
F0 females gestation: 7.3 / 35.9 / 130.4
F0 females lactation: 18.7 / 88.5 / 300.1

HEMATOLOGY
Hemoglobin values were decreased in the rats of both sexes of the high dose group (2000 ppm). In males, this was the only altered red blood cell parameter; the decrease was marginal (mean hemoglobin - 2.3% compared to controls). Therefore, this decrease was regarded as treatment-related, but not adverse. In females of high dose group (2000 ppm), the hemoglobin decrease was a bit more pronounced when compared to that of the males (mean hemoglobin - 6.9% compared to controls). In addition, hematocrit values were decreased and relative reticulocyte counts increased in these rats as compared to the controls. Therefore in this sex, a compound-related, adverse effect was assumed.
In males of high dose (2000 ppm), absolute and relative monocyte counts were decreased, but the values were within historical control ranges (absolute monocyte counts 0.06-0.16 Giga/L; relative monocyte counts 1.3-2.6 %). Therefore, the alterations were regarded as incidental and not treatment-related.

CLINICAL BIOCHEMISTRY
Some clinical chemistry parameters were changed compared to controls, but the altered values were within historical control ranges and therefore, the changes were regarded as incidental and not treatment-related. This is true for decreased glucose values in rats of both sexes of the high dose group (2000 ppm) and additionally in females of the mid dose (500 ppm) as well as for decreased total bilirubin levels and increased total protein and albumin levels in females of the high dose (2000 ppm) (males glucose 5.11-7.16 mmol/L; females glucose 4.93-6.22 mmol/L; females total bilirubin 1.98-3.46 μmol/L; females total protein 62.13-71.91g/L; females albumin 39.50-42.59 g/L).
In females of the mid and high dose group (500 and 2000 ppm) cholesterol concentrations were increased. The mean of the mid dose group was within the historical control range (cholesterol 1.06-2.27 mmol/L,), but that of the high dose group was above the range. Although elevated cholesterol was the only altered parameter in the females of the high dose group (2000 ppm), these results mirrored the alteration in cholesterol levels of the females of the high dose group (2000 ppm) in the F1 generation. Therefore, this increase was assumed to be a treatment-related adverse effect.

ORGAN WEIGHT/ORGAN-TO-BODY-WEIGHT RATIOS
The increased mean absolute and relative adrenal weights in females of mid dose (500 ppm) and high dose group (2000 ppm) and the decreased mean absolute and relative weights of ovaries in females of high dose (2000 ppm) were considered to be treatment-related.
The decreased mean absolute prostate weight, as well as the increased mean relative weights of adrenal glands, cauda epididymis, epididymidis, testes, brain, liver, spleen and thyroid weights in males of high dose group (2000 ppm) were related to the slightly but not significantly decreased (-6%) terminal body weight in this treatment group. Furthermore, there were no histopathological correlates for the decreased absolute prostate weight, as well as the increased relative weights of adrenal glands, cauda epididymis, epididymidis, and testes.
The mean relative heart weight was significantly decreased in females of the high dose group (2000 ppm). Because the absolute heart weight in these females was only slightly (-4%) reduced and the absolute and relative heart weights in F1 parental females were comparable between control and treatment groups, the decreased relative heart weight was regarded to be incidental. This is in line with the lack of an effect on troponin, a clinical chemistry parameter used to investigate injury of the heart muscle.
The absolute spleen and thyroid weights were significantly increased in females of mid dose (500 ppm) and high dose group (2000 ppm). There was no dose-response relationship, the relative weights were not statistically significantly changed and in F1 generation parental females there was no comparable effect. Therefore, the weight changes were considered to be incidental.
Due to the lack of a dose-response relationship, the increased absolute brain weight in females of the mid dose group (500 ppm) and the increased relative brain weight in females of the low dose group (100 ppm) were regarded to be incidental.

GROSS PATHOLOGY
All gross lesions observed in test animals occurred singularly or non-dose-related. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

HISTOPATHOLOGY
Non-neoplastic: In the adrenal cortex, seven females of the high dose group (2000 ppm) showed a minimally increased vacuolation. In control females, this finding was observed in 3 animals. A treatment-related effect could not be ruled out.
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.

TROPONIN
The heart was previously demonstrated to be a target organ in F344 rats treated with the test substance. Therefore, troponin, an indicator for heart muscle damaged was measured. Troponin levels in parental animals of both generations were comparable between controls and treated animals.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE
Estrous cycle data, generated during the last 3 weeks prior to mating for the F1 litter, revealed regular cycles in the females of all test groups including the control. The mean estrous cycle duration in the different test groups was similar: 4.5 days in control, 5.0 in the low-dose group, 4.3 in the mid-dose group and 4.2 in the high-dose group.

REPRODUCTIVE FUNCTION: SPERM PARAMETERS
Concerning the motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as the sperm head counts in the cauda epididymidis no treatment related effects were observed. The sperm head counts in the testis of males of the high dose group (2000 ppm) were marginally lower compared to those of the controls, but the mean was still in the historical control range. Therefore, these slightly lower sperm head counts in males of the high dose group were regarded as incidental and not treatment-related.

REPRODUCTIVE PERFORMANCE
Male mating index: The mating index was comparable between all dose groups in both generations.
Male fertility index: Fertility was proven for almost all F0 parental males with confirmed copulation. One low dose male, three mid dose males and three high dose males did not generate F1 pups. Thus, the male fertility index ranged between 88% and 100% without showing any effect of dosing. These values reflect the normal range of biological variation inherent in the strain of rats used for this study. The apparently infertile male rats did not show histopathological findings that could explain the infertility.
Female mating index: The female mating index calculated after the mating period for F1 litter was 100% in all test groups The mean duration until sperm was detected (GD 0) varied between 2.4 and 2.7 days without any relation to dosing.
Female fertility index: The fertility index varied between 88% (500 and 2000 ppm), 96% (100 ppm) and 100% (control). Among the F0 females which showed the evidence of copulation, three high dose, three mid dose and one low dose female did not become pregnant. The non-pregnant females did not show a histomorphological correlate to explain the apparent infertility.
The mean duration of gestation values varied between 22.2 and 22.3 days without any relation to dosing. The gestation index was 100% in the control, high and mid dose groups and 96% in the low dose group. These values reflect the normal range of biological variation inherent in the strain of rats used for this study. All respective values are within the range of the historical control data of the test facility.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (12.0 / 12.3 / 12.9 and 11.6 implants/dam in the controls and test groups from low to high dose, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo- /fetolethality since the postimplantation loss did not show any significant differences between the groups, and the mean number of F1 pups delivered per dam remained unaffected (11.3 / 11.6 / 12.3 and 10.9 pups/dam in in the controls and test groups from low to high dose, respectively).
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 99.2% (high dose group), 99.6% (control and low dose group) and 100% (mid dose group). Moreover, the number of stillborn pups was comparable between the groups.

Key result

Dose descriptor:

NOAEL

Remarks:

General, systemic toxicity

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

Remarks on result:

other: Dose corresponding to 37 mg/kg bw/d (males) and 55 mg/kg bw/d (females)

Key result

Dose descriptor:

NOAEL

Remarks:

Fertility/Reproductive performance

Effect level:

>= 2 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment-related adverse effects observed

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see "Details on results"

Mortality:

no mortality observed

Description (incidence):

see "Details on results"

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

see "Details on results"

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see "Details on results"

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

see "Details on results"

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see "Details on results"

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see "Details on results"

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

see "Details on results"

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see "Details on results"

Other effects:

no effects observed

Description (incidence and severity):

see "Details on results"

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

see "Details on results"

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

see "Details on results"

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

see "Details on results"

CLINICAL SIGNS / MORTALITY
No mortality was observed. No clinical signs or changes of general behaviour, which may be attributed to the test substance. There were no test substance related clinical findings in the F1 females of all dose groups during the gestation period for F2 litter. One sperm positive female of the high dose group, as well as one sperm positive and two sperm negative females of the mid dose group did not deliver F2 pups. There were no test substance-related clinical findings in the F1 females of all dose groups during the lactation period for F2 litter. Two dams in the F1 high dose group had a complete litter loss (PND 4 and PND 5, respectively). Furthermore one high dose female animal showed insufficient maternal care during PND 1 - 7. These observations were considered to be associated with the test compound.

BODY WEIGHT AND WEIGHT CHANGES
High dose F1 parental males (2000 ppm) had statistically significantly lower body weights during the entire study. The most distinct effect was noted at the beginning of the premating period (up to 31% below the concurrent control). The body weights of the mid and low dose parental males (500 ppm and 100 ppm) were comparable to the concurrent control group throughout the entire study period.
The body weight change of the high dose parental males was statistically significantly below the concurrent control during several study periods: premating weeks 0 - 3 (up to 11%), weeks 6 - 7 (about 18%), if summarized for premating weeks 0 - 10 (about 7%) and during post mating days 2 - 9 (about 48%).
The body weight change of the high dose males during mating and the body weight change of the mid and low dose males during the whole study period were comparable to the concurrent control group. The statistically significantly decreased body weight change in the low dose males during premating weeks 6 - 7 was considered to be spontaneous in nature.
High dose F1 parental females had statistically significantly lower body weights on premating weeks 0 - 2 (up to 26%), on GD 20 (about 6%) and during PND 1 - 14 (up to 10%) below the concurrent control values. The body weights of the mid and low dose females during the whole study period were comparable to the concurrent control group.
High dose parental females had statistically significantly lower body weight change during premating weeks 7 - 8 (about 50%), during GD 14 - 20 (about 17%) and GD 0 - 20 (about 15%) and during PND 1 - 4 (about 45%) below the concurrent control values.
The body weight change of the mid and low dose females was comparable to the concurrent control group throughout the entire study period. The statistically significantly increased body weight change in the high dose females during premating weeks 1 - 4, during several parts of the lactation period and the statistically significantly increased body weight change in the mid dose females during several parts of the premating period were considered to be spontaneous in nature.

FOOD CONSUMPTION AND COMPOUND INTAKE
Food consumption of the high dose F1 males (2000 ppm) was statistically significantly below the concurrent control values during the whole premating period (up to 21%). Food consumption of the male F1 rats in the mid and low dose groups (500 and 100 ppm) was comparable to the concurrent control throughout the entire study.
Food consumption of the high dose F1 females was below the concurrent control during the entire study. The difference gained statistical significance during several study periods including premating weeks 0 – 1, 7 – 8, 9 – 10, gestation days 7 – 20. However, the most severe impairment of food consumption was noted at the beginning of the lactation period (41% below control at PND 1-4). Food consumption of the female F1 rats in the mid and low dose groups was comparable to the concurrent control throughout the entire study.
Mean test substance intake (dose group 100/500/2000 ppm) in mg/kg bw/d:
F1 males: 8.1 / 40.3 / 167.5
F1 females premating: 8.9 / 44.0 / 177.6
F1 females gestation: 7.3 / 35.9 / 143.8
F1 females lactation: 18.9 / 87.8 / 272.5

HEMATOLOGY
In females of the high dose (2000 ppm), red blood cell (RBC) counts, hemoglobin and hematocrit values were only slightly decreased (RBC -4.6%, hemoglobin -8.1%, hematocrit -7.2%) as compared to the controls, whereas relative reticulocyte counts were increased. RBC, hemoglobin and hematocrit means were slightly decreased compared to controls. However, an adverse effect cannot be excluded.
Mean corpuscular hemoglobin content (MCH) and relative basophil counts were decreased in females of the dose groups 500 and 2000 ppm, but the values were within historical control ranges (MCH 1.09-1.24 fmol, relative basophil counts 0.0-1.3 %).
Mean corpuscular volume (MCV) in females of the mid dose (500 ppm) was decreased, but the values were not dose-dependently changed and therefore this alteration was regarded as incidental and not treatment-related.

CLINICAL BIOCHEMISTRY
In rats of both sexes of the high dose (2000 ppm) as well as in females of the mid dose (500 ppm) total bilirubin values were decreased. However, in males of the high dose (2000 ppm) and in females of the mid dose (500 ppm) this was the only altered parameter and therefore the change in these rats was regarded as treatment-related, but not adverse. In females of the high (2000 ppm) the low bilirubin levels can be related to the decreased red blood cell parameter values and were therefore assumed as adverse. Additionally, in females of the high dose (2000 ppm) cholesterol values were increased.
In males of the mid dose (500 ppm) total protein values were increased, but the change was not dose-dependent and therefore it was regarded as incidental and not treatment-related. In males of the high dose (2000 ppm) glucose and triglyceride levels were decreased and urea levels were increased. Urea concentrations were already higher in males of mid dose (500 ppm). However, all mentioned parameter values were within historical control ranges and therefore, these changes were regarded as incidental and not treatment-related (glucose 5.11-7.16 mmol/L; triglycerides 0.55-1.32 mmol/L; urea 4.91-7.42 mmol/L).

ORGAN WEIGHT / ORGAN-TO-BODY-WEIGHT-RATIO
The terminal body weight was significantly decreased (-10%) in males of the high dose group (2000 ppm). This can result in significant decreases in absolute organ weights. As not all organs weighed show close correlation with body weight, some of the resulting statistically significant changes in relative weights are secondary and of no toxicological significance. Thus, decreased mean absolute and increased relative brain weights, decreased mean absolute prostate weight, increased mean relative weight of the cauda epididymis, increased mean relative weights of adrenal glands, pituitary gland, spleen, and thyroid glands were not considered further.
The decreased mean absolute and relative weights of ovaries in females of the high dose (2000 ppm) and the increased adrenal weights in females of mid dose (500 ppm) and high dose (2000 ppm) were considered to be treatment-related but not adverse. For the adrenals a slight increase in the incidence of vacuolation was observed, that did not correlate to the individual increase in organ weight. For the ovaries, no histological correlate was observable and there was no effect on differential ovarian follicle counts.
The relative liver weights were significantly increased in males of mid dose (500 ppm) and high dose (2000 ppm). Because the mean absolute liver weights were nearly comparable between control group (10.097 g) and treatment groups (mid dose: 10.501 g; high dose: 9.955 g), a treatment-related effect is unlikely.
The mean relative kidney weights were significantly increased in males and decreased in females of mid dose (500 ppm) and high dose (2000 ppm). Because the mean absolute kidney weights were nearly comparable between control and treatment groups, a treatment-related effect is unlikely.
The decrease of the absolute and relative weights of the pituitary gland in females of low dose (100 ppm) and high dose (2000 ppm) was not dose-related and there was no histopathological correlate in females of the high dose. Therefore, a treatment-related effect is unlikely.
Due to the lack of a dose-response relationship, the increased absolute thyroid weight in females of mid dose (500 ppm) was considered to be incidental.

GROSS PATHOLOGY
All gross lesions observed in test animals occurred singularly or non-dose-related. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

HISTOPATHOLOGY
Non-neoplastic: In the adrenal cortex, a minimal or slight increased vacuolation was observed in 10 females of the high dose group (2000 ppm).
The increased vacuolation in females of the high dose (2000 ppm) was considered to be treatment-related but not adverse as there was no correlation between incidence of vacuolation and adrenal weight.
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.

DIFFERENTIAL OVARIAN FOLLICLE COUNT
The results of the differential ovarian follicle count – comprising the numbers of primordial and growing follicles, as well as the combined incidence of primordial plus growing follicles – did not reveal significant differences between controls and animals of high dose.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE
Estrous cycle data, generated during the last 3 weeks prior to mating for the F2 litter, revealed regular cycles in the females of all test groups including the control. The mean estrous cycle duration in the different test groups was similar: 4.4 days in control, 4.7 days in the low-dose group, 4.3 days in the mid-dose group and 4.2 days in the high-dose groups.

REPRODUCTIVE FUNCTION: SPERM MEASURES
Concerning the motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as the sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed.

REPRODUCTIVE PERFORMANCE
Mating index: The mating index was comparable between all dose groups in both generations.
Male fertility index: For nearly all F1 parental males, which were placed with females to generate F2 pups, copulation was confirmed. Copulation was not confirmed for two mid dose males with two mid dose females. Thus, the male mating index was 100% in the control, low and high dose groups and 92% in the mid dose group. Fertility was proven for most of the F1 parental males within the scheduled mating interval for F2 litter. Three infertile males did not show histopathological findings that could explain infertility. One apparently infertile male rat showed histopathological findings which might explain its infertility. These included slight to moderately decreased size of epididymides, prostate, seminal vesicle, and testes. Microscopic examinations found debris (grade 3) and aspermia in the left epididymis and grade 4 multifocal tubular degeneration in the left testicle; the prostate and seminal vesicle showed diffuse atrophy (grade 2). No spermatology was available for this individual. Taken together, these findings may explain the apparent infertility; however as they were considered to be incidental, no relation to treatment is assumed.
Female fertility index: Female mating index and duration till mating was comparable between controls and treated animals. Among the F1 females which showed the evidence of copulation, one high dose, and three mid dose females did not become pregnant. The non-pregnant females did not show a histomorphological correlate to explain the apparent infertility. Fertility index and the mean duration of gestation was comparable between treated and control animals.
The fertility index varied between 100% (control and low-dose group), 96% (high-dose group) and 95.7% (mid-dose group). These values reflect the normal range of biological variation inherent in the strain of rats used for this study. The gestation index was 100% (control and low-dose group), 95.8% (high-dose group) and 95.5% (mid-dose group). All respective values are within the range of the historical control data of the test facility and do not show any relation to dosing. The mean duration of gestation was similar in all test groups (i.e. between 22.0 and 22.2 days). All values are within the historical control range of the test facility.
The mean number of implantation sites was statistically significantly reduced in the high dose group (9.8* vs 13.9 in controls). The high dose mean was also marginally lower than the lower limit of the historical control range (10.2 – 13.7). As a consequence of the fewer implants, the mean number of pups delivered was significantly decreased in the high dose group (9.8* vs 12.0 in controls). This mean, however is within the historical control range of the test facility (9.3 – 12.8). While these findings were statistically significant within a dose group, at the litter-level the changes were borderline reductions of a few implants per litter, rather than considerable fluctuation from litter to litter. In addition, mean absolute and relative weights of ovaries were decreased at the high dose level. It should be noted, however, that no morphologic correlate was observed which gave evidence for any effect of the test compound in the ovaries (in particular the number and appearance of primordial and growing follicles), oviducts or uteri. Also, morphology of the male reproductive organs and sperm quality were unaffected. Thus the reason for this small reduction of implants remains indeterminate. A relationship to the treatment cannot be excluded.
There were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any significant differences between the groups. The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 98.6% (control), 99.3% (low dose group), 100% (mid dose group) and 96.4% (high dose group). Moreover, the number of stillborn pups was comparable between the groups.

Key result

Dose descriptor:

NOAEL

Remarks:

General, systemic toxicity

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

Remarks on result:

other: Dose corresponding to 39 mg/kg bw/d (males) and 56 mg/kg bw/d (females)

Key result

Dose descriptor:

NOAEL

Remarks:

Fertility/Reproductive performance

Effect level:

500 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Remarks on result:

other: Dose corresponding to 56 mg/kg bw/d

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see "Details on results"

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

see "Details on results"

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see "Details on results"

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

see "Details on results"

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Gross pathological findings:

effects observed, non-treatment-related

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex ratio, see "Details on results"

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

CLINICAL SIGNS
For 4 male and 1 female pup of the high dose group a reduced nutritional condition was recorded during several part of the lactation period.

NUMBER OF PUPS DELIVERED
No statistically significant difference was noted in the number of F1 pups delivered between the control group and any of the treated groups.
The number of pups found dead was significantly increased in F1 (0 vs. 9) in the high dose. The number of pups cannibalized / dead was significantly increased in the high dose animals of F1 (2 vs. 9) in comparison to the control. At the high dose pups displayed signs of reduced maternal care, non-proper nursing, reduced general condition as well as no stomach contents at necropsy.

VIABILITY/LACTATION INDEX
The viability index indicating pup mortality shortly after birth (PND 0 - 4) varied between 97.6% (high dose), 99.3% (control), 99.6 (mid dose) and 99.7 (low dose) without showing any association with the treatment.
The lactation index indicating pup mortality during further course of lactation (PND 4 – 21) was 100% (control and low dose), 98.9% (mid dose) and 92.0% (high dose). The lower index in the high-dose group does not differ statistically significantly from control, however the value is slightly below the historical control range (95-100%). This higher rate of dead offspring is related to improper nursing of three high-dose dams which consequentially lost parts of their litters or the entire litter.

BODY WEIGHT
Mean body weights of the high dose F1 male and female pups (2000 ppm) were statistically significantly below the concurrent control values during the whole lactation period (up to 30% [males], up to 28% [females] and up to 29% [both sexes combined]). No test compound-related influence on F1 pup body weights were noted in the low and mid dose groups (100 and 500 ppm).
Body weight change of the high dose F1 pups of both sexes was statistically significantly below the concurrent control values during the whole lactation period (up to 42% [males], up to 39% [females] and up to 39% [both sexes combined]). Body weight change of the mid dose F1 pups of both sexes was statistically significantly below the concurrent control values during PND 14 - 21 in both sexes (about 8% [males], about 6% [females] and about 7% [both sexes combined]). No test compound-related influence on F1 pup body weight change was noted in the low dose group.

SEXUAL MATURATION
Vaginal opening: Each female F1 pup, which was selected to become a parental F1 female, was evaluated for commencement of sexual maturity. The first day when vaginal opening was observed was PND 27, the last was PND 39. The mean number of days to reach the criterion in the control and 100, 500 and 2000 ppm test groups amounted to 29.8; 30.4; 30.5 and 33.4** (**:p≤0.01) days. The mean body weight on the day, when vaginal opening was recorded, amounted to 92.2 g, 94.1 g, 96.8 g, and 97.9 g in test groups.
Comparison of ages and body weights of individual F1 females in controls and the 2000 ppm group with the mean body weight development of control F1 females reveals that the distribution of the animals in the high dose group is directly right-shifted with respect to the controls; no corresponding increase in body weight is observed in the older animals. Thus, the delay in vaginal opening must be due to the decreased body weight (slowed general development) in this dose group. These data indicate that the delay in vaginal opening reflects the general toxicity in the pups of this dose, rather than any other specific mechanism.
Preputial separation: Each male F1 pup, which was selected to become a F1 parental male, was evaluated for commencement of sexual maturity. The first day when preputial separation was observed was PND 38, the last was PND 53. The mean number of days to reach the criterion in the control and 100, 500 and 2000 ppm test groups was 41.0, 41.3, 42.5*, and 46.4** (*:p≤0.05 or **:p≤0.01) days. The mean body weight on the day when preputial separation was recorded, amounted to 180.1 g, 177.2 g, 183.8 g, and 177.3 g in the control, low, mid or high dose group, respectively.
Comparison of ages and body weights of individual F1 males in the control and the 2000 ppm dose group with the mean body weight development of control F1 males reveals that, like the females, the distribution of the male animals in the high dose group is directly right-shifted with respect to the controls; no corresponding increase in body weight is observed in the older animals. Thus, the delay in preputial separation must also be due to the decreased body weight (slowed general development) in the pups of this dose group. These data indicate that the delay in preputial separation reflects the general toxicity in the pups of this dose, rather than any other specific mechanism. The delay in preputial separation is a direct effect of just one animal. If this animal is removed as an outlier, the mean age at sexual maturation drops from 42.5 (the edge of the historical control range) to a much more normal 42.1±1.65 days. Thus any delay in preputial separation in this dose group is judged to be incidental and unrelated to treatment.

GROSS NECROPSY
A few F1 pups showed spontaneous findings at gross necropsy, such as post mortem autolysis, red discolored thymus, fluid-filled thorax, empty stomach, dilated renal pelvis, pale discolored liver lobe, dilated ureter, hydroureter, distended urine bladder, yellow discolored intestinal content and hydronephrosis. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were not considered to be associated to the test substance.

ORGAN WEIGHT
The decreased absolute brain weights and increased relative brain weights (high dose group) and the decreased absolute and relative spleen and thymus weights in the high and mid dose F1 pups were considered to be secondary to the lower pup body weights in these groups. The hematologic evaluation of F1 animals did not reveal any indication of an adverse effect on the lymphocyte count.

SEX RATIO
The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 21 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

Key result

Dose descriptor:

NOAEL

Remarks:

General, systemic toxicity

Generation:

F1

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: Dose corresponds to 40.3 mg/kg bw/d (males) or 44.0 mg/kg bw/d (females)

Key result

Dose descriptor:

NOAEL

Remarks:

Fertility

Generation:

F1

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: number of implantation sites, number of pups delivered

Remarks on result:

other: Dose corresponds to 40.3 mg/kg bw/d (males) or 44.0 mg/kg bw/d (females)

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Generation:

F1

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other:

Remarks:

Dose corresponding to 7.4 mg/kg bw/d (males) and 11.5 mg/kg bw/d (females) mg/kg bw/d

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see "Details on results"

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

see "Details on results"

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see "Details on results"

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

see "Details on results"

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

see "Details on results"

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

see "Details on results"

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

CLINICAL SIGNS
Several male (32 animals) and female (27 animals) pups from dams of the high dose group displayed signs of reduced nutritional condition during several time points of lactation.

NUMBER OF PUPS DELIVERED
In F2 the number of pups delivered was significantly reduced from 12.0 in the control to 9.8 in the high dose group (2000 ppm). The rates of stillborn pups was evenly distributed between the groups in F1 and F2.
The number of pups found dead was significantly increased in F2 (0 vs. 9) in the high dose. The number of pups cannibalized / dead was significantly increased in the high dose animals of F2 (0 vs. 28) in comparison to the control. At the high dose pups displayed signs of reduced maternal care, non-proper nursing, reduced general condition as well as no stomach contents at necropsy.

VIABILITY/LACTATION INDEX
The viability index indicating pup mortality during lactation (PND 0 - 4) was statistically significantly decreased in the high dose group and varied between 83.9%** (**:p≤0.01), 99.7% (mid dose) and 100% (low dose and control). The lactation index indicating pup mortality during further course of lactation (PND 4 – 21) was 100% (control, low and mid dose), and 90.2% (high dose). The lower index in the high-dose group does not differ statistically significantly from control, however the value is slightly below the historical control range (95-100%).
These higher rates of died offspring are related to four high-dose dams which suffered from distinct impairments of food consumption/body weight gain during this particular study period and/or were not able to nurse their pups properly. Consequentially, those dams lost parts of their litters or the entire litter. At the high dose pups displayed signs of reduced maternal care, non-proper nursing, reduced general condition as well as no stomach contents at autopsy. This indicates that poor nutritional condition lead to the death/cannibalization of the pups.
This is, in turn, probably secondary to the decreased food consumption by the parental females during the last part of gestation and the early part of lactation. As the quantity and quality of mammary secretions is highly dependent on maternal nutrition, and so is pup growth during the first few weeks of life. To meet the caloric demand, lactating females consume about twice the food than they would otherwise. Thus the parental females, and probably indirectly the offspring, were exposed to twice the effective dosage during this period contributing to the reduced nutritional status of the pups. Furthermore, the doubling of the effective dose during lactation may also be a cause of the small effects on pup body and organ weight seen at the 500 ppm concentration during lactation, as no effects were observed outside of this study period.

BODY WEIGHT
Mean body weights of the high dose F2 male and female pups (2000 ppm) were statistically significantly below the concurrent control values between PND 4 - 21 in both sexes (up to 31%). Body weights of the mid dose F2 pups of both sexes (500 ppm) were slightly but statistically significantly below the concurrent control values on PND 21 (up to 6%). No test compound-related influence on F2 pup body weights was noted in the low dose group (100 ppm). Body weight change of the high dose F2 pups of both sexes was statistically significantly below the concurrent control values during the entire lactation period (up to 54%). Body weight change of the mid dose F2 pups of both sexes was statistically significantly below the concurrent control values during PND 14 - 21 (up to 9%). The change in the last third of lactation was big enough to convey a statistically significant decrease in the average value calculated for the entire lactation period (PND 1 – 21, about 6-7%). No test compound-related influence on F2 pup body weight change was noted in the low dose group.

GROSS NECROPSY
A few F2 pups showed spontaneous findings at gross necropsy, such as situs inversus, red discolored thymus, diaphragmatic hernia, empty stomach and dilated renal pelvis. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, none of these findings was considered to be associated with the test substance.

ORGAN WEIGHT
The decreased absolute brain weights and increased relative brain weights (high dose group) and the decreased absolute and relative spleen and thymus weights in the high and mid dose F2 pups were considered to be secondary to the lower pup body weights in these groups.

SEX RATIO
The sex distribution and sex ratios of live F2 pups on the day of birth and on PND 21 did not show substantial differences between controls and treated groups. All differences observed were regarded to be spontaneous in nature.

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Generation:

F2

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Dose corresponding to 8.1 mg/kg bw/d (males) and 11.7 mg/kg bw/d (females)

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

2 000 ppm

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Reproduction parameters of male rats

Parental generation	F0				F1
Dose [ppm]	0	100	500	2000	0	100	500	2000
Animals per dose	25	25	25	25	24	24	25	25
Male fertility
-placed with females	25	25	25	25	24	24	25	25
-mated[n]	25	25	25	25	24	24	23	25
- mating index [%]	100	100	100	100	100	100	92.0	100
- pregnant [n]	25	24	22	22	24	24	22	24
- Fertility index[%]	100	96	88	88	100	100	88	96

 

Reproduction and gestational parameters of female rats

Parental generation	F0				F1
Dose [ppm]	0	100	500	2000	0	100	500	2000
Animals per dose	25	25	25	25	24	25	25	25
Female fertility
- placed with males	25	25	25	25	24	25	25	25
- mated [n]	25	25	25	25	24	25	23	25
- mating index [%]	96	100	92	100	100	100	92	100
- pregnant [n]	25	24	22	22	24	25	22	24
-Fertility index[%]	100	96	88	88	100	100	95.7	96
Estrous cycle length [days]	4.46	4.99	4.29	4.22	4.44	4.74	4.32	4.23
Mating days until day 0 [days]	2.5	2.4	2.5	2.7	3.2	2.5	2.5	3.2
Duration of gestation [days]	22.3	22.2	22.2	22.2	22.1	22.0	22.0	22.2
Implantation sites, total [n]	299	295	283	255	333	315	256	240
Implantation sites, mean	12	12.3	12.9	11.6	13.9	12.6	11.6	10.0**
Post implantation loss [mean%]	5.56	9.22	4.02	4.87	8.92	3.77	9.37	8.17
Females with liveborn
-Gestation index[%]	100	95.8	100	100	100	100	95.5	95.8
- dams with stillborn pups [n]	1	1	0	2	4	2	0	4
- dams with all stillborn [n]	0	0	0	0	0	0	0	0
Pups delivered [n]	282	267	271	239	289	303	242	225
- per dam [mean n]	11.3	11.6	12.3	10.9	12	12.1	11.5	9.8**
- liveborn [n]	281	266	271	237	285	301	242	217
- stillborn [n]	1	1	0	2	4	2	0	8
- Found dead [n]	0	1	1	9	0	0	1	9
- cannibalized / dead [n]	2	0	1	12	0	0	0	28
Pup mortality [n] - Day 0	0	1	0	0	0	0	1	2
- Day 1 to 4	2	0	1	7	0	0	0	32
- Day 5 to 7	0	0	0	11	0	0	0	3
- Day 8 to 14	0	0	2	3	0	0	0	0
- Day 15 to 21	0	0	0	0	0	0	0	0
Litters not surviving day 21 [n]	0	0	0	1	0	0	0	3
Pups surviving days 0-4 [n]	279	265	270	230	285	301	241	186
Viability index [mean%]	99.3	99.7	99.6	97.6	100	100	99.7	83.9*
Pups surviving days 4 to 21 [n]	194	183	174	160	191	200	166	151
Lactation index [mean%]	100	100	98.9	92	100	100	100	90.2
Clinical conditions
- male
Animals examined	128	119	130	133	144	150	117	127
# with signs	0	2	0	19	3	1	0	48
Reduced nutritional condition	0	0	0	8	0	0	0	32
Died	0	2	0	12	3	1	0	25
- female
Animals examined	154	148	141	106	145	153	125	98
# with signs	1	0	1	11	1	1	0	34
Reduced nutritional condition	0	0	0	5	0	0	0	27
Died	1	0	1	6	1	1	0	14

* p ≤ 0.05; ** p ≤ 0.01

Values may not calculate exactly due to rounding of values

 

Selected organ weights of F0 and F1 male parental animals

	Dose [ppm]	F0 Male		F1 Male
		Absolute weight (mean ± SD)	Relative weight[%of bw]	Absolute weight (mean ± SD)	Relative weight [% of bw]
Terminal weight [g]	0	402.3± 24.4	100	422.4± 40.1	100
	100	405.34± 39.8	100	406.6± 30.3	100
	500	412.7± 26.2	100	417.8± 41.5	100
	2000	377.0± 43.1	100	379.4± 34.3	100
Adrenals [mg]	0	61.28± 7.5	0.015± 0.002	61.32± 6.762	0.015±0.002
	100	61.08± 8.1	0.015± 0.002	59.46± 7.003	0.015± 0.002
	500	63.80± 6.9	0.015± 0.001	64.76± 11.016	0.016± 0.002
	2000	62.60± 8.093	0.017± 0.002*	65.60± 8.986	0.017± 0.002
Brain [g]	0	2.106± 0.075	0.525 ± 0.033	2.148± 0.098	0.512± 0.043
	100	2.124± 0.105	0.527± 0.037	2.138± 0.102	0.528± 0.03
	500	2.097± 0.069	0.510± 0.033	2.141± 0.074	0.517± 0.053
	2000	2.087± 0.106	0.559± 0.057*	2.05± 0.097*	0.543± 0.042*
Cauda epididymis [g]	0	0.436± 0.04	0.109 ± 0.014	0.457± 0.061	0.109± 0.017
	100	0.426± 0.074	0.106± 0.02	0.458± 0.055	0.113± 0.012
	500	0.438± 0.045	0.106± 0.012	0.425± 0.093	0.101± 0.021
	2000	0.451± 0.056	0.120± 0.015*	0.452± 0.054	0.120± 0.014*
Epididymis [g]	0	1.128± 0.062	0.282 ± 0.026	1.223± 0.097	0.292± 0.033
	100	1.099± 0.168	0.272± 0.041	1.18± 0.133	0.29± 0.026
	500	1.118± 0.091	0.271± 0.021	1.148± 0.212	0.274± 0.046
	2000	1.140± 0.11	0.304± 0.026*	1.161± 0.101	0.307± 0.026
Heart	0	1.094± 0.085	0.272 ± 0.017	1.11± 0.122	0.263± 0.022
	100	1.089± 0.099	0.27± 0.02	1.075± 0.106	0.264± 0.015
	500	1.091± 0.076	0.265± 0.017	1.109± 0.111	0.266± 0.017
	2000	1.032± 0.111	0.275± 0.02	1.032± 0.095	0.273± 0.02
Kidneys [g]	0	2.517± 0.227	0.626 ± 0.044	2.532± 2.532	0.601± 0.048
	100	2.474± 0.205	0.612± 0.043	2.464± 0.251	0.607± 0.05
	500	2.597± 0.23	0.630± 0.046	2.618± 0.268	0.628± 0.045*
	2000	2.525± 0.565	0.683± 0.234	2.463± 0.236	0.650± 0.042*
Liver [g]	0	9.314± 0.835	2.315 ± 0.159	10.097± 1.445	2.388± 0.233
	100	9.033± 0.961	2.231± 0.131	9.67± 0.959	2.379± 0.167
	500	9.569± 0.749	2.318± 0.107	10.501± 1.753	2.502± 0.273*
	2000	9.249± 1.172	2.452± 0.105*	9.955± 0.987	2.629± 0.208*
Pituitary gland [mg]	0	11.76± 2.296	0.003 ± 0.001	10.16± 2.495	0.002± 0.001
	100	11.48± 2.7	0.003± 0.001	9.833± 2.099	0.002± 0.001
	500	12.2± 2.198	0.003± 0.001	10.28± 3.035	0.002± 0.001
	2000	11.04± 2.051	0.003± 0.001	11.16± 2.824	0.003± 0.001
Prostate [g]	0	1.13± 0.141	0.282 ± 0.039	1.054± 0.201	0.25± 0.047
	100	1.151± 0.202	0.286± 0.052	1.051± 0.182	0.258± 0.038
	500	1.123± 0.219	0.272± 0.048	1.028± 0.25	0.245± 0.054
	2000	0.974± 0.194*	0.258± 0.044	0.919± 0.117*	0.243± 0.027
Seminal vesicle [g]	0	1.239± 0.17	0.309 ± 0.046	1.243± 0.225	0.295± 0.052
	100	1.287± 0.175	0.321± 0.055	1.3± 0.242	0.319± 0.048
	500	1.253± 0.228	0.304± 0.059	1.142± 0.254	0.273± 0.06
	2000	1.134± 0.211	0.301± 0.049	1.12± 0.184
Spleen [g]	0	0.633± 0.055	0.158 ± 0.014	0.7± 0.101	0.166± 0.022
	100	0.632± 0.081	0.156± 0.015	0.665± 0.087	0.164± 0.02
	500	0.647± 0.075	0.157± 0.016	0.696± 0.101	0.167± 0.021
	2000	0.659± 0.101	0.175± 0.021	0.714± 0.094	0.189± 0.027*
Testes [g]	0	3.673± 0.258	0.916 ± 0.086	3.994± 0.355	0.952± 0.105
	100	3.495± 0.593	0.863± 0.143	3.942± 0.407	0.97± 0.08
	500	3.605± 0.224	0.875± 0.05	3.782± 0.625	0.903± 0.139
	2000	3.691± 0.299	0.988± 0.105*	3.812± 0.385	1.01± 0.113
Thyroid gland [mg]	0	25.92± 5.291	0.006 ± 0.001	24.32± 5.886	0.006± 0.001
	100	24.80± 4.6	0.006± 0.001	23.79± 3.514	0.006± 0.001
	500	26.84± 4.516	0.007± 0.001	24.16± 5.281	0.006± 0.001
	2000	26.88± 4.167	0.007± 0.001	25.72± 4.722	0.007± 0.001*
* p ≤ 0.05

 

 

Selected organ weights of F0 and F1 female parental animals

	Dose [ppm]	F0 Female		F1 Female
		Absolute weight (mean ± SD)	Relative weight [% of bw]	Absolute weight (mean ± SD)	Relative weight [% of bw]
Terminal weight [g]	0	240.4± 17.28	100	237.4 ± 20.7	100
	100	237.5± 14.2	100	233.7 ± 15.737	100
	500	252.4± 19.9	100	243.9 ± 16.747	100
	2000	245.8± 16.1	100	240.3 ± 19.8	100
Adrenals [mg]	0	78.16± 9.384	0.033 ± 0.004	78.375 ± 7.878	0.033 ± 0.004
	100	81.12± 12.283	0.034 ± 0.004	75.1 ± 11.341	0.032 ± 0.004
	500	89.40± 10.79*	0.036 ± 0.005	85.7± 8.473*	0.035± 0.004*
	2000	94.12± 15.501*	0.038 ± 0.004	85.3± 11.866*	0.036 ± 0.005
Brain [g]	0	1.956± 0.081	0.818 ± 0.074	1.953 ± 0.098	0.826 ± 0.046
	100	1.996± 0.073	0.842± 0.041*	1.979 ± 0.092	0.85 ± 0.061
	500	1.999± 0.084*	0.796 ± 0.065	1.957 ± 0.067	0.805 ± 0.042
	2000	1.941± 0.064	0.792 ± 0.038	1.919 ± 0.09	0.804 ± 0.073
Heart [g]	0	0.928± 0.062	0.387 ± 0.028	0.851 ± 0.072	0.36 ± 0.029
	100	0.93± 0.085	0.392 ± 0.034	0.858 ± 0.087	0.368 ± 0.036
	500	0.957± 0.09	0.38 ± 0.036	0.852 ± 0.067	0.35 ± 0.035
	2000	0.892± 0.097	0.362 ± 0.025	0.855 ± 0.092	0.356 ± 0.03
Kidneys [g]	0	1.916± 0.138	0.798 ± 0.046	1.815 ± 0.146	0.768 ± 0.062
	100	1.928± 0.173	0.812 ± 0.059	1.77 ± 0.156	0.759 ± 0.067
	500	2.016± 0.182	0.801 ± 0.071	1.773 ± 1.49	0.727 ± 0.045
	2000	1.928± 0.189	0.783 ± 0.043	1.787 ± 0.234	0.749 ± 0.132
Liver [g]	0	7.649± 1.084	3.179 ± 0.363	7.384 ± 0.753	3.121 ± 0.308
	100	7.812± 1.427	3.285 ± 0.549	7.184 ± 0.826	3.07 ± 0.244
	500	8.231± 1.55	3.265 ± 0.584	7.336 ± 0.802	3.016 ± 0343
	2000	8.634± 1.708	3.492 ± 0.556	7.764 ± 1.133	3.228 ± 0.382
Ovaries [mg]	0	110.0± 15.411	0.046 ± 0.006	109.708 ± 21.8	0.046 ± 0.009
	100	109.4± 14.989	0.046 ± 0.006	103.7 ± 18.569	0.044 ± 0.007
	500	114.8± 14.666	0.046 ± 0.007	108.2 ± 16.128	0.045 ± 0.008
	2000	95.8± 18.141*	0.039± 0.008*	92.4± 18.225*	0.038± 0.006*
Pituitary gland [mg]	0	13.52± 3.111	0.006 ± 0.001	14.167 ± 2.408	0.006 ± 0.001
	100	13.12± 2.833	0.006 ± 0.001	11.12 ± 3.18	0.005 ± 0.001
	500	14.36± 3.29	0.006 ± 0.001	13.72 ± 1.99	0.006 ± 0.001
	2000	12.08± 2.272	0.005 ± 0.001	12.48 ± 3.016	0.005 ± 0.001
Spleen [g]	0	0.513± 0.065	0.214 ± 0.026	0.488 ± 0.084	0.206 ± 0.031
	100	0.512± 0.058	0.216 ± 0.024	0.465 ± 0.094	0.199 ± 0.039
	500	0.552± 0.056*	0.219 ± 0.018	0.488 ± 0.087	0.200 ± 0.035
	2000	0.548± 0.066*	0.223 ± 0.024	0.493 ± 0.102	0.206 ± 0.042
Thyroid glands [mg]	0	17.68± 3.705	0.007 ± 0.001	18.92 ± 3.063	0.008 ± 0.001
	100	18.4± 3.488	0.008 ± 0.001	18.92 ± 4.122	0.008 ± 0.002
	500	20.88± 5.167	0.008 ± 0.002	21.92 ± 4.061	0.009 ± 0.002
	2000	20.76± 4.648	0.008 ± 0.002	20.48 ± 3.698	0.008 ± 0.001
Uterus [g]	0	0.722± 0.2	0.303 ± 0.091	0.673 ± 0.251	0.285 ± 0.109
	100	0.69± 0.191	0.29 ± 0.077	0.631 ± 0.183	0.271 ± 0.081
	500	0.798± 0.349	0.319 ± 0.14	0.708 ± 0.27	0.288 ± 0.099
	2000	0.736 ± 0.29	0.3 ± 0.12	0.586 ± 0.244	0.245 ± 0.105
* p≤0.05

 

Selected hematology parameters of F0 and F1 male parental animals

Parameter	Dose	F0 Male	F1 Male
		Mean ± SD	Mean ± SD
HGB [mmol/L]	0	9.0± 0.3	9.0± 0.2
	100	9.1± 0.4	9.0± 0.3
	500	9.1± 0.2	9.0± 0.3
	2000	8.8± 0.2*	8.9± 0.4
MONOA [giga/L]	0	0.13± 0.05	0.13± 0.04
	100	0.12± 0.04	0.14± 0.03
	500	0.12± 0.04	0.14± 0.05
	2000	0.07± 0.03**	0.13± 0.05
MONO[%]	0	2.4± 0.7	2.2±0.9
	100	2.2± 0.8	2.1± 0.4
	500	2.1± 0.5	2.2± 0.5
	2000	1.5± 0.6*	2.1± 1.0

* p≤0.05; ** p≤0.01

 

 

Selected hematology parameters of F0 and F1 female parental animals

Parameter	Dose	F0 Female	F1 Female
		Mean ± SD	Mean ± SD
RBC [tera/L]	0	8.89 ± 0.39	8.71 ± 0.38
	100	8.77 ± 0.46	8.93 ± 0.38
	500	8.71 ± 0.34	8.88 ± 0.44
	2000	8.39 ± 0.46	8.31± 0.48*
HGB [mmol/L]	0	10.1 ± 0.4	9.9 ± 0.3
	100	10 ± 0.4	9.9 ± 0.3
	500	10 ± 0.4	9.7 ± 0.3
	2000	9.4± 0.4**	9.1± 0.3*
HCT [L/L]	0	0.475 ± 0.024	0.471 ± 0.013
	100	0.473 ± 0.020	0.474 ± 0.020
	500	0.465 ± 0.019	0.461 ± 0.013
	2000	0.443± 0.020**	0.437± 0.016*
MCV [fL]	0	53.4 ± 1.9	54.2 ± 2.1
	100	54.0 ± 2.0	53.1 ± 1.6
	500	53.4 ± 1.7	52.0± 1.9**
	2000	52.9 ± 2.3	52.6 ± 1.6
MCH [fmol]	0	1.14 ± 0.04	1.14 ± 0.05
	100	1.15 ± 0.04	1.11 ± 0.03
	500	1.14 ± 0.04	1.09± 0.06*
	2000	1.13 ± 0.05	1.09± 0.05*
RET [%]	0	0.2 ± 0.1	0.3 ± 0.2
	100	0.2 ± 0.1	0.3 ± 0.1
	500	0.3 ± 0.2	0.2 ± 0.1
	2000	0.7± 0.3*	0.9± 0.7**
Baso [%]	0	0.3 ± 0.1	0.8 ± 0.3
	100	0.3 ± 0.1	0.8 ± 0.3
	500	0.3 ± 0.1	0.6± 0.3*
	2000	0.3 ± 0.2	0.6± 0.2*

* p≤0.05; ** p≤0.01

 

Selected biochemical parameters of F0 and F1 male parental animals

Parameter	Dose	F0 Male	F1 Male
		Mean ± SD	Mean ± SD
Urea [mmol/L]	0	7.1 ± 0.94	5.64± 0.97
	100	7.1 ± 0.88	5.89± 0.55
	500	7.27 ± 0.95	6.48± 0.68*
	2000	7.67 ± 0.80	6.72± 0.84*
GLUC [mmol/L]	0	7.21 ± 0.73	7.82± 1.05
	100	6.91 ± 0.81	7.48± 0.76
	500	6.77 ± 0.75	7.35± 0.57
	2000	5.84± 0.84**	6.28± 0.74**
TBIL [mmol/L]	0	2.07 ± 0.44	2.06± 0.42
	100	2.22 ± 0.33	2.12± 0.26
	500	1.89 ± 0.37	1.77± 0.39
	2000	1.72± 0.48	1.24± 0.35**
TPROT[g/L]	0	64.68 ± 2.65	62.83± 1.58
	100	65.59 ± 1.73	62.91± 1.77
	500	65.83 ± 1.56	64.79± 1.51**
	2000	66.55 ± 1.70	63.32± 2.29
TRIG [mmol/L]	0	0.82 ± 0.41	1.31± 0.64
	100	0.82 ± 0.44	1.14± 0.29
	500	0.81 ± 0.27	1.10± 0.28
	2000	0.53± 0.17	0.61± 0.17**

* p≤0.05; ** p≤0.01

 

Selected biochemical parameters of F0 and F1 female parental animals

Parameter	Dose	F0 Female	F1 Female
		Mean ± SD	Mean ± SD
GLUC [mmol/L]	0	6.17± 0.58	5.30± 0.95
	100	5.88± 0.88	5.29± 0.90
	500	5.30± 0.52**	5.33± 0.50
	2000	5.29± 0.57**	4.66± 0.64
TBIL [mmol/L]	0	3.24± 0.77	1.71± 0.35
	100	2.94± 0.95	1.85± 0.35
	500	2.65± 0.46	1.20± 0.33**
	2000	2.10± 0.34**	1.05± 0.31**
TPROT[g/L]	0	65.49± 2.32	64.38± 3.45
	100	66.82± 3.09	64.28± 2.36
	500	67.84± 2.47*	64.81± 2.83
	2000	68.47± 1.75**	65.09± 1.55
ALB [g/L]	0	39.04± 1.39	36.02± 1.91
	100	39.54± 1.73	36.31± 1.41
	500	40.09± 1.60	36.12± 0.95
	2000	40.52± 0.58**	36.25± 1.44
CHOL[g/Ll]	0	1.99± 0.37	1.88± 0.31
	100	1.89± 0.40	1.96± 0.46
	500	2.19± 0.26*	1.79± 0.49
	2000	2.63± 0.47**	2.53± 0.50**

* p≤0.05; ** p≤0.01

 

F1: Selected histopathological findings

Adrenal cortex	Female animals
Test group
[ppm]	0	100	500	2000
Organs examined	25	25	25	25
Vacuolation increased	1	1	0	10
•  Grade 1	1	1		8
•  Grade 2				2

 

F1: Differential ovarian follicle count

Dose	Number of animals	Priomordial		Growing		Primordial + growing
		Absolute values	Mean values	Absolute values	Mean values	Absolute values	Mean values
0	25	5006	200.24	551	22.04	5557	222.28
2000	25	5745	229.80	538	21.52	6283	251.32

 

Pup survival, sex-ratio, and body weights

Pup generation	F1				F2
Dose [ppm]	0	100	500	2000	0	100	500	2000
Number of litters	25	23	22	22	24	23	21	23
Pups delivered [n]	282	267	271	239	289	303	242	225
- per dam [mean n]	11.3	11.6	12.3	10.9	12	12.1	11.5	9.8**
- liveborn [n]	281	266	271	237	285	301	242	217
- stillborn [n]	1	1	0	2	4	2	0	8
- Found dead [n]	0	1	1	9	0	0	1	9
- cannibalized / dead [n]	2	0	1	12	0	0	0	28
Pup mortality [n]
- Day 0	0	1	0	0	0	0	1	2
- Day 1 to 4	2	0	1	7	0	0	0	32
- Day 5 to 7	0	0	0	11	0	0	0	3
- Day 8 to 14	0	0	2	3	0	0	0	0
- Day 15 to 21	0	0	0	0	0	0	0	0
Litters not surviving day 21 [n]	0	0	0	1	0	0	0	3
Pups surviving days 0-4 [n]	279	265	270	230	285	301	241	186
Viability index [mean%]	99.3	99.7	99.6	97.6	100	100	99.7	83.9
Pups surviving days 4 to 21 [n]	194	183	174	160	191	200	166	151
Lactation index [mean%]	100	100	98.9	92	100	100	100	90.2
Clinical conditions
- male
Animals examined	128	119	130	133	144	150	117	127
# with signs	0	2	0	19	3	1	0	48
Reduced nutritional condition	0	0	0	8	0	0	0	32
Died	0	2	0	12	3	1	0	25
- female
Animals examined	154	148	141	106	145	153	125	98
# with signs	1	0	1	11	1	1	0	34
Reduced nutritional condition	0	0	0	5	0	0	0	27
Died	1	0	1	6	1	1	0	14
Sex ratio [% live males]
- Day 0	45.6	44.4	48	55.3	49.5	49.5	48.3	56.7
Male pup weight[g]
- lactation day 1	7.0	7.0	7.0	6.5*	6.8	6.7	6.7	6.4
- lactation day 4	10.6	10.6	10.5	8.6**	10.3	10.2	10.2	8.0**
- lactation day 7	17.2	17.1	17.0	12.9**	16.7	16.5	16.2	12.1**
- lactation day 14	34.5	34.7	34.3	25.6**	33.6	33.6	32.3	24.2**
- lactation day 21	54.9	54.5	52.9	38.3**	52.8	52.7	49.7*	36.8**
Body weight gain: day 1 to 21	47.8	47.5	45.9	31.8**	46.0	46.0	43.0*	30.4**
Female pup weight[g]
- lactation day 1	6.7	6.6	6.6	6.2*	6.4	6.3	6.4	6.0
- lactation day 4	10.3	10.1	10.1	8.4**	9.8	9.8	9.9	7.5**
- lactation day 7	16.8	16.5	16.5	12.6**	16.0	15.8	15.7	11.4**
- lactation day 14	33.7	33.8	33.4	25.5**	32.5	32.3	31.3	23.2**
- lactation day 21	52.6	52.3	51.0	37.9**	50.8	50.1	48.1*	35.0**
Body weight gain: day 1 to 21	45.9	45.6	44.3	31.6**	44.4	43.8	41.7*	28.9**

* p ≤ 0.05; ** p ≤ 0.01

 

F1: Absolute pup organ weights [g]

Test group [ppm]	0	100	500	2000
Brain (male)	1.538	1.561	1.560	1.491*
Brain (female)	1.491	1.507	1.500	1.444**
Brain (male+female)	1.518	1.534	1.530	1.464**
Spleen (male)	0.268	0.260	0.245	0.131**
Spleen (female)	0.263	0.259	0.236*	0.126**
Spleen (male+female)	0.266	0.260	0.240*	0.128**
Thymus (male)	0.266	0.250	0.247	0.166**
Thymus (female)	0.274	0.263	0.248*	0.177**
Thymus (male+female)	0.270	0.257	0.247*	0.169**

* p ≤ 0.05; ** p ≤ 0.01

 

 

F1: Relative pup organ weights, compared to control (=100%)

Test group [ppm]	100	500	2000
Brain (male)	101%	103%	135%**
Brain (female)	103%	103%	134%**
Brain (male+female)	102%	103%	135%**
Spleen (male)	96%	92%*	68%**
Spleen (female)	100%	92%	66%**
Spleen (male+female)	98%	92%*	67%**
Thymus (male)	94%	94%*	86%**
Thymus (female)	98%	93%*	89%**
Thymus (male+female)	96%	94%*	87%**

* p ≤ 0.05; ** p ≤ 0.01

 

 

F2: Absolute pup organ weights [g]

Test group [ppm]	0	100	500	2000
Brain (male)	1.525	1.555	1.520	1.447*
Brain (female)	1.487	1.495	1.461	1.373**
Brain (male+female)	1.506	1.525	1.491	1.410**
Spleen (male)	0.251	0.262	0.217**	0.126**
Spleen (female)	0.255	0.254	0.215**	0.115**
Spleen (male+female)	0.253	0.258	0.216**	0.120**
Thymus (male)	0.249	0.244	0.220*	0.157**
Thymus (female)	0.242	0.245	0.233	0.159**
Thymus (male+female)	0.246	0.245	0.226*	0.158**

* p ≤ 0.05; ** p ≤ 0.01

 

 

F2: Relative pup organ weights, compared to control (=100%)

Test group [ppm]	100	500	2000
Brain (male)	102%	105%**	137%**
Brain (female)	102%	102%	135%**
Brain (male+female)	102%	104%*	136%**
Spleen (male)	104%	91%*	71%**
Spleen (female)	101%	88%**	65%**
Spleen (male+female)	102%	89%**	68%**
Thymus (male)	98%	93%*	88%**

* p ≤ 0.05; ** p ≤ 0.01

 

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

51 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP and Guideline conform study.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Key study: Two-generation reproductive toxicity 2013/1347949

In this 2-generation reproduction study performed according to OECD 416, rats (24/sex/dose) were given 0, 100, 500 or 2000 ppm (equivalent to 0, 8, 39 and 155 mg/kg bw/d for males and 0, 12.0, 56 and 197 mg/kg bw/day for females, based on intake by F0 animals during pre-mating) starting 10 weeks before mating (F0) or at weaning (F1) until scheduled necropsy. No dose adjustment was performed during gestation and lactation. This lead to a significantly higher exposure during lactation equivalent to 18.7 (F0: 12.2 / F1: 23.0), 88.5 (F0: 61.0 / F1: 107.2) and 300.1 (F0: 206.5 / 356.8) mg/kg bw/day at 100, 500, and 2000 ppm, respectively.

A clear effect on the number of pups cannibalized and pup mortality was visible in both F1 and to a greater extent the F2 generation at 2000 ppm. Investigation of the affected pups revealed clear indications of reduced maternal care and insufficient nursing, including empty stomachs. The study concluded that significant pup effects observed in the high dose animals are secondary to lactation and thus maternal toxicity.

The NOAEL (no observed adverse effect level) for general, systemic toxicity is 500 ppm (51 mg/kg bw/d) for the F0 and F1 parental rats, based on decreased food consumption and body weight/body weight gain observed at 2000 ppm (186 mg/kg bw/d) in all F0 and F1 parental animals, as well as effects on hematology and clinical chemistry. The NOAEL for fertility and reproductive performance for the parental rats is 500 ppm (51 mg/kg bw/d) due to the reduction in implantation sites and pups delivered in the F1 parents of the 2000 ppm (186 mg/kg bw/d) dose group. The NOAEL for developmental toxicity in the F1 and F2 progeny is 100 ppm (11 mg/kg bw/d), due to the decrease in the pre-weaning pup body weights/pup weight gains observed at the 500 ppm (51 mg/kg bw/d) dose.

There are two final comments regarding the developmental NOAEL of 100 ppm (11 mg/kg bw/d) from the definitive rat 2-generation study using the LOAEL based on reduced pup body weight at 500 ppm (51 mg/kg bw/d).

1. The effects that set this NOAEL were only observed during the lactation portion of the study when the effective maternal dose is doubled. Therefore, the relevance of using this endpoint as a NOAEL to define an independent adverse developmental finding is questionable.

2. Later pharmacokinetic studies in the rat indicated that doses exceeding 15 mg/kg bw/d follow non-linear kinetics - as oral doses increase, there is a disproportionate increase in plasma concentration of the test substance (also refer to IUCLID chapter 7.1). All of the pup effects that were seen in the 2-generation reproduction studies (and the later discussed developmental toxicity studies) were at doses that exceed a kinetically derived maximum tolerated dose (MTD or KMD). The effective internal exposure at the LOAEL was measured in the PK study to be more than 25X the exposure expected with linear kinetics.

This unproportioned dose-multiplying effect is particularly relevant during the lactational phase of the study, where the maternal rats are already consuming two fold higher levels of the test substance, plus the additional systemic exposure due to saturated elimination kinetics.

Therefore, though the study NOAEL of 100 ppm (11 mg/kg bw/d) is accurate, in the context of risk assessment it does not represent meaningful evidence of increased pup sensitivity but rather an effect that is limited to a high-dose restricted mode(s) of action that is not relevent to humans.

 

 

Supporting study: Two-generation reproduction toxicity 2013/8001785

The test substance was administered to BrlHan:WIST@Jcl(GALAS) rats (24 males and 24 females per group) via the diet at dose levels of 0, 100, 300, and 1000 ppm over two successive generations to evaluate the potential effects on reproductive performance of parental animals and on growth and development of their offspring.

 

No changes in general appearance or deaths related to the test substance administration were observed in either sex of F0 and F1 parental animals in any of the treated groups.

Body weights, body weight gains and food consumption were not affected by the test substance treatment even in males of the 1000 ppm group, as well as in both sexes of parental animals in the 100 and 300 ppm groups. However, food consumption of both F0 and F1 females decreased significantly in the 1000 ppm group during the lactation period.

Postmortem examinations of parental animals also revealed no effects of the test substance administration in the 100 and 300 ppm groups. In the 1000 ppm group, however, the absolute and/or relative liver weights of males and absolute and relative adrenal weights of females increased significantly in both generations. Weight changes were not accompanied by histopathological abnormalities.

 

Reproductive performance of parental animals was not affected in any test substance-treated groups. Although mean days of age at completion of preputial separation in F1 males of the 1000 ppm group significantly exceeded the control value, this was within the historical control range and considered not to be an indication of delay in sexual maturation but merely a suggestion of growth retardation because body weights of F1 males on the day of completion did not differ significantly from the control value even in the 1000 ppm group. The number of primordial follicles in F1 females of the 1000 ppm group was comparable to that of the control group.

 

The test substance treatment did not affect offspring in such parameters as litter sizes, sex ratios, general appearances, viability indices, lactation indices, anogenital distances, body weights, reflex responses, necropsy findings and organ weights in the 100 and 300 ppm groups. In the 1000 ppm group, body weights of male and female pups were significantly lower than their respective controls on postnatal day 21 in both F1 and F2 generations.

 

These results lead to the conclusion that the NOAEL is 300 ppm (corresponding to 24.1 mg/kg bw/d) for systemic toxicity on parental animals and for growth and development of their offspring. It is also concluded that the test substance does not disturb parental reproductive performance up to the highest dose level of 1000 ppm (corresponding to 79.0 mg/kg bw/d) under the present study conditions.

 

 

Supporting study: Dose-range finding study for two-generation reproduction toxicity 2009/7010953

In the scope of a range finding study for the 2-generation study (2013/8001785) the test substance was administered to groups of 8 male and 8 female rats at doses of 0, 150, 1500, 3000, and 6000 ppm. Rats were pre-dosed for 2 weeks, and dosed through mating, gestation and lactation.

Body weight gain was significantly decreased in males of the 3000 and 6000 ppm dose group starting from week 2 or 1 of treatment until week 5, being below control levels until termination. Female rats of the high dose group (6000 ppm) exhibited body weight loss in week 1 and significantly reduced body weight until their termination. Females of the 3000 ppm dose group had significantly decreased body weight starting from day 20 of gestation until termination.

Food consumption was significantly reduced at several time points in males starting from the 3000 ppm dose group. Females showed significantly lower food consumption at several time points starting from 3000 ppm in the premating interval. Significantly lower food consumption was also observed in the female animals during the whole gestation period and the entire lactation period (≥ 1500 ppm). A number of organs showed significant reductions in absolute weight (brain, pituitary, epididymides, seminal vesicles, prostate) and alterations in relative weight (liver, spleen, adrenals, seminal vesicle, prostate) in male rats. Females exhibited a significant increase in absolute and relative liver weights as well as significantly increased relative weights of the thymus and adrenals.

Estrous cyclicity was significantly increased in high dose dams. Dams of the 6000 ppm dose group showed increased length of gestation, reduced numbers of implantations and number of pups delivered and all pups died between lactation day 0 and 4. The number of implantations was also reduced in dams of the 3000 ppm dose group. Sex ratio and viability index at birth were not affected. The number of pups lost or found dead increased in a dose-dependent manner.

Both male and female pups had significantly decreased body weight at doses 1500 ppm, although body weight at birth was comparable. At necropsy male pups showed decrease absolute (3000 ppm) and relative (≥ 1500 ppm) weight of the brain of absolute thymus weight (≥ 1500 ppm) and absolute and relative weight of the spleen (≥ 1500 ppm). Female pups showed decreased absolute (3000 ppm) and relative (≥ 1500 ppm) weight of the brain of absolute thymus weight (≥ 3000 ppm) and absolute and relative weight of the spleen (≥ 1500 ppm) as well as a decrease of absolute uterus weight (3000 ppm).

Based on these findings doses of 100, 300, and 1000 ppm were chosen for the two-generation reproductive toxicity study (2013/8001785).

Supporting: One-generation reproductive toxicity 2012/1090369

The objective of this study was to compare the possible adverse effects of two batches (standard and high-purity batch) of the test substance on reproduction. The test substance was administered to groups of 25 male and 25 female healthy young Wistar rats (F0 parental generation) as a constant homogeneous addition to the food at concentrations of 0 and 1500 ppm. At least 75 days after the beginning of treatment, F0 animals were mated to produce a litter (F1 generation). Mating pairs were from the same dose group. The study was terminated with the terminal sacrifice of the F0 parental animals. Test diets containing containing the test substance were offered continuously throughout the study.

The overall mean dose administered to the male and female Wistar rats during the entire study period was approx. 154 mg/kg bw/d (standard batch) and approx. 156 mg/kg bw/d (high purity batch).

 

Under the conditions of the present one-generation study no substantial difference was observed between the two batches of the test substance when tested for effects on general systemic toxicity, fertility and reproductive performance, as well as developmental toxicity in pre-weaning F1 offspring. The tested dose of 1500 ppm caused systemic toxicity in the F0 parents (reduced food consumption, clinical-pathological changes) and a corresponding slight inhibition of preweaning pup development.

 

 

Supporting: Cross Fostering 2012/1016029

The test substance was administered to groups of 20 male and 20 female healthy young Wistar rats (F0 parental generation) during different study phases as a constant homogeneous addition to the food in different concentrations (0 and 1500 ppm). At least 76 days after the beginning of treatment, F0 animals were mated to produce a litter (F1 generation). Mating pairs were from the same dose group. As information was sought on whether exposure of the offspring during in utero and/or lactational windows resulted in neonatal morbidity or mortality, litters of unexposed dams were cross-fostered with litters of dams which were exposed to the test substance during these specific periods of reproduction. The diets containing the test substance were offered continuously throughout the different study phases.

The overall mean dose administered to the male and female Wistar rats during the entire study period was approx. 156 mg/kg bw/d in the 1500 ppm dose groups. A supplementary control group was added to this study to clarify the decreased viability index in cross-fostered pups after cesarean section. For this purpose, 50 time-mated animals of the same rat strain and source were delivered on gestation day 0 (GD 0) aged 10-12 weeks. The animals remained untreated. The study was terminated with the terminal sacrifice of control weanlings and control parental females.

 

Under the conditions of the present cross-fostering reproduction toxicity study the NOAEL for general, systemic toxicity is below 1500 ppm for the F0 parental rats, based on decreased food consumption observed at 1500 ppm, particularly during lactation in the F0 parental female animals.

 

The NOAEL for fertility and reproductive performance for the F0 parental rats is at least 1500 ppm, the highest dose tested. Postnatal survival and viability was unaffected by treatment.

 

The NOAEL for developmental toxicity in the F1 progeny is below 1500 ppm, due to the reduced pup body weight parameters in animals exposed during lactation. Importantly, no developmental toxicity occurred in the offspring fostered, which were exposed in utero, but not during lactation. In addition, no difference was detected between the developmental effects in the pups fostered in groups exposed postnatal via the milk and postnatal via milk after dams were pre-treated prenatally. Taken together, the study demonstrates that there are no direct or indirect consequences to the offspring of dosing during the premating or gestation periods and that the effect on pups is clearly a post-natal effect that occurs during the lactation period.

Effects on developmental toxicity

Description of key information

Multiple studies were conducted. In the key rabbit developmental study (2011/8000161) there were no adverse effects from the test substance in maternal animals at any of the doses tested (0, 8, 16, 32 mg/kg bw/d). Additionally, there were no treatment-related adverse effects on fetuses at any of the doses tested. Under the conditions of this study, both the maternal and fetal NOAEL was determined to be 32 mg/kg bw/d.

A dose finding supporting study (2016/1112766) with rabbits orally treated via gavage with doses of 0 and 70 mg/kg bw/d was conducted. At 70 mg/kg bw/d marked general toxicity was observed (body weight effects, feeding arrest and an abortion). In combination with the rabbit kinetic study (2020/2080793) it was  demonstrated that the super linear increase of the internal dose is already present at 12.5 mg/kg. This indicated that the internal metabolism or the excretion of the test substance is saturated leading to a kinetically derived maximum dose. Therefore, 70 mg/kg bw/d is clearly not a dose suitable for testing of a teratogenic potential. Thus, the maternal and fetal NOAEL of 32 mg/kg bw/d from the key study (2011/8000161) is confirmed.

In the key rat developmental study (2014/8000288) the test substance was administered orally, via gavage, at a dosage of 0, 50, 100, or 200 mg/kg bw/d. The dose level of 200 mg/kg bw/d produced overt maternal toxicity, as evidenced by body weight loss and one death. The dose level of 100 mg/kg bw/d also produced a slight but statistically significant reduction in food consumption during gestation days 6 -9. For fetal parameters, treatment-related effects were limited to a statistically significant increase in the litter incidences of several skeletal variations as supernumerary rib and zygomatic bone fused with maxilla in the 200 mg/kg bw/d group. These findings were only observed at a maternally toxic dose level that exceeds a kinetically derived maximum tolerated dose. The NOAEL for maternal and fetal (developmental) toxicity was 50 and 100 mg/kg bw/d, respectively.

On the basis of the result from the availabe developmental toxicity studis it is concluded that the test substance did not cause malformations in the rabbit or the rat, and therefore is not considered teratogenic.

A separate whitepaper summarizing some of the particular findings in the rat and rabbit developmental toxicity studies is available and attached in IUCLID Chapter 13.

 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From: June 12, 2013 To: September 25, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: MAFF in Japan, 12-Nousan-No. 8147, 2-1-18, 2000

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Lot number: 080722
Expiry: July 25, 2015

Species:

rat

Strain:

Wistar

Remarks:

(BrlHan:WIST@Jcl[GALAS])

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Source: Fuji Breeding Center, CLEA Japan, Inc. (Shizuoka, Japan)
Age at study initiation: about 13-14 weeks
Weight at study initiation: 209-246 g for females
Housing: animals were housed in suspended wire-mesh stainless steel cages (width 310 x depth 440 x height 230 mm) in groups of 4 males/cage and 5 females/cage. Aluminum cages with wire-mesh floors and fronts (width 260 mm x depth 400 mm x height 240 mm) were used for the mating (1 pair/cage). Copulated females wer ehoused individually.
Diet: ad libitum males were supplied with certified solid feed (MF; Oriental Yeast Co., Ltd., Tokyo, Japan), and females with certified pulverized feed (MF Mash; Oriental Yeast Co., Ltd., Tokyo, Japan)
Water: ad libitum local tap water (Joso-shi Water Supply, Ibaraki, Japan)
Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
Temperature: 22 ± 2 °C
Humidity: 50 ± 20 %
Air changes: at least 10 times per hour
Photoperiod: 12 h light / 12 h dark; lights on at 7:00 a.m. and off at 7:00 p.m.

IN-LIFE DATES: From: June 25, 2013 To: October 10, 2013

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1%

Details on exposure:

The test substance was administered to the animals at approx. the same time in the morning. The volume administered each day was 10 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance was confirmed in a previous study in which the test substance was stable for at least 15 days after preparation. For verification of stability the samples were retained at ambient temperature of the animal room for 1 day, after storing them in a sealed cylinder in a cold and dark place for 14 days.

Samples were taken from the middle layer of the graduated cylinder and verified that the test substance was presented at the target concentrations in each dosing formulation. Dosing formulations for the low- and high-dose levels were further analyzed for homogeneity of the test substance at the first preparation.

The results of homogeneity analyses demonstrated that relative standard deviations (RSD) values of mean concentrations of the test substance in dosing formulations for the low- and high-dose groups were 0.6 % and 0.4 %, respectively, indicating that the test substance was uniformly distributed in the dosing formulations.
The results of the concentration analyses showed that the test substance was detected in the samples from each dosing formulation, which was administered to animals, at a range of 103-106 % of the nominal concentrations.

Details on mating procedure:

Vaginal smears were taken from females for microscopic examination. Then, females showing proestrus or estrus vaginal smears were paired overnight with males on a 1:1 basis. The females were examined next morning for the presence of vaginal plugs and sperm in vaginal smears, and those showing vaginal plugs and/or sperm were considered to have copulated. These mating procedures were repeated for 4 days.

Duration of treatment / exposure:

Gestation day (GD) 6-19

Frequency of treatment:

Daily

Duration of test:

20 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24 pregnant rats/dose

Control animals:

yes, concurrent vehicle

Details on study design:

PREPARATION OF DOSING SOLUTION
Dosing formulations were prepared for each dose level by suspending a specified amount of the test substance (mg/10 mL/kg body weight) in an aqueous suspension of 1 % sodium carboxymethylcellulose. Dosing formulations were prepared twice at an interval of approximately 14 days.

Maternal examinations:

CAESAREAN SECTION
- All surviving females wer euthanised by carbon dioxide inhalation on GD 20. Gravid uteri were removed and fetuses examined.

CAGE SIDE OBSERVATION
- Clinical signs and mortality: twice a day during the dosing period.
- Detailed physical examination was conducted when they were weighed or dosed.

BODY WEIGHT
- Each female was weighed on GD 0, 6, 9, 12, 15, 18 and 20.
- Adjusted body weights were calculated by subtracting the gravid uterine weight from the body weight on GD 20.

BODY WEIGHT GAINS
- Calculated by subtracting the body weight value on GD 6 from each value determined on GD 9, 12, 15, 18 and 20.
- Adjusted body weight gains were calculated by subtracting the gravid uterine weight from the body weight gain during GD 6-20.

FOOD CONSUMPTION
- The amount of food supplied and/or unconsumed was determined on the day of body weight measurement.
- Daily food consumption (g/rat/day) was calculated by dividing values of the total food consumption by the number of days.

NECROPSY AND TISSUE PRESERVATION
Females euthanized on GD 20 were necropsied and pathological findings were recorded. One deceased female was necropsied immediately after discovery and findings were recorded. The organs and tissues were not preserved.

Ovaries and uterine content:

- Gravid uterine weight, numbers of corpora lutea and implants were determined and recorded.


The gravid uteri of females surviving to GD 20 were weighed. When no conceptus was grossly evident, the apparently non-pregnant uterus was stained with 10 % ammonium sulfide solution to detect very early resorptions. The data (clinical signs, body weights, body weight gains, food consumption, gross pathological findings, gravid uterine weights, the numbers of corpora lutea and implants, and percent pre-implantation losses) from females, which had no grossly observable
conceptus in the uteri, was excluded from statistical evaluation.

Fetal examinations:

NUMBER OF LIVE FETUSES AND PERCENT INCIDENCE OF RESOPRTIONS AND FETAL DEATHS
The numbers of live and dead fetuses were recorded with their positions in the uterus. Resorbed embryos or dead fetuses were classified into implantation sites, placental remnants, or macerated fetuses (including dead term fetuses) according to the developmental stage in which resorptions or deaths occurred; an implantation site was a metrial gland with no remnant of placenta or embryo, a placental remnant was the placenta being resorbed with little or no embryonic tissue, a macerated fetus was an embryo or fetus being resorbed or a fetus that died shortly before necropsy.

SEX RATIO, FETAL BODY WEIGHTS AND PLACENTAL WEIGHTS
Sex of each live fetus was determined, and fetal body weights and placental weights were recorded and calculated.

EXTERNAL, VISCERAL AND SKELETAL EXAMINATION
Live fetuses in a litter were individually identified by their uterine position. Then, they were examined for external and orifice abnormalities. Animals were euthanised by an intra-peritoneal injection of a pentobarbital sodium solution (Somnopentyl).

Then the fetuses were assigned serial numbers within a litter beginning at the nearest site of the right ovary in order down the right uterine horn, across cervix, and up the left horn to the nearest site of the left ovary. In odd-numbered fetuses, the thoracic and abdominal soft tissue was examined for visceral abnormalities according to the fresh visceral examination method of Stuckhardt and Poppe. The fetuses were then fixed in Bouin's solution along with the placentas. After fixation for one week or more, sections of the decapitated head were made using Wilson's razor blade sectioning technique, and the eyes, brain, nasal passages, and tongue were observed. The examined tissue of the head was preserved in Bouin's solution along with the other tissue. Even-numbered fetuses were fixed in 70 % ethanol, stained with alizarin red S and alcian blue, and cleared in 70 % glycerin for making the skeletal specimens. After examination, skeletal specimens were stored.

Statistics:

Statistical significance: α=0.05 or 0.01

Body weights, adjusted body weights, body weight gains, adjusted body weight gains, and food consumption of maternal animals, the numbers of corpora lutea, implants, and live fetuses, and the weights of gravid uteri, fetuses, and placentas: Equality of variances was first evaluated by Bartlett's test. If homogeneous, a parametric analysis of variance in one-way classifications was used to determine if any statistical differences exist among groups. If the analysis of variance would be significant, Dunnett's multiple comparison test was performed to detect any statistically significant differences between the treated groups and their corresponding controls. When Bartlett's test indicates that the variances would not be homogeneous, Kruskal-Wallis test was used for detecting any statistical differences among groups and if significant, Dunnett-type nonparametric multiple comparison test was performed to detect statistical differences between the treated groups and their corresponding controls.

Percent pre-implantation losses, percent incidences of resorptions and fetal deaths, and percent litter incidences of fetal malformations or variations were evaluated as follows: Kruskal-Wallis test was used for detecting any statistical differences among groups and if significant, Dunnett-type nonparametric multiple comparison test was performed to detect statistical differences between the treated groups and their corresponding controls. As for the data on the incidences of clinical and gross pathological findings in maternal animals, incidences of maternal animals having fetuses with malformations or variations, and fetal sex ratio, chi-square test was used when all expected values of control and treated groups were 5 or more, and Fisher's exact probability test was used when any expected values of control and treated groups were less than 5.

Indices:

- Percent pre-implantation losses = [(number of corpora lutea-number of implants)/number of corpora lutea] x100

- Percent resorptions and fetal deaths (percent post-implantation losses) = (total number of resorbed embryos and dead fetuses/number of implants) x100
- Sex ratio = total number of male fetuses/total number of live fetuses

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related findings in clinical observations, except for the dead female.

Incidental findings noted during the study included loose stool in 1 female in the control group (dosing period); and loss of fur in 1-3 females among the 50, 100 and 200 mg/kg bw/d groups (dosing and post-dosing periods).

Mortality:

mortality observed, treatment-related

Description (incidence):

One female in the 200 mg/kg bw/d group was found dead, expectorating bloody liquid, shortly after the treatment on gestation day 19. Necropsy findings suggest that this death was treatment-related. No other deaths occurred during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

BODY WEIGHT
In the 200 mg/kg group, a statistically significant decrease in maternal weight gains was seen during gestation days 6-9, during which a weight loss occurred in 6 out of 22 females. Subsequent mean values in the high-dose group were apparently comparable to those in the control group. However, this was not a clear indication of recovery, but rather being attributable to the suppression of body weight gains in the control females.

BODY WEIGHT GAINS
Body weight gains in the 50 and 100 mg/kg groups were generally comparable to or greater than those in the control group throughout the study, with the increase being statistically significant in the 100 mg/kg group during gestation days 6-15 and 6-18.
There was no significant difference in adjusted body weight gains of females between the control and any treated group

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the 200 mg/kg group, food consumption was moderately reduced during gestation days 6-9 when compared to the control and continued to be low until gestation days 12-15 (74-93% of control values). Statistical analysis revealed a significant decrease in food consumption by high-dose females during gestation days 6-9, 9-12 and 12-15. In the 100 mg/kg group, a slight but statistically significant decrease in food consumption was seen during gestation days 6-9 (86% of control value), although subsequent values were comparable to those in the control group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Aside from uterus weight, organ weights were not collected.

There were no statistically significant differences in the numbers of corpora lutea and implants, or gravid uterine or placental weights between the control and any treated group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The dead animal in the 200 mg/kg group showed a red discolored mucosa of the ileum and lung congestion at necropsy. The red discoloration of ileum suggests that there was an intestinal hemorrhage. Despite the lung congestion, there was no evidence of esophageal and/or tracheal injury that might be attributable to gavage error.

There were no treatment-related findings observed in the necropsy of surviving females in the other dose groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

No differences between treated and control groups.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No differences between treated and control groups.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No differences between treated and control groups.

Early or late resorptions:

no effects observed

Description (incidence and severity):

No differences between treated and control groups.

Dead fetuses:

no effects observed

Description (incidence and severity):

No differences between treated and control groups.

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): No differences between treated and control groups.

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in fetal weights of both sexes between treated and control groups.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

All surviving females except two animals in the 200 mg/kg bw/d group had live fetuses; the total number of pregnant females examined was 24, 24, 24 and 21 in the control, 50, 100 and 200 mg/kg bw/d groups, respectively. Two females having no live fetuses did not have grossly visible conceptuses in their uteri. Subsequent examination by 10 % ammonium sulfide staining did not detect any early resorption sites in their uteri, indicating that these females were non-pregnant. The dead female in the 200 mg/kg bw/d group had some fetuses in the uterus, although not evaluated in this study.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the sex ratio between treated and control groups.

Changes in litter size and weights:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Among all live fetuses, external malformations occurred in a total of 4 fetuses. The findings included omphalocele in 1 fetus from the control group (0.28 %), local edema in 1 fetus from the 100 mg/kg bw/d group (0.32 %), and cleft palate in 2 fetuses from a single litter of 200 mg/kg bw/d group (0.79 %). There was no statistically significant difference in the litter incidences of external malformations between the control and any treated group.

Cleft palates occurred in two high-dose fetuses from one single litter. These fetuses weighed 2048 mg and 1906 mg while their normal littermates weighed 2231-3560 mg, and were recognized as the two smallest fetuses in this litter. Hence, the affected fetuses were much smaller than their littermates and weighed less than 60 % of their groupmates (average high-dose fetal weight 3571 mg).

From this notable weight and size decrement it is likely that the process of palatal closure was disrupted perhaps by the accidental delay in fetal development and could not be caught up in these two fetuses after passing the critical window. Hence these cleft palates are considered to be a manifestation of a very distinct developmental delay rather than as a specific malformation. It is regarded to be highly unlikely that these cleft palates were caused by a specific teratogenic potential of the test substance.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

MALFORMATIONS
Skeletal malformations were found in 4 and 1 fetuses from the control and 100 mg/kg bw/d groups, respectively. In the control group, 1 fetus exhibited multiple malformations in cervical and thoracic skeleton (including fused cervical centrum cartilage, fused sternebra and 1st rib cartilage not fused to sternum) together with fused phalanx. Another fetus in the same group had fused sternebra and fused rib cartilage, while the remaining 2 fetuses exhibited 1st rib cartilage not fused to sternum. In the 100 mg/kg bw/d group, the affected fetus had fused cervical centrum cartilage accompanied by fused cervical arch. Mean litter incidence of each skeletal malformation in the control and 100 mg/kg group ranged from 0.00 % to 1.89 % and from 0.00 % to 0.60 %, respectively. Mean litter incidence of fetus having any skeletal malformations in these two groups were 2.48 % and 0.60 %, respectively. Statistical analysis also showed significantly lower litter incidences of 1st rib cartilage not fused to sternum and fetus having any skeletal malformations, for the 50, 100 and/or 200 m/kg bw/d groups, when compared to the control: however, these are thought to be toxicologically meaningless fluctuations.

VARIATIONS
There were also many skeletal variations found in all groups including controls. Although these skeletal variations consisted exclusively of those commonly observed in term rat fetuses such as cervical rib, discontinuous rib cartilage, and 27 presacral vertebrae, a few findings listed below showed increased litter incidences in the 200 mg/kg bw/d fetuses.

94.36 % of fetuses in the 200 mg/kg bw/d group had one or more skeletal variations, one of which was supernumerary rib in almost all cases. In addition, a significantly increased incidence was noted for zygomatic bone fused with maxilla in the 200 mg/kg bw/d group, although the incidence was much lower than that of the supernumerary rib.

No other statistically significant difference was found in the litter incidence of any skeletal variation between the control and treated group.

There was no statistically significant difference between the control and any treated group in the incidence for either females having fetuses with malformations or those having fetuses with variations.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

MALFORMATIONS
There were no visceral malformations in all fetuses examined (half of the live fetuses obtained) except one in the 100 mg/kg bw/d group. The fetus that had local edema further exhibited the following visceral malformations: microphthalmia, small nasal cavity and complex cardiovascular malformations consisting of right-sided aortic arch, overriding aorta, narrowed pulmonary trunk and narrowed left subclavian, with the mean litter incidences of 0.60 %.

VARIATIONS
Visceral variations were found in in all groups including the control; these included left umbilical artery (13.79 %-18.50 %), dilated renal pelvis (0.00 %-0.68 %) and thymic remnant in the neck (0.00 %-0.69 %). Statistical analysis revealed no significant difference in either the litter incidences of visceral malformations or those of visceral variations between the control and any treated group.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: slightly increased incidence of skeletal variations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: rib

Description (incidence and severity):

variation: increased incidence of supernumerary rib at maternal toxic dose of 200 mg/kg bw/d

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: skull

Description (incidence and severity):

variation: increased incidence of zygomatic bone fused with maxilla at maternal toxic dose of 200 mg/kg bw/d

Key result

Developmental effects observed:

no

Maternal food consumption

Dose level [mg/kg]	0	50	100	200
Day 0 - 6 SD	17.4 1.5	17.7 1.4	17.3 1.6	17.9 2.1
Day 6 - 9 SD	18.7 2.3	18.0 1.6	16.1** 2.5	13.8** 2.3
Day 9-12 SD	19.2 3.5	19.2 2.0	18.4 1.7	16.7** 2.4
Day 12-15 SD	19.4 3.0	19.6 1.7	18.9 1.8	18.1* 2.1
Day 15-18 SD	21.5 1.7	21.7 1.5	21.6 1.5	21.5 2.7
Day 18-20 SD	21.7 2.1	21.8 1.9	21.0 2.0	19.8 4.0

* p < 0.05, ** p < 0.01 (Dunnett test, two-sided)

Body weight [g]

Dose level [mg/kg]	0	50	100	200
Day 0 SD	229 11	229 12	229 11	228 11
Day 6 SD	250 10	249 12	249 11	248 13
Day 9 SD	260 12	258 11	258 11	250* 12
Day 12 SD	270 16	273 13	271 10	267 15
Day 15 SD	284 15	287 12	288 11	281 14
Day 18 SD	318 15	321 15	324 11	314 19
Day 20 SD	348 16	351 19	354 14	342 23

* p < 0.05, ** p < 0.01 (Dunnett test, two-sided)

Body weight gain [g]

Dose level [mg/kg]	0	50	100	200
Day 6-9 SD	9 4	9 3	8 5	2** 6
Day 6-12 SD	20 11	23 4	22 6	19 7
Day 6-15 SD	33 11	38 4	39* 7	33 8
Day 6-18 SD	68 9	72 6	75* 9	66 14
Day 6-20 SD	98 10	102 11	105 11	93 20

* p < 0.05, ** p < 0.01 (Dunnett test, two-sided)

Mean gravid uterus weights and net body weight change of pregnant rats
administered the test substance during Days 6 to 19 of gestation

Dose level [mg/kg bw/d]	0	50	100	200
Gravid uterus (g)	73 ± 9	72 ± 9	76 ± 6	71 ± 15
Carcass (g)	275±12	279±16	278±12	271±17
Net weight change from Day 6 (g)	25±7	29±8	29±9	22±12
* p< 0.05

Caesarean section data

Dose level [mg/kg bw/d]	Unit of measure	0	50	100	200
Rats tested	N	24	24	24	24
Pregnant	N (%)	24	24	24	22
Dams with viable fetuses	N	24	24	24	21
Rats pregnant and caesarean-sectioned on day 20 of gestation	N	24	24	24	21
Corpora Lutea	Mean ± S.D.	15±1.5	14.6±1.1	15.0±1.0	14.8±1.1
Implantations	Mean ± S.D.	13.9±1.9	13.7±1.0	14.3±0.9	13.2±2.4
% pre-implantation losses	Mean	7.7	6.1	4.6	10.6
Litter Size	Mean ± S.D.	13.3±2.0	13.0±1.7	13.7±1.0	12.5±2.8
Live Fetuses	Mean ± S.D.	13.3±2.0	13.0±1.7	13.7±1.0	12.5±2.8
Resorptions and fetal deaths	Mean ± S.D.	0.7±4.8	0.7±5.4	0.5±3.8	0.7±5.2
Macerated fetus	Mean	0.0	0.0	0.1	0.0
Placental remnants	Mean	0.1	0.1	0.1	0.1
Implantation Sites	Mean	0.5	0.6	0.4	0.6
Dams with resorptions	N (%)	9 (37.5)	10 (41.7)	8 (33.3)	4 (19.0)
Dams with all conceptuses resorbed	N (%)	0	0	0	0
Litter Observations
% Live male fetuses/litter	Mean ± S.D.	0.519	0.497	0.505	0.529
Live Fetal body weights (mg/litter)
Male	Mean ± S.D.	3594±231	3653±223	3589±212	3571±324
Female	Mean ± S.D.	3414±210	3430±181	3420±181	3448±302
Placental weight (mg)	Mean ± S.D.	470±46	454±33	463±34	476±48

Incidences of external malformations

Dose level [mg/kg]	Measure	0	50	100	200
Litters Evaluated	N	24	24	24	21
Fetuses Evaluated	N	318	312	329	263
Litters with external malformations	N	1	0	1	1
Fetuses with external malformations	N	1	0	1	2
Omphalocele
Number of affected litters	N (%)	1 (4 %)	0	0	0
Number of affected fetuses	N	1	0	0	0
Mean incidence of affected fetuses in litters ± SD	(%)	0.28±1.36	0	0	0
Local edema
Number of affected litters	N (%)	0	0	1 (4 %)	0
Number of affected fetuses	N	0	0	1	0
Mean incidence of affected fetuses in litters ± SD	(%)	0	0	1	0
Cleft palate
Number of affected litters	N (%)	0	0	0	1
Number of affected fetuses	N	0	0	0	2
Mean incidence of affected fetuses in litters ± SD	(%)	0	0	0	0.79±3.64

Incidence of visceral malformation

Dose level [mg/kg]	Measure	0	50	100	200
Litters Evaluated	N	24	24	24	21
Fetuses Evaluated	N	165	150	169	135
Litters with visceral malformations	N	0	0	1	0
Fetuses with visceral malformations	N	0	0	1	0
Right-sided aortic arch
Number of affected litters	N (%)	0 (0%)	0 (0%)	1 (4 %)	0 (0 %)
Number of affected fetuses	N	0	0	1	0
Mean incidence of affected fetuses in litters ± SD	(%)	0	0	0.6±2.92	0
Overriding aorta
Number of affected litters	N (%)	0 (0 %)	0 (0 %)	1 (4%)	0 (0 %)
Number of affected fetuses	N	0	0	1	0
Mean incidence of affected fetuses in litters ± SD	(%)	0	0	0.6±2.92	0
Narrowed pulmonary trunk
Number of affected litters	N (%)	0 (0 %)	0 (0 %)	1 (4%)	0 (0 %)
Number of affected fetuses	N	0	0	1	0
Mean incidence of affected fetuses in litters ± SD	(%)	0	0	0.6±2.92	0
Narrowed left subclavian
Number of affected litters	N (%)	0 (0 %)	0 (0 %)	1 (4%)	0 (0 %)
Number of affected fetuses	N	0	0	1	0
Mean incidence of affected fetuses in litters ± SD	(%)	0	0	0.6±2.92	0
Small nasal cavity
Number of affected litters	N (%)	0 (0 %)	0 (0 %)	1 (4%)	0 (0%)
Number of affected fetuses	N	0	0	1	0
Mean incidence of affected fetuses in litters ± SD	(%)	0	0	0.6±2.92	0
Microphthalmia
Number of affected litters	N (%)	0 (0%)	0 (0%)	1 (4%)	0 (0%)
Number of affected fetuses	N	0	0	1	0
Mean incidence of affected fetuses in litters ± SD	(%)	0	0	0.6±2.92	0

Incidence of visceral variations

Dose level [mg/kg]	Measure	0	50	100	200
Litters Evaluated	N	24	24	24	21
Fetuses Evaluated	N	165	162	169	135
Litters with variations	N	17	16	17	15
Fetuses with variations	N	28	25	31	19
Left umbilical artery
Number of affected litters	N (%)	17 (71 %)	16 (67 %)	17 (71 %)	14 (67 %)
Number of affected fetuses	N	28	25	31	18
Mean incidence of affected fetuses in litters ± SD	(%)	16.75 ± 13.59	15.58 ±14.89	18.50 ±14.64	13.79 ±12.68
Dilated renal pelvis
Number of affected litters	N (%)	1 (4%)	0	0	1 (5%)
Number of affected fetuses	N	1	0	0	1
Mean incidence of affected fetuses in litters ± SD	(%)	0.52 ±2.55	0.00 ±0.00	0.00 ±0.00	0.68 ±3.12
Thymic remnant in the neck
Number of affected litters	N (%)	1 (4%)	1 (4%)	0	0
Number of affected fetuses	N	1	1	0	0
Mean incidence of affected fetuses in litters ± SD	(%)	0.69 ±3.40	0.60 ±2.92	0.00 ±0.00	0.00 ±0.00

Incidence of skeletal malformations

Dose level [mg/kg]	Measure	0	50	100	200
Litters Evaluated	N	24	24	24	21
Fetuses Evaluated	N	153	150	160	128
Litters with skeletal malformations	N	4	0	1	0
Fetuses with skeletal malformations	N	4	0	1	0
Fusion between occipital and first cervical centrum
Number of affected litters	N (%)	1	0	0	0
Number of affected fetuses	N	1	0	0	0
Mean incidence of affected fetuses in litters ± SD	(%)	0.69±3.40	0	0	0
Fused cervical centrum cartilage
Number of affected litters	N (%)	1	0	1	0
Number of affected fetuses	N	1	0	1	0
Mean incidence of affected fetuses in litters ± SD	(%)	0.69±3.40	0	0.60±2.92	0
Fused cervical arch
Number of affected litters	N (%)	0	0	1	0
Number of affected fetuses	N	0	0	1	0
Mean incidence of affected fetuses in litters ± SD	(%)	0	0	0.60±2.92	0
Split cartilage of ventral arch
Number of affected litters	N (%)	1	0	0	0
Number of affected fetuses	N	1	0	0	0
Mean incidence of affected fetuses in litters ± SD	(%)	0.69±3.40	0	0	0
Fused sternebra
Number of affected litters	N (%)	2	0	0	0
Number of affected fetuses	N	2	0	0	0
Mean incidence of affected fetuses in litters ± SD	(%)	1.29±4.38	0	0	0
Rib cartilage not fused to sternum (1st)
Number of affected litters	N (%)	3	0	0	0
Number of affected fetuses	N	3	0	0	0
Mean incidence of affected fetuses in litters ± SD	(%)	1.89±5.11	0	0	0
Fused rib cartilage
Number of affected litters	N (%)	1	0	0	0
Number of affected fetuses	N	1	0	0	0
Mean incidence of affected fetuses in litters ± SD	(%)	0.60±2.92	0	0	0
Fused phalanx
Number of affected litters	N (%)	1	0	0	0
Number of affected fetuses	N	1	0	0	0
Mean incidence of affected fetuses in litters ± SD	(%)	0.69±3.40	0	0	0

Mean % litter incidences of major skeletal variations

Skeletal findings	Dose levels (mg/kg/day)
	0	50	100	200
Fetuses with one or more variations	62.64	74.82	81.29	94.36**
Supernumerary rib	51.35	67.26	72.82	89.95**
Zygomatic bone fused with maxilla	3.87	0.69	5.30	13.14**

** significant different from control at p ≤ 0.01

Incidences of skeletal malformations

Dose level [mg/kg]	Measure	0	50	100	200
Litters Evaluated	N	24	24	24	21
Fetuses Evaluated	N	153	150	160	128
Litters with skeletal variations	N	23	24	24	21
Fetuses with skeletal variations	N	95	112	132	120
Zygomatic bone fused with maxilla
Number of affected litters	N (%)	3	1	6	11
Number of affected fetuses	N	6	1	8	17
Mean incidence of affected fetuses in litters ± SD	(%)	3.87±11.72	0.69±3.40	5.30±10.09	13.14±18.83**
Cervical rib
Number of affected litters	N (%)	0	2	3	3
Number of affected fetuses	N	0	2	3	3
Mean incidence of affected fetuses in litters ± SD	(%)	0	1.19±4.03	1.98±5.38	2.34±6.00
Bipartite ossification of cervical centrum
Number of affected litters	N (%)	0	1	0	1
Number of affected fetuses	N	0	1	0	1
Mean incidence of affected fetuses in litters ± SD	(%)	0	0.60±2.92	0	0.60±2.73
Bipartite ossification of sternebra
Number of affected litters	N (%)	0	0	1	0
Number of affected fetuses	N	0	0	1	0
Mean incidence of affected fetuses in litters ± SD	(%)	0	0	0.69±3.40	0
Misaligned sternebra
Number of affected litters	N (%)	1	1	2	0
Number of affected fetuses	N	1	1	2	0
Mean incidence of affected fetuses in litters ± SD	(%)	0.60±2.92	0.69±3.40	1.29±4.38	0
Discontinuous rib cartilage (false rib)
Number of affected litters	N (%)	21	38	16	19
Number of affected fetuses	N	48	19	39	38
Mean incidence of affected fetuses in litters ± SD	(%)	31.98±19.27	25.81±23.85	23.81±22.02	31.09±18.30
Branched rib cartilage (false rib)
Number of affected litters	N (%)	3	6	1	3
Number of affected fetuses	N	4	3	1	4
Mean incidence of affected fetuses in litters ± SD	(%)	3.51±11.05	4.58±13.18	0.60±2.92	2.72±7.31
Supernumerary rib
Number of affected litters	N (%)	22	24	23	21
Number of affected fetuses	N	79	101	119	114
Mean incidence of affected fetuses in litters ± SD	(%)	51.35±33.31	67.26±31.91	72.82±30.42	89.95±15.18**
Dumbbell ossification of thoracic centrum
Number of affected litters	N (%)	0	0	3 (12.5%)	1 (4.8%)
Number of affected fetuses	N	0	0	3	1
Mean incidence of affected fetuses in litters ± SD	(%)	0	0	1.89±5.11	0.79±3.64
Bipartite ossification of thoracic centrum
Number of affected litters	N (%)	0	1 (4.1%)	0	2 (8.3%)
Number of affected fetuses	N	0	1	0	2
Mean incidence of affected fetuses in litters ± SD	(%)	0	0.60±2.92	0	1.36±4.30
27 presacral vertebrae
Number of affected litters	N (%)	6 (25%)	12 (50%)	7 (29%)	2 (9.5%)
Number of affected fetuses	N	9	17	14	5
Mean incidence of affected fetuses in litters ± SD	(%)	6.13±12.46	11.67±16.11	8.63±16.45	3.40±10.98
Lumbosacral transitional vertebra
Number of affected litters	N (%)	9 (37.5%)	10 (41.7%)	9 (37.5%)	12 (57.1%)
Number of affected fetuses	N	15	13	12	18
Mean incidence of affected fetuses in litters ± SD	(%)	9.70±14.49	8.61±12.02	7.44±10.86	13.87±13.82

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and guideline compliant

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Key study (rat): Developmental toxicity 2014/8000288

A teratogenicity study in rats was conducted to evaluate the potential maternal toxicity and prenatal developmental toxicity of the test substance. The test substance was administered orally, via gavage, to groups of 22-24 pregnant female Wistar Hannover (BrlHan:WIST@Jcl[GALAS]) rats once per day from days 6 to 19 of gestation at a dosage of 0, 50, 100, or 200 mg/kg/day.

The dose level of 200 mg/kg/day produced overt maternal toxicity, as evidenced by body weight loss in some individual females during gestation days 6-9, a statistically significant decrease in mean body weights on gestation day 9, and mild to moderate reductions in food consumption during gestation days 6 through 15. One treatment-related death occurred in the 200 mg/kg group. The necropsy of the dead animal revealed red-colored ileum as well as lung congestion. The dose level of 100 mg/kg/day also produced a slight but statistically significant reduction in food consumption during gestation days 6-9. There was no treatment-related change in maternal parameters examined in the 50 mg/kg group.

Examination of ovary and uterus revealed no treatment-related effects on gravid uterine weights, the numbers of corpora lutea and implantation sites, or percent pre- and post-implantation losses in any treated group.

For fetal parameters, treatment-related effects were limited to a statistically significant increase in the litter incidences of such skeletal variations as supernumerary rib and zygomatic bone fused with maxilla in the 200 mg/kg group. No other statistically significant change was found in any fetal parameters examined in the 200 mg/kg group or other treated groups. No evidence of an increased incidence of particular malformations was detected in any treated groups through external, visceral or skeletal examination. Only sporadic malformations were observed.

Based on the results of present study, it is concluded that a dose level of 50 mg/kg bw/day of the test substance is the no-observed-adverse-effect-level (NOAEL) for maternal toxicity, based on reduced food consumption and/or body weight gain at 100 mg/kg bw/day and above. The NOAEL for developmental toxicity is 100 mg/kg bw/day of the test substance, based on a slightly increased incidence of skeletal variations at 200 mg/kg bw/d. No fetal findings were evident at any dose level that did not cause maternal toxicity. The test substance is not teratogenic under the conditions of this study.

Key study (rabbit): Developmental toxicity 2011/8000161

Female rabbits were artificially inseminated and dosed with 0, 8, 16 or 32 mg/kg bw/d of the test substance from day 6-27 of gestation. Rabbits were necropsied on gestation day 28 and subject to macroscopic examination and assessment of fetal external, visceral and skeletal abnormalities.

There were no adverse effects from the test substance in maternal animals at any of the doses tested. Additionally, there were no treatment-related adverse effects on fetuses at any of the doses tested.

Under the conditions of this study, both the maternal and fetal NOAEL was determined to be 32 mg/kg/day.

Supporting study (rat): Developmental toxicity 2013/8001786

The purpose of this study was to provide information for the selection of dosages to be used in a later study of developmental toxicity in the rat (embryo-fetal toxicity and teratogenic potential).

The test substance was administered orally via gavage to BrlHan:WIST@Jcl(GALAS) rats. There were 8 mated females per group which were dosed from Days 6 through 19 of gestation at dose levels of 0, 20, 100, 500 and 1000 mg/kg/day.

On the basis of these results, oral administration of the test substance at doses of 500 mg/kg/day and more was not considered suitable for the main study because approximately half of the maternal rats died or were euthanized due to moribundity during the dosing period, bodyweight and food consumption were reduced and there were histological changes in the heart and liver.

Absolute and relative weights of the liver and absolute weight of the heart, both of which are target organs in general toxicity studies, were significantly increased in the maternal rats at a dose of 100 mg/kg/day, although there were no corresponding histopathological changes.

Consequently, the test substance, administered at doses of approximately 100 mg/kg/day, was suggested to be suitable as the highest dose level in the main study.

Supporting study (rat): Developmental toxicity 2013/8001787

Female rats were mated (1:1) and dosed with 0, 10, 30 or 100 mg/kg bw/d from day 6-19 of gestation. Rats were necropsied on gestation day 20 and subject to macroscopic examination and assessment of fetal visceral and skeletal abnormalities.

The maternal NOAEL is set at 30 mg/kg bw/d based on transiently lowered bodyweight gains and food consumption and increased adrenal weight. The NOAEL for developmental effects is set conservatively at 30 mg/kg bw/d based on an increased incidence of lumbar ribs (a skeletal variation). Since no irrversible stuctural effects occurred the dose of 30 mg/kg bw/d repesents rather a NOEL than a NOAEL and the true NOAEL for developmental effects of the study can be considered to be 100 mg/kg bw/d.

Supporting study (rabbit): Developmental toxicity 2011/8000281

This study was conducted to select dose levels of the test substance prior to the initiation of a teratogenicity study in rabbits. The test substance was administered orally by gavage to pregnant female Japanese White (Kbl:JW) rabbits, 8 per group, once a day on days 6 through 27 of gestation at a dosage of 0, 10, 30, 100, or 300 mg/kg/day to evaluate the potential effects on maternal animals and their fetuses.

In the 10 mg/kg dose group, no effects on maternal animals or on fetal abnormalities or alterations were reported. In the 30 mg/kg dose group, body weights of maternal animals were lower and there were significantly lower values reported for body weight gain and mean food consumption. At 30 mg/kg, there were also slight increases reported for mean percent resorptions and fetal death. Fetal weight of females and placental weights were slightly lower at 30mg/kg when compared to controls.

At doses of 100 mg/kg and higher, there was a decrease in body weight and food consumption. There was also maternal death, abortion and premature delivery at the higher doses, which resulted in an insufficient number of live fetuses for evaluation following cesarean section.

Based on these results, a dose level of 30-40 mg/kg/day is recommended for the high-dose level in a definitive teratogenicity study in rabbits, which is expected to get enough live fetuses for teratological evaluation without resulting in critical effects on maternal pregnancy.

NOAELs were not established by this study.

Toxicity to reproduction: other studies

Description of key information

No study available

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008.

No test substance specific adverse effects were seen on fertility, reproductive performance and developmental toxicty which would justify a classification according to Regulation (EC) 1272/2008. The effects observed on pup development in the generational studies were post-natal effects that occurred during lactation at doses exceeding the kinetic MTD. They are considered non-relevant for classification.

In summary, the substance is not considered to be classified for toxicity to reproduction under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/918.