Registration Dossier
Registration Dossier
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EC number: 701-385-4 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
No adverse effects on reproductive organs were observed in the subchronic toxicity study in rats.
Under the conditions of the present Reproduction/Developmental Toxicity Screening Test (10 week-premating treatment) the oral administration of Reaction products of benzeneamine, N-phenyl with nonene (branched) to male and female Wistar(Han) rats via the diet revealed signs of systemic toxicity at 1500 and 5000 ppm, in concentration-dependency.
Based on altered liver parameters in clinical pathology and pathology, the no observed adverse effect level (NOAEL) for general systemic toxicity was 500 ppm for male (40 mg/kg bw/d) and female rats (44 mg/kg bw/d).
The NOAEL for reproductive performance and fertility was 1500 ppm for male (122 mg/kg bw/d) and female (133 mg/kg bw/d) rats.
The NOAEL for developmental toxicity in the offspring was 1500 ppm (133 mg/kg bw/d in parental females).
During the recovery period, the signs of systemic toxicity observed in clinical examinations decreased in degree over time. The treatment-related and adverse findings in clinical pathology of the main groups were not observed at the end of the two weeks recovery
period.
Link to relevant study records
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2019-2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- yes
- Remarks:
- ten weeks premating administration period, estrous cycle determination performed at the end of the premating administration period (in a three weeks period before the start of mating), recovery groups for high and control group
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Justification for study design:
- 10 weeks premating was requested by ECHA for the analogue substance for the e1Gen (OECD 443) study. This study was set up as the bridging study for this endpoint (COLLA).
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
BASF and 0016046440
- Expiration date of the lot/batch:
07 Jan 2020
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient (room temperatures)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: not applicable
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None
- Preliminary purification step (if any): None
FORM AS APPLIED IN THE TEST: test substance addition to the diet - Species:
- rat
- Strain:
- Wistar
- Remarks:
- Crl:Wl(Han)
- 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: 35±1 d (arrival at test facility)
- Weight at study initiation: (P) Males: 116g; Females: 96 g
- Housing: group housed - up to 5 animals/cage (pretreatment), 2 animals/cage (premating), 2 male animals/cage (mating and postmating)
- Polysulfonate cages Typ 2000P (H-Temp)
- Diet: Mouse and rat maintenance diet "GLP", Granovit AG, Kaiseraugst, Switzerland; ad libitum
- Water: Drinking water ad libitum
- Acclimation period: 7 d
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45 - 65
- Air changes (per hr): 15
- Photoperiod (hrs dar k / hrs light): 12/12
IN-LIFE DATES: From: 2019-05-21 To: 2019-09-25 (2019-10-10 recovery females) - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- PREPARATION OF DIET:
The required quantity of test substance was weighed in a beaker depending on the dose group and thoroughly mixed with a small amount of food. Then further amounts of food were
added to this premix and thoroughly mixed for 3 minutes. Afterwards, further amounts of food, depending on the dose group, were added to this premix in order to obtain the desired
concentrations.
DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with food.
- Storage temperature of food:
The food used was mouse and rat maintenance diet “GLP”, meal, supplied by Granovit AG, Kaiseraugst, Switzerland.
- Details on mating procedure:
- - M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 (GD 0) of pregnancy
- After successful mating each pregnant female was caged: individually - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- At the beginning and towards the end of the premating phases, once during gestation and once during lactation of the Study each 3 samples were taken from the lowest and highest
concentration for potential homogeneity analyses. These samples were used as aconcentration control at the same time. At the above mentioned time points additionally one
sample from the mid concentration was taken for concentration control analysis.
The test item was a mixture of components. Thus, data evaluation was based on the integration of extracted ion chromatograms, being representative for the test item.
Furthermore, spiking experiments had shown that recovery of the test item was 91% or 107% for low (~750 mg/kg) or high (~3000 mg/kg) concentrations, respectively.
These recovery rates were in the range of the error of the method and therefore the content found was not corrected by the recovery rates. The concentration control was considered
as achieved when the mean recovery of a given sample was 90% ≤ x ≤ 110%. The samplewas considered as homogeneous when the standard deviation was RSD/MW ≤ 5%.
These requirements were fulfilled for all investigated samples and thus the concentration control as well as homogeneity was achieved - Duration of treatment / exposure:
- 12 weeks (males) (10 weeks premating+2 weeks mating)
16 weeks (females) (10 weeks premating+2 weeks mating + 3 weeks gestation + 13 days lactation) - Frequency of treatment:
- daily for 7 days a week
- Details on study schedule:
- - Age at mating of the mated animals in the study: 15 weeks
- Dose / conc.:
- 500 ppm
- Remarks:
- dose applied to animals during the premating and mating phase (males and females), postmating (males) and gestation phase and after weaning (females)
- Dose / conc.:
- 1 500 ppm
- Remarks:
- dose applied to animals during the premating and mating phase (males and females), postmating (males) and gestation phase and after weaning (females)
- Dose / conc.:
- 5 000 ppm
- Remarks:
- dose applied to animals during the premating and mating phase (males and females), postmating (males) and gestation phase and after weaning (females);
dose was also applied to recovery animals - Dose / conc.:
- 250 ppm
- Remarks:
- dose applied to F0 females during lactation phase to maintain dams at desired target dose of the test substance: adjustment to historical body weight and food consumption data
- Dose / conc.:
- 750 ppm
- Remarks:
- dose applied to F0 females during lactation phase to maintain dams at desired target dose of the test substance: adjustment to historical body weight and food consumption data
- Dose / conc.:
- 2 500 ppm
- Remarks:
- dose applied to F0 females during lactation phase to maintain dams at desired target dose of the test substance: adjustment to historical body weight and food consumption data
- No. of animals per sex per dose:
- 10
(plus 10 for control and high dose for recovery group) - Control animals:
- yes
- Details on study design:
- - Dose selection rationale:
requested by the sponsor
- Rationale for animal assignment: rats will be randomized according to their weight and allocated to the dose groups
- Fasting period before blood sampling for clinical biochemistry: 16-20 hours - Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule for moribund and dead animals: twice daily (Mon-Fri), once daily (Sat, Sun and public holidays)
- Time schedule for clinical signs: at least once daily, if signs occur: several times daily
DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:
BODY WEIGHT: Yes
- Time schedule for examinations: once a week for animals in the main group and the recovery group
- not determined in females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period
- during mating, females will be weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20
- females with litter will be weighed on the day after parturition (PND1) and on PND 4, 7, 10 and 13
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption will be determined once a week for the male and female parental animals of main groups as well as the males and females of recovery groups.
- not be determined during mating, in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period
- females with evidence of sperm: food consumption will be determined for GD 0-7, 7-14 and 14-20
- females which gave birth to a litter: food consumption will be determined for PND 1-4, 4-7, 7-10 and 10-13
OTHER: - Oestrous cyclicity (parental animals):
- - in all parental females in the premating phase: estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 3 weeks prior to mating and throughout cohabitation until there was evidence of sperm in the vaginal smear
- in all females of the recovery groups: estrous cycle length and normality will be evaluated in the 2 weeks of the recovery period
- in all parental females: estrous status was determined on the day of scheduled sacrifice - Sperm parameters (parental animals):
- Parameters examined in F0 male parental generations and males of the recovery group:
- after organ weight determination, parameters determined in the right testis or right epididymis:
- Cauda epididymis sperm motility
- Sperm morphology
- Spermatid head count in the testis
- Sperm head count in the cauda epididymis
Parameters exmined for the control and highest dose group:
- Sperm morphology and sperm head count (cauda epididymis and testis) - Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.
- Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: All surviving animals after the end of the administration period the last litters in each generation were produced.
- Female animals: All surviving animals after the last litter of each generation was weaned (PND 14).
- Recovery animals: All surviving animals will be maintained for at least two weeks (recovery period) longer compared to the respective main groups.
GROSS NECROPSY
- Gross necropsy performed for all animals in the main and the recovery group
- consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera
- special attention being given to reproductive organs
HISTOPATHOLOGY / ORGAN WEIGHTS
Organs/Tissues weighed for...:
- all animals (main test and recovery group): anesthetized animals (final body weight), Epididymides, Ovaries, Prostate (ventral and dorsolateral part together, fixed), Seminal vesicles with coagulating glands (fixed), Testes, Thyroid glands (with parathyroid glands) (fixed), Uterus with cervix
- 5 animals/sex/test group (main test and recovery group): Adrenal glands (fixed), Brain, Heart, Kidneys, Liver, Spleen, Thymus (fixed)
Organs/Tissues fixed (4% neutral buffered formaldehyde) for...:
- all parental animals (main test and recovery group): All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Esophagus, Extraorbital lacrimal glands, Epididymides, left (modified Davidson's solution), Eyes with optic nerve (modified Davidson's solution), Femur with knee joint, Heart, Ileum, Jejunum (with Peyer's patches), Kidneys, Larynx - Postmortem examinations (offspring):
- SACRIFICE
- One selected male and one female pup/litter were sacrificed at 13 days of age (PND 13) for blood sampling and thyroid/parathyroid glands fixation.
- All F1 offspring
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: - Statistics:
- Beside means and standard deviations, details statistics are given in table 1.
- Offspring viability indices:
- Pups Surviving days 0-4
Pups Surviving days 4-13 - Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Decreased body weight in males during in-life, including premating, from study day
21 to 91 with a maximum of -12.1% on study day 77 and -12.0% at the end of the
administration period in the main group
• Decreased body weight in females during premating from study day 28 to 70 with a
maximum of -11.5 % on study day 56 and -10.8% at the end of the administration
period in the main group
• Decreased body weight in recovery females during in-life phase from study day
21 to 119 with a maximum of -16.8 % (on study day 119) and during recovery
phase from study day 121 to 134 with a maximum of -15.2 % on study day 121 and
-10.6% at the end of the recovery period
• Decreased body weight in females from gestation day 0 to 20 with a maximum of
-18.5% on gestation day 20 and from lactation day 1 to 13 with a maximum of
-17.6% on lactation day 1 and resulting in -16.9% on lactation day 13
• Decreased body weight changes in males during in-life from study day 0 to 91
(-16.5%) and in females from study day 0 to 70 (-20%) in the main group
In the satellite male animals a decreased body weight gain was observed only during study day 56-63 and a non-statistically significant decrease from study day 0 to 91 (-7%) of the administration period followed by an increased body weight gain over the entire 14-day recovery period (+82%).
In the satellite (non pregnant) females a decreased body weight gain was observed during the administration period (-26%) and an increased body weight gain in the 14-day recovery period (+155%). - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Decreased food consumption in females from study day 7 onwards (up to -26.8% between study day 42 and 49) resulting in -11.6% from study day 0 to 70 in the main group
Decreased food consumption in satellite females during in-life phase (administration period) on study days 0 to 119 (-15.6%)
Decreased food consumption in females during gestation days 0 to 20 (-20.1%) and lactation days 1 to 13 (-23.5%) - Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- On day 94 (males) Increase in ALT (1.03 vs 0.78 ukat/L) and ALP (2.28 vs 1.33 ukat/L) at the high dose group, reversible within 14-day recovery period
On day 113 (females): Increase in ALP ( 4.16 vs 0.91 ukat/L) and GGT_C (45 vs 25 nkat/L) at the high dose group, reversible within 14-day recovery period
Reduction in bile acids
Increase in triglycerides - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Description (incidence and severity):
- No effects on thyroid hormones
- Reproductive function: oestrous cycle:
- effects observed, treatment-related
- Description (incidence and severity):
- Estrous cycle data, generated during the premating phase, revealed regular cycles in the females of all test groups including the control. The mean number of estrous cycle was 4.2 / 4.3 / 4.5 and 3.9 in test groups 0 - 3. The estrous cycle length in the different test groups was identical: 4.0 days in the test groups 0 – 2. In test group 3 (5000 ppm) the cycle length was 4.3 days which was not statistically significant different to the current control but outside of the historical control range 3.82-4.03 days.
All animals were cycling normally (3-6 days). No animal had a long estrous (3 days). One animal of the high dose group had a long distrous (4 days) - Reproductive function: sperm measures:
- no effects observed
- Description (incidence and severity):
- After the administration period, regarding the incidence of abnormal sperms in the cauda epididymidis, sperm head counts in the testis and in the cauda epididymidis no treatmentrelated
effects were observed. The sperm motility in males of test group 1 (5000 ppm) was significantly lower compared to controls, but this change was not dose-dependent and
therefore it was regarded as incidental and not treatment-related.
After the recovery period, in males of test group 13 (5000 ppm) motility of the sperms and sperm head counts in the testis were significantly decreased. However, the values were
within historical control ranges (males, motility 79-93 %, sperm head counts in the testis 87- 126 Mio/g testis). Therefore, these alterations were regarded as incidental and not
treatment-related. Sperm head counts in the cauda epididymidis and incidences of abnormal sperms were not changed. - Dose descriptor:
- NOEL
- Effect level:
- 500 ppm (analytical)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- clinical biochemistry
- organ weights and organ / body weight ratios
- histopathology: non-neoplastic
- Dose descriptor:
- NOAEL
- Effect level:
- 1 500 ppm
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- reproductive function (oestrous cycle)
- reproductive performance
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 1 500 ppm
- System:
- hepatobiliary
- Organ:
- liver
- Treatment related:
- yes
- Dose response relationship:
- yes
- Clinical signs:
- no effects observed
- Mortality / viability:
- mortality observed, non-treatment-related
- Description (incidence and severity):
- Viability index (days 0-4) groups 0 -3: 100 / 100 / 98.1 / 96.1
Viability index (days 4-13) groups 0-3: 100 / 100 / 100 / 100 - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Offspring body weight (male and female) in the high dose group is lower than that of the control group starting day 7. On day 13, the mean body weight is 26.4g versus 32.5g (-18.8%)
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Urinalysis findings:
- not examined
- Anogenital distance (AGD):
- no effects observed
- Nipple retention in male pups:
- no effects observed
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- No changes in absolute organ weights in males (slight reduction in heart weight at the high dose group)
Reduction in absolute weight of ovaries (Control 111 mg, LD 104 mg, MD 91 mg and HD 67 mg)
Relative organ weights returned to normal within the 14-day recovery period. - Gross pathological findings:
- no effects observed
- Histopathological findings:
- not examined
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 1 500 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 5 000 ppm
- System:
- other: Apart from decreased body weight gain - none
- Organ:
- other: no obvious target identified (screening level study)
- Treatment related:
- yes
- Dose response relationship:
- yes
- Reproductive effects observed:
- yes
- Lowest effective dose / conc.:
- 5 000 ppm
- Treatment related:
- yes
- Relation to other toxic effects:
- not specified
- Dose response relationship:
- yes
Reference
Table 1: Estrous cycle pre-mating
| Group 0 | Group 1 | Group 2 | Group 3 | |||
| 0 ppm | 500 ppm | 1500 ppm | 5000 ppm | |||
| Number of Cycles | Mean | 4.2 | k | 4.3 | 4.5 | 3.9 |
| S.d. | 0.4 | 0.5 | 0.5 | 0.9 | ||
| N | 10 | 10 | 10 | 10 | ||
| Cycles Length (days) | Mean | 4.0 | v | 4.0 | 4.0 | 4.3 |
| S.d. | 0.0 | 0.0 | 0.1 | 0.5 | ||
| N | 10 | 10 | 10 | 10 | ||
| Cycling Normally (3-6 Days) | N | 10 | 10 | 10 | 10 | |
| % | 100.0 | 100.0 | 100.0 | 100.0 | ||
| Long Estrous (3 Days) | N | 0 | 0 | 0 | 0 | |
| % | 0.0 | 0.0 | 0.0 | 0.0 | ||
| Long Diestrous (4 Days) | N | 0 | 0 | 0 | 1 | |
| % | 0.0 | 0.0 | 0.0 | 10.0 |
Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics
k=KRUSKALL-WALLIS; v=KRUSKALL-WALLIS-WILCOX
Table 2: Summary mating report
| Group 0 | Group 1 | Group 2 | Group 3 | |||
| 0 ppm | 500 ppm | 1500 ppm | 5000 ppm | |||
| No. of females mated | N | 10 | 10 | 10 | 10 | |
| - Inseminated | N | 10 | f- | 10 | 10 | 10 |
| Female mating index | % | 100.0 | 100.0 | 100.0 | 100.0 | |
| -- Pregnant | N | 9 | f- | 10 | 9 | 10 |
| Female fertility index | % | 90.0 | 100.0 | 90.0 | 100.0 | |
| No. of males mated | N | 10 | 10 | 10 | 10 | |
| - With inseminated females | N | 10 | f- | 10 | 10 | 10 |
| Male mating index | % | 100.0 | 100.0 | 100.0 | 100.0 | |
| - With pregnant females | N | 9 | f- | 10 | 9 | 10 |
| Male fertility index | % | 90.0 | 100.0 | 90.0 | 100.0 | |
| Females with defined Day 0 pc | N | 10 | 10 | 10 | 10 | |
| Mating days until Day 0 pc | Mean | 2.4 | x+ | 4.0 | 2.4 | 2.6 |
| S.d. | 1.1 | 3.2 | 1.2 | 1.4 | ||
| N | 10 | 10 | 10 | 10 | ||
| Days 0 To 4 | N | 10 | 9 | 10 | 9 | |
| % | 100.0 | 90.0 | 100.0 | 90.0 | ||
| Days 5 To 9 | N | 0 | 0 | 0 | 1 | |
| % | 0.0 | 0.0 | 0.0 | 10.0 | ||
| Days 10 To 14 | N | 0 | 1 | 0 | 0 | |
| % | 0.0 | 10.0 | 0.0 | 0.0 |
Statistic Profile = Fisher's exact test (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics
f=FISHER-EXACT; x=WILCOX
Table 3: Summary of pregnancy status report
| Group 0 | Group 1 | Group 2 | Group 3 | ||
| 0 ppm | 500 ppm | 1500 ppm | 5000 ppm | ||
| No. of females at start | N | 10 | 10 | 10 | 10 |
| No. of females mated | N | 10 | 10 | 10 | 10 |
| Without evidence of mating | N | 0 | 0 | 0 | 0 |
| Females with defined Day 0 pc | N | 10 | 10 | 10 | 10 |
| Pregnant | N | 9 | 10 | 9 | 10 |
| - sacrificed scheduled | N | 9 | 10 | 9 | 10 |
| Not pregnant | N | 1 | 0 | 1 | 0 |
| - sacrificed scheduled | N | 1 | 0 | 1 | 0 |
| Pregnant, not delivering | N | 0 | 0 | 0 | 0 |
| Delivering | N | 9 | 10 | 9 | 10 |
| -- With liveborn pups | N | 9 | 10 | 9 | 10 |
| % | 100.0 | 100.0 | 100.0 | 100.0 | |
| -- With all pups stillborn | N | 0 | 0 | 0 | 0 |
| % | 0.0 | 0.0 | 0.0 | 0.0 |
Table 4 Summary delivery report
| Group 0 | Group 1 | Group 2 | Group 3 | ||
| 0 ppm | 500 ppm | 1500 ppm | 5000 ppm | ||
| Gestation Index | % | 100.0 | 100.0 | 100.0 | 100.0 |
| Gestation days | Mean | 22.3 n | 21.9 | 21.7** | 22.1 |
| S.d. | 0.5 | 0.3 | 0.5 | 0.3 | |
| N | 9 | 10 | 9 | 10 | |
| -- With stillborn pups | N | 0 f+ | 2 | 0 | 2 |
| % | 0.0 | 20.0 | 0.0 | 20.0 | |
| -- With all pups stillborn | N | 0 f+ | 0 | 0 | 0 |
| % | 0.0 | 0.0 | 0.0 | 0.0 |
Statistic Profile = Fisher's exact test (one-sided-), Dunnett test (two-sided), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics
f=FISHER-EXACT; n=DUNNETT
Table 5: Summary Litter report - pup status
| Group 0 | Group 1 | Group 2 | Group 3 | ||||
| 0 ppm | 500 ppm | 1500 ppm | 5000 ppm | ||||
| Total Number of Pregnant Females | N | 9 | 10 | 9 | 10 | ||
| Total number of litters | N | 9 | 10 | 9 | 10 | ||
| With liveborn pups | N | 9 | f- | 10 | 9 | 10 | |
| % | 100.0 | 100.0 | 100.0 | 100.0 | |||
| With stillborn pups | N | 0 | f+ | 2 | 0 | 2 | |
| % | 0.0 | 20.0 | 0.0 | 20.0 | |||
| With all pups stillborn | N | 0 | f+ | 0 | 0 | 0 | |
| % | 0.0 | 0.0 | 0.0 | 0.0 | |||
| Implantation Sites | N | 130 | 131 | 98 | 99 | ||
| Historical Control Range 9.8-14.2 | Mean | 14.4 | x- | 13.1 | 10.9** | 9.9** | |
| S.d. | 1.9 | 2.1 | 1.6 | 2.3 | |||
| N | 9 | 10 | 9 | 10 | |||
| Pups delivered | N | 113 | 129 | 92 | 87 | ||
| Historical Control Range 9.0 - 13.2 | Mean | 12.6 | x- | 12.9 | 10.2* | 8.7** | |
| S.d. | 2.2 | 2.1 | 1.7 | 2.1 | |||
| N | 9 | 10 | 9 | 10 | |||
| Postimplantation Loss | Mean% | 12.1 | x+ | 1.6 | 5.7 | 10.4 | |
| S.d. | 15.7 | 3.5 | 11.1 | 16.1 | |||
| N | 9 | 10 | 9 | 10 | |||
Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-),
Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics
f=FISHER-EXACT; x=WILCOX
Table 6: Thyroid hormones, F0 males
| Group 0 | Group 1 | Group 2 | Group 3 | ||
| 0 ppm | 500 ppm | 1500 ppm | 5000 ppm | ||
| T4 | Mean | 62.84 k | 63.11 | 63.39 | 57.75 |
| [nmol/L] | S.d. | 9.44 | 6.72 | 10.77 | 8.75 |
| day 94 | N | 10 | 10 | 10 | 10 |
| Median | 67.60 | 62.18 | 63.14 | 60.31 | |
| Deviation Vs Control [%] | 0.43 | 0.88 | -8.09 | ||
| TSH | Mean | 5.94 k | 6.51 | 8.58 | 8.64 |
| [µg/L] | S.d. | 1.74 | 2.57 | 3.23 | 2.94 |
| day 94 | N | 10 | 10 | 10 | 10 |
| Median | 6.12 | 6.01 | 7.93 | 8.78 | |
| Deviation Vs Control [%] | 9.50 | 44.41 | 45.37 | ||
Table 7: Absolute and relative organ weights
| Relative weights | Males | Females | ||||
| Test group (ppm) | 1 (500) |
2 (1500) |
3 (5000) |
1 (500) |
2 (1500) |
3 (5000) |
| Brain | 102% | 107% | 116%** | 99% | 101% | 114%* |
| Kidneys | 116%** | 110%* | 104% | 102% | 93% | 114%** |
| Liver | 103% | 112%** | 127%** | 105%* | 112%** | 125%** |
| Ovaries | 93% | 85% | 69%** | |||
| Thyroid glands | 101% | 100% | 119%* | |||
| Absolute weights | Males | Females | ||||
| Test group (ppm) | 1 (500) |
2 (1500) |
3 (5000) |
1 (500) |
2 (1500) |
3 (5000) |
| Final body weight | 97% | 96% | 87%** | 102% | 97% | 87%** |
| Epididymides | 98% | 94% | 91%* | |||
| Heart | 97% | 93% | 87%* | 103% | 93%* | 87%* |
| Kidneys | 111%* | 101% | 87%* | 103% | 89%* | 97% |
| Ovaries | 94% | 82%* | 60%** | |||
* : p <= 0.05, **: p <= 0.01
Effect on fertility: via oral route
- Endpoint conclusion:
- no study available (further information necessary)
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
Reaction products of benzeneamine, N-phenyl with nonene (branched) was administered via diet to groups of 10 male and 10 female Wistar rats (F0 animals) at concentrations of 0 ppm (test group 0), 500 ppm (test group 1), 1500 ppm (test group 2) and 5000 ppm (test group 3).
The duration of treatment covered a 10-week premating period and a 2-week mating period in both sexes (mating pairs were from the same test group) as well as entire gestation period as well as 13 days of lactation period in females up to one day prior to the day of scheduled sacrifice of the animals. Additional treated but not mated animals (recovery animals) to groups of 10 male and 10 female animals at nominal doses of 0 and 5000 ppm was maintained for a subsequent period of at least 14 days of no test substance administration in order to observe reversibility of the findings.
In deviation from the OECD 421 test guideline a ten week instead of a two weeks premating exposure duration for the parental generations was included.
In addition to the OECD 421 test guideline additional investigations were implemented in the study on request of the Sponsor: sperm and spermatid examinations, determination of organ weights of brain, heart, kidneys, liver, spleen and thymus, several organ or tissue fixations, and histopathology of liver.
The following test substance-related effects/findings were noted:
Test group 3 / 13: 5000 ppm
(397 mg/kg bw/d in males, 419 mg/kg bw/d in females)
F0 PARENTAL ANIMALS
Clinical Examinations
• Decreased food consumption in females from study day 7 onwards (up to -26.1%
between study day 42 and 49) resulting in -11.6% from study day 0 to 70 in the
main group
• Decreased food consumption in recovery females during in-life phase
(administration period) on study days 0 to 119 (-15.6%)
• Decreased food consumption in females during gestation days 0 to 20 (-20.1%) and
lactation days 1 to 13 (-23.5%)
• In recovery male animals a decreased food consumption was observed only during
study day 77-84 (-9,8%)
• Decreased body weight in males during in-life, including premating, from study day
21 to 91 with a maximum of -12.1% on study day 77 and -12.0% at the end of the
administration period in the main group
• Decreased body weight in females during premating from study day 28 to 70 with a maximum of -11.5 % on study day 56 and -10.8% at the end of the administration period in the main group
• Decreased body weight in recovery females during in-life phase from study day 21 to 119 with a maximum of -16.8 % (on study day 119) and during recovery phase from study day 121 to 134 with a maximum of -15.2 % on study day 121 and -10.6% at the end of the recovery period
• Decreased body weight in females from gestation day 0 to 20 with a maximum of -18.5% on gestation day 20 and from lactation day 1 to 13 with a maximum of -17.6% on lactation day 1 and resulting in -16.9% on lactation day 13
• Decreased body weight changes in males during in-life from study day 0 to 91 (-16.5%) and in females from study day 0 to 70 (-20%) in the main group
• In recovery male animals a decreased body weight gain was observed only during
study day 56-63 and a non-statistically significant decrease from study day 0 to 91 (-7%) of the administration period followed by an increased body weight gain over the entire recovery period (+82%).
• In recovery females a decreased body weight gain was observed during the
administration period (-26%) and an increased body weight gain in the recovery
period (+155%).
• Decreased body weight changes in females during gestation from study day 0 to 20
(-31%)
Reproductive Performance
• Decreased number of pups delivered (8.7 to control 12.6)
Clinical Pathology
• Shortened prothrombin time (HQT) in dams and nulliparous females
• Increased alkaline phosphatase (ALP) activities in males, dams and nulliparous
females
• Increased γ-glutamyl transferase (GGT) activities and cholesterol values in dams and nulliparous females
• Increased triglyceride values in dams and nulliparous females
• Decreased total protein and albumin values in dams
• Decreased albumin values in nulliparous females
• Decreased glucose values in males
• Decreased glucose and cholesterol values in males
• No treatment-related adverse findings in the animals of the recovery groups
Pathology
• Decreased final body weight in males (-13%) and females (-13%)
• Increased relative liver weight in males (+27%) and females (+25%)
• Liver cell hypertrophy, centrilobular: in all males and all females (minimal to
moderate)
• Fatty change in liver, midzonal in 7 out of 10 males (minimal to moderate) and
periportal in 7 out of 10 females (minimal to moderate)
• Single cell necrosis/apoptosis in the liver of 7 out of 10 males (minimal)
• No treatment-related adverse findings in the animals of the recovery groups
F1 PUPS
Clinical Examinations/ Gross Findings
• Decreased body weight in male and female pups as well as both sexes combined
on PND 7 (-15.7% combined) and PND 13 (-18.8% combined)
• Decreased body weight changes in male and female pups during the postnatal
period (PND 1-13 both sexes combined (-22.5%) starting on PND 4
Test group 2: 1500 ppm
(122 mg/kg bw/d in males, 133 mg/kg bw/d in females)
F0 PARENTAL ANIMALS
Clinical Examinations
• Decreased body weight in females on lactation day 13 (-7.1%)
• Decreased body weight changes in females between lactation days 10 to 13
(-105%)
Reproductive Performance
• No test substance-related, adverse findings were noted.
Clinical Pathology
• Increased alkaline phosphatase (ALP) activities in dams
• Increased triglyceride values in dams
• Decreased total protein and albumin values in dams
Pathology
• Increased relative liver weight in males (+12%) and females (+12%)
• Liver cell hypertrophy, centrilobular in 5 out of 10 males (minimal to slight) and 3 out
of 10 females (minimal)
• Fatty change in liver, midzonal in 5 out of 10 males (minimal to slight) and periportal
in 2 out of 10 females (minimal)
• Single cell necrosis/apoptosis in the liver of 7 out of 10 males (minimal)
F1 PUPS
Clinical Examinations/Gross Findings
• No test substance-related, adverse findings were noted.
Test group 1: 500 ppm
(40 mg/kg bw/d in males, 44 mg/kg bw/d in females)
F0 PARENTAL ANIMALS
Clinical Examinations, Reproductive Performance, Clinical pathology and Pathology
• No test substance-related, adverse findings were noted.
F1 PUPS
Clinical Examinations/Gross Findings
• No test substance-related, adverse findings were noted.
Effects on developmental toxicity
Description of key information
Two prenatal developmental toxicity studies according to OECD 414 were performed in rats and rabbits. No direct developmental toxicity, teratogenic or embryotoxic effects were observed in the studies.
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2014
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: OECD 414 and GLP compliant study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: (age at delivery 10 weeks)
- Weight at study initiation: (weight range at delivery 177-196 g)
- Fasting period before study: none
- Housing: individual cages (during gestation)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 18 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 55% +/- 15%
- Air changes (per hr): 15 -20
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 2014-05-20 To: 2014-06-16 - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: The formulations were prepared daily and the concentrations were calculated and expressed in terms of test item as supplied.
VEHICLE
- Justification for use and choice of vehicle: The substance is miscible in corn oil and insoluble in water.
- Concentration in vehicle: 12.5, 37.5 and 125 mg/mL
- Amount of vehicle: 4 ml/kg bw - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The proposed formulation procedure for the test item was checked in the range from 12.5 to 125 mg/mL by chemical analysis (concentration and
homogeneity) during the pre-treatment period to confirm that the method was suitable. Final results for all levels were within the acceptability limits
for concentration (90-110%). Stability after 24 hours at room temperature was verified in the range from 1 to 300 mg/mL in the validation study.
Samples of the formulations prepared on week 1 and Last Week were analysed to check the homogeneity and concentration. - Details on mating procedure:
- Females were paired one to one in the home cage of the male and left overnight. Vaginal smears were taken daily in the morning from the day after pairing until a positive identification of matingwas made. The day of mating, as judged by the presence of sperm in the vaginal smear or by the presence of a copulation plug, was considered as Day 0 of gestation (or Day 0 post coitum).
- Duration of treatment / exposure:
- Day 6 through Day 19 post coitum
- Frequency of treatment:
- daily
- Duration of test:
- Day 6 through Day 20 post coitum
- Remarks:
- Doses / Concentrations:
50, 150 and 500 mg/kg bw
Basis:
actual ingested - No. of animals per sex per dose:
- 24
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Range-finding study with pregnant rats. The highest dose group of 500 mg/kg was expected to cause maternal toxicity as indicated by adverse effects on body weights and evidence on liver toxicity as indicated by clinical chemistry parameters. For details it is referred to the robust study summary of the maternal toxicity study.
- Rationale for animal assignment: Females were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights - Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: mortality
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum
FOOD CONSUMPTION : Yes
- Time schedule for examinations: Days 3, 6, 9, 12, 15, 18 and 20 post coitum starting from Day 0 post coitum
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: ovaries and uteri - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: gross evaluation of placentae, number of intra-uterine deaths,. Uteri or individual uterine horns without visible implantations were immersed
in a 20% solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation. - Fetal examinations:
- - External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter] - Statistics:
- For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t-test, depending on the
homogeneity of data. Statistical analysis of non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences
between the control and treated groups assessed by a non-parametric version of the Williams test. The mean values, standard deviations and statistical
analysis were calculated from actual values in the computer without rounding off. - Indices:
- Preimplantation loss
Postimplantation loss
Total implantation loss
Sex ratios - Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
A slight decrease in body weight was noted in all treated females when compared to controls, reaching a statistical significance in females receiving
500 mg/kg bw/day (up to 7%), starting from Day 9 post coitum until the end of the study.
Statistically significant decrease was also recorded in body weight gain of females of the same group on Day 9 post coitum (109%; body weight loss) and Day 12 post coitum (22%). Starting from Day 15 post coitum the mean values of body weight gain were comparable between control and high dose group.
Statistically significant decrease (up to 22%) in food consumption was observed in treated females receiving 500 mg/kg bw/day, starting from Day 9 post coitum until the end of the study.
A slight trend to decrease was observed in terminal body weight of all treated females with respect to the control. This change was about -6% in the high
dose group, without statistical significance. A statistically significant decrease in corrected body weight (up to 6%) and corrected body weight gain (up
to 50%) was noted in treated females receiving 150 and 500 mg/kg bw/day. Gravid uterus weight was similar between control and treated groups.
There were no adverse findings at the macroscopic examination at necropsy. - Dose descriptor:
- NOAEL
- Effect level:
- 150 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Details on embryotoxic / teratogenic effects:
- Litter data, mean foetal weight and sex ratio were unaffected by treatment.
A total of 15 small foetuses (foetal weight < 2.7 g) were detected: 2 out of 269 in the control group, 2 out of 214 in the low dose group, 1 out of 269 in the mid-dose group and 10 out of 254 in the high dose group. One foetus in the high dose group showed malrotation of the hindlimb, considered incidental. Of the ten small fetuses in the high dose group, 7 were from the dam which suffered most strongly from maternal toxicity as indicated by the lowest corrected body weight gain of minus 9.8g. This dam was also the only dam showing hunched posture and piloerection on gestation day 20. The higher incidence of small foetuses is therefore considered to be related to the lower maternal body weight gain.
No relevant findings that could be considered treatment-related were observed at visceral examination of foetuses in the treated groups, compared to controls.
The alterations recorded at skeletal examinations of foetuses were noted both in control and treated groups with a similar incidence. - Dose descriptor:
- NOAEL
- Effect level:
- >= 500 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: No effects observed
- Abnormalities:
- no effects observed
- Developmental effects observed:
- no
- Conclusions:
- The substance is not teratogenic and not embryotoxic in rats. It causes maternal toxicity at a dose level of 500 mg/kg bw.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2018-03-28 until
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- 22 Jan 2001
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Landesamt fuer Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Strasse 7, 55116 Mainz, Germany
- Species:
- rabbit
- Strain:
- New Zealand White
- Remarks:
- Crl:KBL(NZW)
- Route of administration:
- oral: gavage
- Vehicle:
- CMC (carboxymethyl cellulose)
- Remarks:
- 0.5% CMC suspension in deionized water
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
- specific amount of the test substance was weighed, topped up with vehicle (with 10 mg/100 mL Cremophor EL) in a calibrated beaker and intensely mixed with a magnetic stirrer
- test substance preparations were warmed up to approximately 30 degrees Celsius
- before and during administration, the preparations were kept homogeneous with a magnetic stirrer and kept at approximately 30 degrees Celsius with a heating sleeve or with a magnetic stirrer with heating plate
VEHICLE
- Justification for use and choice of vehicle: stability and homogenity could be proven in the vehicle
- Amount of vehicle: the preparation was administered as a 10 mL/kg bw - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- HPLC method with UV/VIS detection
- Details on mating procedure:
- - Impregnation procedure: artificial insemination
- GD0: day of insemination - Duration of treatment / exposure:
- gestation days 6 through 28
- Frequency of treatment:
- daily
- Dose / conc.:
- 10 mg/kg bw/day (nominal)
- Dose / conc.:
- 30 mg/kg bw/day (nominal)
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 25
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: futher details are given in the dose-range finder in the the supporting study record.
- Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily and if effects were observed, several times daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: (during GD 6-28) daily check, as well as within 5 hours after the administration
BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: uteri, ovaries - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened) - Fetal examinations:
- - External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter - Statistics:
- - Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means: Food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight
- Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions: Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings
- Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians: Proportions of fetuses with malformations, variations and/or unclassified observations in each litter - Indices:
- conception rate = number of pregnant animals / number of fertilized animals * 100
preimplantation loss, for each individual pregnant animal which underwent scheduled sacrifice = (number of corpora lutea – number of implantations) / number of corpora lutea * 100
postimplantation loss, for each individual pregnant animal which underwent scheduled sacrifice = (number of implantations – number of live fetuses) / number of implantations * 100 - Historical control data:
- - mean maternal bw during gestation, reproduction data, placenta weights, mean maternal weights, fetal external malformations/variations, fetal soft tissue malformations/variations, fetal skeletal malformations/variations
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- For details, see to table 1.
- 0/10/30/100 mg/kg bw/d dose group: 4/2/11/20 females showed reduced defecation, respectively
- no defecation was observed in 2/0/4/4 females, respectively
- exceptionally high incidence of reduced defecation in the high dose group, along with reduced food consumption indicates a treatment-related effect
For details, see to table 5.
- high-dose (100 mg/kg bw/d) group: mean food consumption distinctly and statistically significantly reduced from GD 7-23 (up to -59% in comparison to the control)
- high-dose does: 31% less food consumption compared to the concurrent control (GD 6-28)
- low- and mid-dose groups (10 and 30 mg/kg bw/d): comparable food consumption to control throughout the entire study period - Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- One female of the control group (No.21) died after a gavage error.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- For details, see to table 3 and 4 and the attached figure
- mean body weights (BW); average body weight gain (BWC)
- high-dose (100 mg/kg bw/d) group: BW statistically significant reduced on GD 14-25 and BWC statistically significant reduced on GD 9-11
- high-dose rabbits: lost weight overall (-24.0 g vs. +104.4 g in control) during the treatment period (GD 6-28)
- no effects observed in the low- and mid-dose groups (10 and 30 mg/kg bw/d) - Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry 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):
- - no effects observed for the mean gravid uterus compared to the control group
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- For details, see to table 6.
spontaneous effects were observed in the control and the test group:
- watery feces or no feces in rectum: one control doe, one mid-dose doe and one high-dose doe
- rudimentary appearance of uterus and ovaries in one control doe
- granulated surface of liver in one low-dose doe
- malpositioned kidney in combination with a short ureter in one low-dose and one mid-dose doe
- absence of uterine horn(s) in one low-dose doe
- findings after gavage error (thoracic cavity filled with blood) in one control doe - Description (incidence and severity):
- Two control (Nos. 7 [GD 20] and 11 [GD 28]) and four high-dose females (Nos. 78 [GD 24], 79 [GD 26], 84 [GD 28] and 94 [GD 27] - 100 mg/kg bw/d) were sacrificed after abortion ahead of schedule. Although spontaneous abortions in single does are not uncommon findings in the strain of rabbits used for this study, the high-dose cases may already represent exaggerated maternal toxicity, given also the distinct drop in food consumption and body weight gain as well as an exceptionally high number of does showing reduced defecation.
For details, see to table 2. - Number of abortions:
- effects observed, treatment-related
- Description (incidence and severity):
- For details, see to table 2.
- abortions were observed in two control females and four high-dose females
- the females were sacrificed and excluded from calculations of mean maternal food consumption, body weight and body weight change, mean gravid uterine weights, mean organ weights, corrected (net) body weight gain and summary of reproduction data - Pre- and post-implantation loss:
- no effects observed
- Description (incidence and severity):
- For details, see to table 2.
- no test substance-related and/or biologically relevant differences between the different test groups observed - Early or late resorptions:
- no effects observed
- Description (incidence and severity):
- For details, see to table 7.
- no test substance-related and/or biologically relevant differences between the different test groups in the numbers of resorptions observed - Dead fetuses:
- no effects observed
- Description (incidence and severity):
- For details, see to table 7.
- no test substance-related and/or biologically relevant differences between the different test groups in the numbers of viable fetuses
One dead fetus was found at cesarean section of one low-dose doe which may occur spontaneously in this rabbit strain. - Changes in number of pregnant:
- no effects observed
- Description (incidence and severity):
- For details, see to table 2.
- conception rate was 92% in the control group and 96% in the low-, mid- and high-dose groups
- no test substance-related and/or biologically relevant differences between the different test groups in the conception rate observed - Other effects:
- no effects observed
- Description (incidence and severity):
- All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age for historical control data.
- Dose descriptor:
- NOAEL
- Remarks:
- systemic toxicity
- Effect level:
- 30 mg/kg bw/day (nominal)
- Basis for effect level:
- body weight and weight gain
- clinical signs
- number of abortions
- other: distinct decrease of food consumption
- Dose descriptor:
- NOEL
- Remarks:
- developmental toxicity
- Effect level:
- > 100 mg/kg bw/day (nominal)
- Basis for effect level:
- other: no test substance-related and/or biologically relevant differences for conception rate, mean numbers of corpora lutea and implantation sites or for pre- and post-implantation losses, the numbers of resorptions and viable fetuses
- Abnormalities:
- no effects observed
- Fetal body weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- For details, see to table 8.
- high-dose (100 mg/kg bw/d) group: mean fetal weight statistically significantly lower than control in male fetuses and -12% for both sexes in comparison to the concurrent control
- mean weight of the female high-dose fetuses was also slightly lower (not statistically significanct)
- low- and mid-dose group: not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group - Changes in sex ratio:
- no effects observed
- Description (incidence and severity):
- The sex distribution of the fetuses in all three test groups was comparable to the control fetuses. Any observable differences were without biological relevance.
- External malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- - no statistically significant differences of overall incidences were noted between the groups
- Skeletal malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- - detected in single fetuses of all test groups including the control
All findings were considered to be spontaneous in origin and not treatment-related.
No statistically significant differences between the groups were noted. The overall incidences were well within the historical control range of the test facility. - Visceral malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- - soft tissue malformations occurred in all test groups including the control
- distribution of the findings about the test groups does not indicate an association to the treatment and no statistically significant differences between the groups were noted
- total incidence of soft tissue malformations in treated animals did not differ significantly from the control group - Other effects:
- effects observed, non-treatment-related
- Description (incidence and severity):
- VARIATIONS/UNCLASSIFIED OBSERVATIONS:
EXTERNAL (refer to table 9)
- paw hyperflexion: recorded in four fetuses of one high-dose litter, occurred together with multiple external malformations
Paw hyperflexion can be found in the historical control data at comparable incidences.
- discolored placentae: recorded in one fetus of the low-dose test group
This finding is not considered to be related to treatment.
SOFT TISSUE (refer to table 10)
- absent lung lobe (Lobus inferior medialis) in all test groups including the control observed
- for the high-dose group: not statistically significant, however, it was outside the historical control range (historical control data [HCD]: mean% 0.9, range 0.0 - 2.0); these findings were clustered in only 3 litters = litter incidence of 15% which is inside the historical control range (HCD: mean% 7.0, range 0.0 – 17.4)
Overall, this finding is a common anatomical variant in this rabbit strain. Thus, the slightly higher high-dose incidence it is not considered to be of toxicological relevance.
- other variations (e.g. cerebral ventricle, malpositioned carotid branches narrowed pulmonary trunk, dilated aorta and dilated renal pelvis) occurred in individual fetuses of low- and/or high-dose test animals as well as in control groups
The incidences of these variations were neither statistically significantly different from control nor dose-dependent and, therefore, not considered biologically relevant.
- blood coagulum around urinary bladder: in four mid-dose and three high-dose fetuses
This finding can be found in the historical control data at comparable incidences, therefore, it was neither assessed as treatment-related nor as adverse.
- empty stomach (devoid of amniotic fluid): in four fetuses of the same high-dose litter
These fetuses had multiple other findings, discussed in the section for details.
SKELETAL (refer to table 11)
- skeletal variations of different bone structures: observed in all test groups with or without effects on corresponding cartilages
The observed skeletal variations were related to several parts of fetal skeletons and appeared in the majority of cases without a relation to dosing.
- irregular ossification of interparietal: in the mid- and high-dose groups increased and outside the historical control range
This finding represents small irregularities in the shape of the ossification nuclei in the interparietal. As desmal ossification of the neurocranium continues during later development and interparietal membrane as well as surrounding bones were intact, a completely regular ossification of this bone can be expected to occur postnatally. Thus, this finding was considered to be of little, if any toxicological relevance.
- ‘unossified talus (with present cartilage)’: statistically significantly increased and outside the historical control range in the high-dose group
This finding may represent slight delays of ossification which did not affect morphology, as the underlying cartilage model was completely intact in all these cases. This assessment is supported by the fact, that the mean fetal weight of all 10 fetuses showing this finding (i.e. 18.8 g) was clearly below the mean fetal weight of all fetuses in test group 3 (33.6 g), which indicates a delay in overall development going along with the delay in ossification.
The other increased incidences of skeletal variations were either not related to dose and/or inside the historical control range. Thus, they are not considered to be associated with treatment.
The observed unclassified cartilage findings were related to the sternum and the ribs and did not show any relation to dosing. Therefore, they were assessed as not treatment-related. - Details on embryotoxic / teratogenic effects:
- ASSESSMENT OF ALL OBSERVATIONS
- distribution of total malformations about the groups was not related to dose
One fetus of the control, two fetuses of the low-dose, four fetuses of the mid-dose and four fetuses of the high-dose group had more than one malformation or were multiple-malformed across the different examination areas.
For details, see table 12.
- a cluster of 4 fetuses in litter No.76 showed a spectrum of malformations and a number of less severe findings, e.g. paw hyperflexion and empty stomach (devoid of amniotic fluid)
All these findings contributed to apparently higher high-dose rates of external malformations and variations as well as unclassified soft tissue observations.
The clustered appearance in one litter and the almost identical spectrum of ontogenetically different findings in all those fetuses strongly suggests an origin of these anomalies which is unrelated to treatment. Consequently, the higher incidence of high-dose findings in their respective sections is also considered to be unrelated to treatment.
- other malformations appeared in individual fetuses of test groups 1 or 3
- no ontogenetic pattern is recognizable for the individual malformations, nor was there any cluster of any of these individual malformations
- malformations were not dose-related and all of them can be found in the historical control data at comparable or higher frequency
Overall, an association of all these findings to the treatment is not assumed.
- spontaneous origin is assumed for the external variations, soft tissue variations and the skeletal variations as observed in all test groups and the control group
If all different types of variations are summarized, none of the incidences showed a relation to dosing and can be found in the historical control data at a comparable frequency.
- spontaneous origin is assumed for the unclassified external, unclassified soft tissue and unclassified skeletal cartilage observations, observed in several fetuses of all groups
The distribution and type of these findings do not suggest any relation to treatment.
Finally, fetal examinations revealed that there is no adverse effect of the compound on the respective morphological structures up to the highest dose tested (100 mg/kg bw/d). - Dose descriptor:
- NOAEL
- Effect level:
- 30 mg/kg bw/day (nominal)
- Sex:
- male/female
- Basis for effect level:
- fetal/pup body weight changes
- Abnormalities:
- no effects observed
- Description (incidence and severity):
- Four fetuses in one litter showed malformations and a number of less severe findings such as (among others) paw hyperflexion and empty stomach (devoid of amniotic fluid). These findings contributed to apparently higher high-dose rates of external malformations and variations as well as unclassified soft tissue observations.
The clustered appearance in one litter and the almost identical spectrum of ontogenetically different findings in all those fetuses strongly suggests an origin of these anomalies which is unrelated to treatment. Consequently, the higher incidence of high-dose findings in their respective sections is also considered to be unrelated to treatment. - Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 100 mg/kg bw/day (nominal)
- Treatment related:
- yes
- Relation to maternal toxicity:
- developmental effects as a secondary non-specific consequence of maternal toxicity effects
- Dose response relationship:
- no
- Relevant for humans:
- no
- Conclusions:
- The oral administration of the test substance to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) caused evidence of systemic maternal toxicity at the high-dose level of 100 mg/kg bw/d, such as a slightly higher incidence of abortions and reduced defecation in almost all females of this group, along with a distinct decrease of food consumption as well as body weight/body weight gain.
In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 30 mg/kg bw/d.
The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is also set at 30 mg/kg bw/d. The mean fetal body weights were significantly lower in the 100 mg/kg bw/d group compared to the control group (33.6 g versus 38.6 g). Considering the immediate effect of dosing on maternal food consumption and body weight loss during GD 6-28, the reduced fetal body weights are a result of the poor nutritional condition of the does. The substance has no direct developmental toxicity. No teratogenicity was observed.
Referenceopen allclose all
Table 1: TERMINAL BODY WEIGHT, UTERUS WEIGHT, CORRECTED BODY WEIGHT AND CORRECTED BODY WEIGHT GAIN OF FEMALES - GROUP MEAN DATA
| Group | Terminal body weight (g) | Gravid uterus weight (g) | Body weight at necropsy minus gravid uterus weight (corrected body weight) (g) | Body weight at necropsy minus gravid uterus weight, minus body weight on GD6 (corrected body weight gain) (g) | ||
| 1 (control) | Mean | 336.38 | 66.40 | 269.98 | 29.87 | |
| SD | 15.84 | 8.80 | 14.79 | 7.67 | ||
| (n) | 23 | 23 | 23 | 23 | ||
| 2 | Me | 323.9 | 60.42 | 263.5 | 29.45 | |
| SD | 23 | 18 | 18 | 16 | ||
| (n) | 20 | 20 | 20 | 20 | ||
| 3 | Me | 323.8 | 66.20 | 257.6* | 20.5* | |
| SD | 28 | 12 | 18 | 9. | ||
| (n) | 23 | 23 | 23 | 23 | ||
| 4 | Me | 315.9 | 62.53 | 253.3* | 14.83* | |
| SD | 24 | 14 | 23 | 16 | ||
| (n) | 22 | 22 | 22 | 22 | ||
* = Statistically significantly different from control group value at p< 0.05
Table 2: FOOD CONSUMPTION (g/animal/day) - GROUP MEAN
| Group | gestation day 3 | gestation day 6 | gestation day 9 | gestation day 12 | gestation day 15 | gestation day 18 | gestation day 20 | |
| 1 (control) | n | 23 | 23 | 23 | 23 | 23 | 23 | 23 |
| Mean | 18.13 | 20.66 | 18.72 | 20.99 | 21.78 | 23.68 | 23.06 | |
| SD | 2.37 | 2.54 | 2.05 | 1.98 | 2.10 | 2.20 | 1.99 | |
| 2 | n | 20 | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean | 17.99 | 20.97 | 18.32 | 20.64 | 21.04 | 23.43 | 22.83 | |
| SD | 2.3 | 3.03 | 2.46 | 2.19 | 2.68 | 2.99 | 4.33 | |
| 3 | n | 23 | 23 | 23 | 23 | 23 | 23 | 23 |
| Mean | 18.7 | 20.23 | 17.36 | 19.28 | 20.90 | 22.32 | 21.56 | |
| SD | 1.99 | 2.53 | 2.59 | 2.90 | 2.40 | 2.91 | 3.14 | |
| 4 | n | 22 | 22.00 | 22 | 22.00 | 22.00 | 22.00 | 22.00 |
| Mean | 18.24 | 20.97 | 15.33** | 17.1** | 19.01** | 20.12** | 18.02** | |
| SD | 2.62 | 2.55 | 1.98 | 2.76 | 2.68 | 3.02 | 2.96 |
** = mean value of group is significantly different from control at p < 0.01
Table 3: Pregnancy status overview
| Group | 1 (control) | 2 | 3 | 4 |
| Initial group size (n) | 24 | 24 | 24 | 24 |
| Not pregnant (n) | 1 | 4 | 1 | 2 |
| Unilateral implantation (n) | 0 | 1 | 0 | 0 |
| With live foetuses at gestation Day 20 (n) | 23 | 20 | 23 | 22 |
Table 4: Litter data and sex ratios - group mean data
| Corpus lutea | Implantations | Early Uterine Deaths | Late Uterine Deaths | Total Uterine Deaths | Viable Young (total) | Viable males | Viable females | % Males | Preimplantation loss (%) | Postimplantation loss | Total implantation loss(%) | Litter weight (g) | Mean fetal weight (g) | ||
| 1 | Mean | 12.87 | 12.26 | 0.52 | 0.04 | 0.57 | 11.7 | 6 | 5.7 | 50.96 | 4.68 | 4.15 | 8.52 | 43.1 | 3.69 |
| SD | 1.63 | 1.63 | 1.47 | 0.21 | 1.5 | 1.77 | 2.24 | 2.05 | 17.27 | 5.13 | 10.27 | 11.78 | 6.71 | 0.34 | |
| (n) | 23 | 23 | 23 | 23 | 23 | 23 | 23 | 23 | 23 | 23 | 23 | 23 | 23 | 23 | |
| 2 | Mean | 12.26 | 11.47 | 0.21 | 0.05 | 0.26 | 11.21 | 5.67 | 5.84 | 49.82 | 6.56 | 4.12 | 10.05 | 39.88 | 3.63 |
| SD | 2.58 | 2.44 | 0.54 | 0.23 | 0.56 | 2.84 | 2.22 | 2.29 | 14.18 | 6.13 | 11.67 | 13.68 | 9.32 | 0.4 | |
| (n) | 19 | 19 | 19 | 19 | 19 | 19 | 18 | 19 | 18 | 19 | 19 | 19 | 19 | 19 | |
| 3 | Mean | 13.09 | 12.22 | 0.52 | 0 | 0.52 | 11.7 | 5.78 | 5.91 | 449.19 | 6.28 | 4.73 | 10.86 | 42.41 | 3.65 |
| SD | 2.7 | 2.35 | 0.73 | 0 | 0.73 | 2.55 | 1.93 | 1.98 | 12.79 | 6.68 | 6.74 | 7.39 | 8.85 | 0.28 | |
| (n) | 23 | 23 | 23 | 23 | 23 | 23 | 23 | 23 | 23 | 23 | 23 | 23 | 23 | 23 | |
| 4 | Mean | 12.27 | 11.73 | 0.14 | 0 | 0.14 | 11.59 | 5.36 | 6.23 | 46.95 | 6.11 | 1.18 | 7.23 | 40.11 | 3.51 |
| SD | 2.66 | 3.06 | 0.35 | 0 | 0.35 | 3.08 | 2.06 | 2.25 | 12.97 | 10.6 | 3.07 | 10.96 | 9.86 | 0.39 | |
| (n) | 22 | 22 | 22 | 22 | 22 | 22 | 22 | 22 | 22 | 22 | 22 | 22 | 22 | 22 |
Table 1: Excerpt of the results of maternal clinical signs during gestation
|
|
Day of gestation |
|
|
|||||||||||||||||||||||||||||
|
Group# |
0 |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
13 |
14 |
15 |
16 |
17 |
18 |
18 |
19 |
20 |
21 |
22 |
23 |
24 |
25 |
26 |
27 |
28 |
TOTAL |
|
# OF FEMALES EXAMINED |
0 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
24 |
24 |
24 |
24 |
24 |
24 |
24 |
24 |
24 |
24 |
24 |
23 |
23 |
23 |
23 |
23 |
23 |
23 |
23 |
22 |
|
|
|
1 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
|
|
|
2 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
|
|
|
3 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
25 |
24 |
24 |
23 |
22 |
21 |
|
|
BLOOD IN BEDDING BEFORE |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
1 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
|
TREATMENT |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
|
2 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
|
3 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
ABORTION AFTER TREATMENT |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
|
|
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
|
2 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
|
3 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
1 |
|
ABORTION BEFORE TREATMENT |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
1 |
|
|
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
|
2 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
|
3 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
1 |
0 |
3 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Day of gestation |
||||||||||||||||||||||||||||||
|
Group# |
0 |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
13 |
14 |
15 |
16 |
17 |
18 |
18 |
19 |
20 |
21 |
22 |
23 |
24 |
25 |
26 |
27 |
28 |
TOTAL |
STOOL/URINE
|
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
2 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
2 |
1 |
0 |
4 |
REDUCED DEFECATION BEFORE |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
2 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
2 |
1 |
0 |
4 |
TREATMENT |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
0 |
0 |
0 |
0 |
2 |
|
2 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
2 |
3 |
2 |
2 |
2 |
3 |
3 |
1 |
0 |
1 |
2 |
5 |
3 |
1 |
3 |
0 |
11 |
|
3 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
1 |
4 |
7 |
17 |
10 |
9 |
6 |
9 |
8 |
8 |
5 |
6 |
6 |
5 |
3 |
5 |
5 |
0 |
20 |
REDUCED DEFECATION AFTER |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
2 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
2 |
1 |
0 |
4 |
TREATMENT |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
0 |
0 |
0 |
0 |
2 |
|
2 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
2 |
3 |
2 |
2 |
2 |
3 |
3 |
1 |
0 |
1 |
2 |
5 |
3 |
1 |
3 |
0 |
11 |
|
3 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
1 |
4 |
7 |
17 |
10 |
9 |
6 |
9 |
8 |
8 |
5 |
6 |
6 |
5 |
3 |
5 |
5 |
0 |
20 |
NO DEFECATION BEFORE |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
2 |
0 |
2 |
TREATMENT |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
2 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
1 |
1 |
1 |
2 |
3 |
1 |
0 |
4 |
|
3 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
3 |
4 |
3 |
3 |
3 |
2 |
2 |
2 |
1 |
0 |
0 |
0 |
0 |
0 |
4 |
NO DEFECATION AFTER |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
0 |
2 |
TREATMENT |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
2 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
1 |
1 |
1 |
2 |
3 |
1 |
0 |
4 |
|
3 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
3 |
4 |
3 |
3 |
3 |
2 |
2 |
2 |
1 |
0 |
0 |
0 |
0 |
0 |
4 |
Table 2: Summary of reproduction data
|
TEST GROUP 0 0 mg/kgbw/d |
TEST GROUP 1 10 mg/kgbw/d |
TEST GROUP 2 30 mg/kgbw/d |
TEST GROUP 3 100 mg/kgbw/d |
Females Mated (N) |
25 |
25 |
25 |
25 |
Pregnant (N) |
23 |
24 |
24 |
24 |
Conception Rate (%) |
92 |
96 |
96 |
96 |
Aborted (N) |
2 |
0 |
0 |
4 |
Premature Births (N) |
0 |
0 |
0 |
0 |
Dams with Viable Fetuses (N) |
20 |
24 |
24 |
20 |
Dams with all Resorptions (N) |
0 |
0 |
0 |
0 |
Female Mortality (N) |
3 Fi |
0 |
0 |
4 |
(%) |
12 |
0.0 |
0.0 |
16 |
Pregnant at Terminal Sacrifice (N) |
20 Fi |
24 |
24 |
20 |
(%) |
80 |
96 |
96 |
80 |
Corpora Lutea MEAN |
9.8 D |
9.8 |
10.8 |
11.0 |
S.D. |
1.74 |
2.45 |
2.50 |
2.66 |
TOTAL |
195 |
235 |
259 |
220 |
Implantation Sites MEAN |
9.1 D |
8.4 |
9.2 |
9.6 |
S.D. |
2.24 |
2.93 |
2.69 |
3.35 |
TOTAL |
181 |
201 |
221 |
192 |
Preimplantation Loss MEAN (%) |
7.9 D |
15.0 |
14.6 |
14.8 |
S.D. |
13.88 |
22.83 |
19.55 |
18.32 |
Postimplantation Loss MEAN (%) |
3.7 D |
6.6 |
6.8 |
6.2 |
S.D. |
8.47 |
9.64 |
8.66 |
11.52 |
Statistics: D = Dunnett-test (two-sided), * : p<=0.05 ** : p<=0.01; Fi =Fisher's exact test (one-sided)
Table 3: Mean maternal body weights during gestation (g)
|
|
|
TEST GROUP 0 0 mg/kg bw/d |
TEST GROUP 1 10 mg/kg bw/d |
TEST GROUP 2 30 mg/kg bw/d |
TEST GROUP 3 100 mg/kg bw/d |
|
DAY |
0 |
MEAN |
3755 |
D |
3735 |
3738 |
3722 |
|
|
S.D. |
161.8 |
|
174.4 |
165.0 |
163.5 |
|
|
N |
22 |
|
24 |
24 |
24 |
DAY |
2 |
MEAN |
3820 |
D |
3803 |
3813 |
3804 |
|
|
S.D. |
163.3 |
|
174.3 |
159.5 |
157.9 |
|
|
N |
22 |
|
24 |
24 |
24 |
DAY |
4 |
MEAN |
3869 |
D |
3851 |
3847 |
3845 |
|
|
S.D. |
177.5 |
|
176.5 |
167.6 |
156.0 |
|
|
N |
22 |
|
24 |
24 |
24 |
DAY |
6 |
MEAN |
3932 |
D |
3914 |
3908 |
3910 |
|
|
S.D. |
175.9 |
|
192.7 |
170.9 |
157.3 |
|
|
N |
22 |
|
24 |
24 |
24 |
DAY |
9 |
MEAN |
3969 |
D |
3939 |
3944 |
3911 |
|
|
S.D. |
191.9 |
|
186.3 |
194.7 |
179.9 |
|
|
N |
22 |
|
24 |
24 |
24 |
DAY |
11 |
MEAN |
3982 |
D |
3967 |
3965 |
3885 |
|
|
S.D. |
174.7 |
|
191.0 |
191.4 |
196.7 |
|
|
N |
22 |
|
24 |
24 |
24 |
DAY |
14 |
MEAN |
4036 |
D |
4022 |
4020 |
3888* |
|
|
S.D. |
167.4 |
|
215.5 |
206.3 |
189.2 |
|
|
N |
22 |
|
24 |
24 |
24 |
DAY |
16 |
MEAN |
4060 |
D |
4062 |
4043 |
3875* |
|
|
S.D. |
167.9 |
|
245.2 |
209.3 |
211.7 |
|
|
N |
22 |
|
24 |
24 |
24 |
DAY |
19 |
MEAN |
4012 |
D |
4034 |
4006 |
3824** |
|
|
S.D. |
154.6 |
|
216.7 |
183.5 |
219.0 |
|
|
N |
22 |
|
24 |
24 |
24 |
DAY |
21 |
MEAN |
4020 |
D |
4006 |
3969 |
3808** |
|
|
S.D. |
142.0 |
|
233.8 |
185.8 |
228.7 |
|
|
N |
21 |
|
24 |
24 |
24 |
|
|
|
|
|
|
|
|
DAY |
23 |
MEAN |
4026 |
D |
4036 |
3994 |
3816** |
|
|
S.D. |
153.5 |
|
222.1 |
208.6 |
248.6 |
|
|
N |
21 |
|
24 |
24 |
24 |
DAY |
25 |
MEAN |
4016 |
D |
4030 |
4002 |
3848** |
|
|
S.D. |
163.9 |
|
197.0 |
232.9 |
244.3 |
|
|
N |
21 |
|
24 |
24 |
23 |
DAY |
28 |
MEAN |
4030 |
D |
4071 |
4018 |
3864 |
|
|
S.D. |
202.8 |
|
216.6 |
255.6 |
291.3 |
|
|
N |
21 |
|
24 |
24 |
20 |
|
|
|
|
|
|
|
|
DAY |
29 |
MEAN |
4066 |
D |
4082 |
4038 |
3897 |
|
|
S.D. |
179.2 |
|
213.0 |
273.2 |
289.9 |
|
|
N |
20 |
|
24 |
24 |
20 |
Statistics: D=Dunnett-test (two-sided), * : p<=0.05 ** : p<=0.01
Table 4: Mean maternal body weight change during gestation (g)
|
|
|
|
|
TEST GROUP 0 0 mg/kg bw/d |
TEST GROUP 1 10 mg/kg bw/d |
TEST GROUP 2 30 mg/kg bw/d |
TEST GROUP 3 100 mg/kg bw/d |
DAYS |
0 |
TO |
2 |
MEAN |
65.6 D |
67.5 |
75.1 |
81.9 |
|
|
|
|
S.D. |
32.00 |
40.53 |
36.07 |
33.40 |
|
|
|
|
N |
22 |
24 |
24 |
24 |
DAYS |
2 |
TO |
4 |
MEAN |
48.1 D |
47.7 |
33.8 |
40.5 |
|
|
|
|
S.D. |
36.94 |
36.09 |
36.35 |
33.23 |
|
|
|
|
N |
22 |
24 |
24 |
24 |
DAYS |
4 |
TO |
6 |
MEAN |
63.2 D |
63.0 |
60.8 |
65.0 |
|
|
|
|
S.D. |
23.22 |
46.02 |
36.95 |
45.39 |
|
|
|
|
N |
22 |
24 |
24 |
24 |
DAYS |
6 |
TO |
9 |
MEAN |
37.7 D |
25.7 |
35.8 |
1.7 |
|
|
|
|
S.D. |
56.02 |
79.94 |
67.63 |
68.06 |
|
|
|
|
N |
22 |
24 |
24 |
24 |
DAYS |
9 |
TO |
11 |
MEAN |
12.9 D |
27.3 |
21.5 |
-26.6* |
|
|
|
|
S.D. |
53.91 |
27.89 |
50.28 |
48.89 |
|
|
|
|
N |
22 |
24 |
24 |
24 |
DAYS |
11 |
TO |
14 |
MEAN |
53.6 D |
55.8 |
54.3 |
3.0 |
|
|
|
|
S.D. |
54.88 |
60.38 |
90.43 |
80.20 |
|
|
|
|
N |
22 |
24 |
24 |
24 |
DAYS |
14 |
TO |
16 |
MEAN |
23.7 D |
39.6 |
23.9 |
-13.4 |
|
|
|
|
S.D. |
69.71 |
60.25 |
58.82 |
51.46 |
|
|
|
|
N |
22 |
24 |
24 |
24 |
DAYS |
16 |
TO |
19 |
MEAN |
-47.8 D |
-28.4 |
-37.8 |
-50.1 |
|
|
|
|
S.D. |
65.23 |
70.23 |
91.54 |
75.09 |
|
|
|
|
N |
22 |
24 |
24 |
24 |
DAYS |
19 |
TO |
21 |
MEAN |
-0.3 D |
-27.5 |
-36.7* |
-16.4 |
|
|
|
|
S.D. |
34.96 |
50.66 |
38.55 |
61.69 |
|
|
|
|
N |
21 |
24 |
24 |
24 |
DAYS |
21 |
TO |
23 |
MEAN |
6.0 D |
29.7 |
25.0 |
7.7 |
|
|
|
|
S.D. |
40.54 |
56.58 |
62.58 |
67.35 |
|
|
|
|
N |
21 |
24 |
24 |
24 |
DAYS |
23 |
TO |
25 |
MEAN |
-10.6 D |
-5.5 |
8.4 |
20.3 |
|
|
|
|
S.D. |
73.17 |
57.69 |
68.65 |
72.82 |
|
|
|
|
N |
21 |
24 |
24 |
23 |
DAYS |
25 |
TO |
28 |
MEAN |
13.7 D |
40.1 |
15.8 |
-4.2 |
|
|
|
|
S.D. |
72.97 |
66.74 |
66.45 |
81.05 |
|
|
|
|
N |
21 |
24 |
24 |
20 |
DAYS |
28 |
TO |
29 |
MEAN |
11.4 D |
11.4 |
19.9 |
32.8 |
|
|
|
|
S.D. |
33.17 |
36.99 |
47.77 |
55.59 |
|
|
|
|
N |
20 |
24 |
24 |
20 |
Statistics: D=Dunnett-test (two-sided), * : p<=0.05 ** : p<=0.01
Table 5: Mean maternal food consumption during gestation
|
|
|
TEST GROUP 0 |
TEST GROUP 1 |
TEST GROUP 2 |
TEST GROUP 3 |
|
|
|
0 mg/kg bw/d |
10 mg/kg bw/d |
30 mg/kg bw/d |
100 mg/kg bw/d |
DAYS |
0 TO 6 |
MEAN OF MEANS |
176.2 |
171.4 |
173.7 |
175.3 |
|
|
S.D. |
4.60 |
6.55 |
5.07 |
7.45 |
|
|
N (days) |
6 |
6 |
6 |
6 |
DAYS |
6 TO 28 |
MEAN OF MEANS |
118.2 |
123.0 |
114.1 |
81.6 |
|
|
S.D. |
29.57 |
25.04 |
28.78 |
31.98 |
|
|
N (days) |
22 |
22 |
22 |
22 |
DAYS |
0 TO 29 |
MEAN OF MEANS |
129.6 |
132.1 |
125.9 |
101.5 |
|
|
S.D. |
35.47 |
30.24 |
35.35 |
47.50 |
|
|
N (days) |
29 |
29 |
29 |
29 |
Table 6: Summary of maternal necropsy observations
FEMALES EXAMINED |
N |
TEST GROUP 0 0 mg/kg bw/d 25 |
TEST GROUP 1 10 mg/kg bw/d 25 |
TEST GROUP 2 30 mg/kg bw/d 25 |
TEST GROUP 3 100 mg/kg bw/d 25 |
NOTHING ABNORMAL DETECTED |
N |
20 |
22 |
22 |
20 |
|
% |
80 |
88 |
88 |
80 |
FINDINGS AFTER GAVAGE ERROR |
N |
1 |
0 |
0 |
0 |
|
% |
4.0 |
0.0 |
0.0 |
0.0 |
LUNGS: ACUTE FIBRINOUS - |
N |
1 |
0 |
0 |
0 |
PURULENT PNEUMONIA |
% |
4.0 |
0.0 |
0.0 |
0.0 |
LIVER: GRANULATED SURFACE |
N |
0 |
1 |
0 |
0 |
|
% |
0.0 |
4.0 |
0.0 |
0.0 |
KIDNEY: MALPOSITIONED |
N |
0 |
1 |
1 |
0 |
|
% |
0.0 |
4.0 |
4.0 |
0.0 |
RECTUM: NO FECES |
N |
1 |
0 |
2 |
1 |
|
% |
4.0 |
0.0 |
8.0 |
4.0 |
WATERY FECES |
N |
2 |
0 |
1 |
1 |
|
% |
8.0 |
0.0 |
4.0 |
4.0 |
URETER: SHORT |
N |
0 |
1 |
1 |
0 |
|
% |
0.0 |
4.0 |
4.0 |
0.0 |
OVARIES: RUDIMENTARY |
N |
1 |
0 |
0 |
0 |
|
% |
4.0 |
0.0 |
0.0 |
0.0 |
Table 7: Summary of reproduction data of the females
|
TEST GROUP 0 0 mg/kg bw/d |
TEST GROUP 1 10 mg/kg bw/d |
TEST GROUP 2 30 mg/kg bw/d |
TEST GROUP 3 100 mg/kg bw/d |
Pregnant at Terminal Sacrifice (N) |
20 |
24 |
24 |
20 |
Resorptions: Total MEAN |
0.3 D |
0.5 |
0.7 |
0.6 |
S.D. |
0.81 |
0.78 |
0.82 |
1.23 |
TOTAL |
7 |
12 |
16 |
13 |
MEAN (%) |
3.7 D |
6.2 |
6.8 |
6.2 |
S.D. |
8.47 |
9.70 |
8.66 |
11.52 |
Early MEAN |
0.3 D |
0.3 |
0.3 |
0.4 |
S.D. |
0.80 |
0.69 |
0.48 |
1.14 |
TOTAL |
6 |
7 |
8 |
8 |
MEAN (%) |
3.2 D |
3.8 |
3.6 |
3.7 |
S.D. |
8.32 |
8.94 |
5.42 |
10.47 |
Late MEAN |
0.1 D |
0.2 |
0.3 |
0.3 |
S.D. |
0.22 |
0.51 |
0.56 |
0.64 |
TOTAL |
1 |
5 |
8 |
5 |
MEAN (%) |
0.6 D |
2.3 |
3.3 |
2.5 |
S.D. |
2.48 |
5.72 |
5.80 |
6.52 |
Dead Fetuses (N) |
0 |
1 |
0 |
0 |
Statistics: D=Dunnett-test (two-sided), *: p<=0.05 ** : p<=0.01
Table 8: Mean placental and fetal body weights (based on litter)
|
|
TEST GROUP 0 0 mg/kg bw/d |
TEST GROUP 1 10 mg/kg bw/d |
TEST GROUP 2 30 mg/kg bw/d |
TEST GROUP 3 100 mg/kg bw/d |
PLACENTAL WEIGHTS UNITS; GRAMS: |
|
|
|
|
|
|
MEAN |
5.0 D |
5.1 |
4.8 |
4.7 |
|
S.D. |
0.80 |
0.94 |
0.77 |
0.87 |
|
N |
20 |
24 |
24 |
20 |
of Male Fetuses |
MEAN |
5.1 D |
5.2 |
4.9 |
4.8 |
|
S.D. |
0.83 |
0.85 |
0.75 |
1.00 |
|
N |
20 |
23 |
23 |
20 |
of Female Fetuses |
MEAN |
4.9 D |
5.0 |
4.7 |
4.7 |
|
S.D. |
0.80 |
0.93 |
0.89 |
0.86 |
|
N |
20 |
23 |
24 |
20 |
FETAL WEIGHTS, UNITS: |
GRAMS |
|
|
|
|
of all Viable Fetuses |
MEAN |
38.3 D |
38.6 |
36.3 |
33.6* |
|
S.D. |
5.23 |
6.72 |
5.07 |
5.93 |
|
N |
20 |
24 |
24 |
20 |
of Male Fetuses |
MEAN |
39.1 D |
39.2 |
36.0 |
33.9* |
|
S.D. |
5.76 |
5.48 |
5.31 |
5.91 |
|
N |
20 |
23 |
23 |
20 |
of Female Fetuses |
MEAN |
37.6 D |
37.3 |
36.0 |
33.6 |
|
S.D. |
5.15 |
6.83 |
5.49 |
6.30 |
|
N |
20 |
23 |
24 |
20 |
Statistics: D=Dunnett-test (two-sided), * : p<=0.05 ** : p<=0.01
Table 9: Total fetal external malformations
|
|
Test group 0 0 mg/kgbw/d |
Test group 1 10 mg/kg bw/d |
Test group 2 30 mg/kg bw/d |
Test group 3 100 mg/kg bw/d |
Litter Fetuses |
N N |
20 174 |
24 189 |
24 205 |
20 179 |
Fetal incidence |
N (%) |
1 (0.6) |
1 (0.5) |
0.0 |
5 (2.8) |
Litter incidence |
N (%) |
1 (5.0) |
1 (4.2) |
0.0 |
2 (10) |
Affected fetuses/litter |
Mean % |
0.5 |
0.4 |
0.0 |
2.4 |
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent
Table 10: Total soft tissue malformations
|
|
Test group 0 0 mg/kgbw/d |
Test group 1 10 mg/kg bw/d |
Test group 2 30 mg/kg bw/d |
Test group 3 100 mg/kg bw/d |
Litter Fetuses |
N N |
20 174 |
24 189 |
24 205 |
20 179 |
Fetal incidence |
N (%) |
2 (1.1) |
3 (1.6) |
3 (1.5) |
1 (0.6) |
Litter incidence |
N (%) |
1 (5.0) |
3 (13) |
2 (8.3) |
1 (5.0) |
Affected fetuses/litter |
Mean % |
0.9 |
1.6 |
1.4 |
0.4 |
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent
Table 11: Total skeletal malformations
|
|
Test group 0 0 mg/kgbw/d |
Test group 1 10 mg/kg bw/d |
Test group 2 30 mg/kg bw/d |
Test group 3 100 mg/kg bw/d |
Litter Fetuses |
N N |
20 174 |
24 189 |
24 205 |
20 179 |
Fetal incidence |
N (%) |
1 (0.6) |
2 (1.1) |
2 (1.0) |
1 (0.6) |
Litter incidence |
N (%) |
1 (5.0) |
2 (8.3) |
2 (8.3) |
1 (5.0) |
Affected fetuses/litter |
Mean% |
0.6 |
1.2 |
1.1 |
0.4 |
Table 12: Fetuses with more than one malformation
Test group |
Doe No.-Fetus No., Sex |
Finding |
0 (0 mg/kg bw/d) |
25-10 F |
domed head, hydrocephaly |
1 (10 mg/kg bw/d) |
26-10 M |
thoracic hemivertebra, misshapen thoracic vertebra |
44-01 F |
malpositioned kidney, short ureter |
|
2 (30 mg/kg bw/d) |
68-07 F |
exoccipital fused with 1st cervical arch, cervical hemivertebra |
69-03 M |
multiple malformations of the great vessels (persistent truncus arteriosus, aortic arch atresia, malpositioned subclavian origin) |
|
75-02 F |
aortic arch atresia, malpositioned kidney |
|
75-06 F |
thoracic hemivertebra, branched rib |
|
3 (100 mg/kg bw/d) |
76-04 M |
multiple external malformations (domed head, cleft palate, small tongue), hydrocephaly |
76-06 F |
multiple external malformations (domed head, cleft palate, small tongue) |
|
76-11 F |
multiple external malformations (domed head, cleft palate, small tongue), severely malformed skull bones |
|
76-12 M |
multiple external malformations (domed head, cleft palate, small tongue) |
mg/kg bw/d = milligram per kilogram body weight per day; No.= number; M = male; F = female
The stability of the test substance in 0.5% CMC suspension in deionized water (with 10 mg/100 mL Cremophor EL) over a maximum of 7 days and warmed up to approximately 30 degrees Celsius was demonstrated. The homogeneous distribution of the test substance in the vehicle was shown. The correctness of the prepared concentrations was shown.
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 500 mg/kg bw/day
- Species:
- rat
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
A developmental toxicity study in rats (OECD 414) is available. Dose levels were 50, 150 and 500 mg/kg bw and corn oil was used as vehicle. Dose levels were chosen based on a range-finding study in pregnant rats with 300 and 1000 mg/kg bw. In the maternal toxicity study, alterations of some liver/metabolic parameters were found specially in the high dose group and included increased values of alkaline phosphatase, alanine aminotransferase, cholesterol, triglycerides, glucose and decreases of creatinine, protein, albumin, globulin, calcium and bile acids. Relative liver weights were increased. Increased absolute and relative adrenals weights and decreased absolute spleen and kidneys weights were found in the high dose group when compared to controls. Values of alkaline phosphatase, triglycerides and bile acids were also different to controls at 300 mg/kg bw/day. Pale discoloration of liver was observed in four dams of the high dose group. Terminal body weight, corrected body weight and corrected body weight gain were significantly reduced in the high dose group. Uterus weight did not differ between groups.
The doses chosen for the main study are sufficient to ensure some but not excessive maternal toxicity. In the main study, statistically significant decrease in body weight (up to 7%) and body weight gain was observed in treated females receiving 500 mg/kg bw/day, starting from Day 9 post coitum. Statistically significant decrease in food consumption (up to 22%) was observed in treated females receiving 500 mg/kg bw/day, starting from Day 9 post coitum. Statistically significant decrease in corrected body weight and corrected body weight gain was noted in treated females receiving 150 and 500 mg/kg bw/day. The difference in corrected body weight at 150 mg/kg bw/day was minor (258 versus 270 g) and therefore not considered adverse. There were no macroscopic findings at necropsy. Considering the effects on food consumption, corrected body weight and corrected body weight gain and the knowledge on clinical chemistry from the range-finding study, the high dose group caused liver toxicity and the NOAEL for maternal toxicity was 150 mg/kg. Litter data, mean foetal weight and sex ratio were unaffected by treatment. No treatment-related differences were seen at visceral examination of foetuses between the control and treated groups. No relevant changes were recorded at the skeletal examination of foetuses in treated groups, compared to controls. An increase in the presence of small foetuses was recorded in the high dose group. This higher number of small foetuses was due to one litter, and the corresponding dam had the smallest corrected body weight gain and was most strongly affected by maternal toxicity.
A prenatal developmental toxicity in New Zealand White rabbits was performed according to OECD 414. The test substance was administered as an aqueous suspension to groups of 25 inseminated female New Zealand White rabbits orally by gavage in doses of 10, 30 and 100 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 28. The vehicle control group, consisting of 25 females, was dosed with the vehicle (0.5% Sodium carboxymethyl cellulose [CMC] suspension in deionized water (with 10 mg/100 mL Cremophor EL) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group.
At terminal sacrifice on GD 29, 20-24 females per group had implantation sites. Food consumption and body weight of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day.
On GD 29, all females were sacrificed and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each doe, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorp-tions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for any external, soft tissue and skeletal (inclusive cartilage) findings.
The stability of the test substance in 0.5% CMC suspension in deionized water (with 10 mg/100 mL Cremophor EL) over a maximum of 7 days and warmed up to approximately 30 degrees Celsius was demonstrated. The homogeneous distribution of the test substance in the vehicle was shown. The correctness of the prepared concentrations was shown.
The following test substance-related adverse effects/findings were noted:
Test group 3 (100 mg/kg bw/d):
Dams: Increased number of abortions (4 vs. 2 in control), reduced food consumption, overall 31% less food than the concurrent control does during GD 6-28, reduced mean body weights (BW) and average body weight gain (BWC), weight loss during the treatment period (-24.0 g vs. +104.4 g in control), lower net weight gain ( 421.0g) in comparison to the concurrent control (-322.2 g).
Fetuses: No test substance-related, independent adverse effects on fetuses.
Test group 2 (30 mg/kg bw/d):
No test substance-related adverse effects on does, gestational parameters or fetuses. The mean fetal body weights were significantly lower in the 100 mg/kg bw/d group compared to the control group (33.6 g versus 38.6 g). Considering the immediate effect of dosing on maternal food consumption and body weight loss during GD 6-28, the reduced fetal body weights are a result of the poor nutritional condition of the does. The substance has no direct developmental toxicity. No teratogenicity was observed.
Test group 1 (10 mg/kg bw/d):
No test substance-related adverse effects on does, gestational parameters or fetuses.
Under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) caused evidence of systemic maternal toxicity at the high- dose level of 100 mg/kg bw/d, such as a slightly higher incidence of abortions and reduced defecation in almost all females of this group, along with a distinct decrease of food consumption as well as body weight/body weight gain. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 30 mg/kg bw/d. Since there was no evidence for independent, toxicologically relevant adverse effects of the test substance on fetal morphology at any dose, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is the highest dose of 100 mg/kg bw/d.
Justification for classification or non-classification
The available data on reproductive organs are considered not sufficient for classification of the substance under Regulation (EC) 1272/2008.
The developmental toxicity studies in rabbit and rat (OECD 414) do not warrant classification of the substance for developmental toxicity under Regulation (EC) 1272/2008.
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