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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

This information will be updated when the final study report is available.

Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

Oral: NOAEL for maternal toxicity and developmental toxicity is 33500 ppm (corresponding to an actual test substance intake of 904 mg/kg/day), rabbit, OECD TG 414, 2021

Oral: NOAEL for maternal toxicity and developmental toxicity was established being at least 1000 mg/kg bw/day, Rat, OECD TG 414, 2018

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 Sep 2020 to 01 Mar 2021
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:
2018
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 18-20 weeks old
- Weight at study initiation: Between 2899 and 4201 g
- Housing: The animals were individually housed in cages with perforated floors equipped with water bottles
- Diet: standard powder diet for rabbits (ad libitum), refreshed daily
- Water: ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Targeted: 17 to 21, actual mean: 18 to 19
- Humidity (%): Targeted: 40 to 70, actual mean: 49 to 64
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 24 Sep 2020 to 01 Mar 2021
Route of administration:
other: oral: feed (powdered diet)
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet: The test substance was mixed without the use of a vehicle, directly with some powder feed (premix) and subsequently mixed with the bulk of the diet. Diets were prepared at least once weekly for use at room temperature for a maximum of one day.
- The same diets remained in the food hopper for a maximum of one day. On the day of weighing the remaining food in the food hopper, diet was replaced with new diet retained from the freezer acclimated to room temperature conditions for at least 1 hour prior to opening the diet bag
- Storage temperature of food: Diets were kept in the freezer (≤-15°C) for a maximum of 8 days prior to use, if not used on the day of preparation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Homogeneity and concentration of the test substance in dietary preparations were determined based on Zinc content since there was no better, feasible analytical method available for this type of molecule. The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 preparations were averaged and utilized as the concentration results. The stability of the test substance in the diet was not determined, since the available analytical method was only capable of measuring the zinc content in the test substance.
- The stability of the test substance in the diet was not determined, since the available analytical method was only capable of measuring the zinc content in the test substance. Since the diets were prepared at least once weekly, stored in daily portions in tightly closed bags in the freezer (≤-15°C) for a maximum of 8 days prior to use and diets were refreshed daily, stability was not considered an issue during the performance of the study and the integrity of the study or its results are not affected
Details on mating procedure:
- Day 0 post-coitum is defined as the day of successful mating
Duration of treatment / exposure:
Day 7 to Day 29 post-coitum, inclusive
Frequency of treatment:
Continuously
Duration of test:
Until day 29 post-coitum
Dose / conc.:
3 350 ppm
Remarks:
Group 2: Corresponded with an intended dose level of 99 mg/kg bw/day
Dose / conc.:
10 050 ppm
Remarks:
Group 3: Corresponded with an intended dose level of 291 mg/kg bw/day
Dose / conc.:
33 500 ppm
Remarks:
Group 4: Corresponded with an intended dose level of 904 mg/kg bw/day
No. of animals per sex per dose:
22 females
Control animals:
yes, plain diet
Details on study design:
Tolerability study (non-pregnant female New Zealand White rabbits) via oral gavage
Method: The study started with one group of 3 animals (Group 1, period 1) dosed with corn oil for 10 days in order to determine the maximum tolerated volume of vehicle (2 mL/kg of corn oil was tested). Upon determination of the maximum tolerated volume of corn oil, the first dose level was confirmed for Group 2, period 1. Animals of Group 2 (period 1) were treated at a dose level of 500 mg/kg for 14 days. In order to evaluate the treatment effects during a similar time-interval as in period 1, Group 1 animals (period 2) were treated at 500 mg/kg for 14 days. A wash-out period of at least 7 days was maintained for Group 1 between Periods 1 and 2. The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test substance. The test substance was administered to the animals by oral gavage as a suspension in corn oil based on the results of the trial preparations and to be in line with the available toxicity testing. The first dose level was selected based on the results of a previously performed teratology toxicity study with the test substance by oral gavage in Wistar Han rats (Test Facility No. 20150567) in which no maternal and developmental toxicity was observed by treatment up to, and including, 1000 mg/kg/day. In addition, the maximum dose level tested in this study was conditioned by the solubility of the test substance in the vehicle (maximum concentration in corn oil of 250 mg/mL) and the maximum volume of vehicle tolerated by the rabbit species that was determined in the current study (up to 2 mL/kg of corn oil was tested).

Results: Animals treated with corn oil at a volume of 2 mL/kg for 10 days presented with reduced food consumption; severe over 1-2 days during the first 6 days of treatment (followed by a partial recovery afterwards), which resulted in a slight and transient body weight loss (up to 3% vs start of dosing) during the first 5-6 days of treatment. No oily vehicle-related diarrhoea was observed in any of the animals. As no severe/persistent toxicity was observed after treatment with corn oil and as the use of a water-based vehicle was not feasible for formulating the test substance, a dose volume of 2 mL corn oil/kg was considered acceptable in the current study to reach the first dose level of 500 mg/kg. A dose level of 500 mg/kg/day was tolerated in 5/6 animals. Severe toxicity was observed only in 1/6 animal which led to discontinuation of treatment on Day 13 due to severely reduced/absent food consumption resulting in persistent body weight loss. However, the remaining 5/6 animals presented only with transient effects in both body weight (slight/moderate body weight loss) and food consumption (up to severe reduction in food intake). Some of these effects observed (in particular in 3/6 animals) were similar to those observed in Group 1 animals (period 1) treated only with corn oil at 2 mL/kg. Therefore, the dose level of 500 mg/kg was established as the maximum dose level for the dose range finder study in pregnant rabbits as 500 mg/kg was tolerated for 14 days and as higher doses could not be further tested due to the solubility of the test substance in the vehicle (maximum concentration in corn oil of 250 mg/mL) and the maximum volume of vehicle tolerated by the rabbit species (2 mL/kg).

Conclusion: Based on the results of this tolerability study, selected dose levels for the dose range finder in pregnant rabbits (Test Facility Study No. 20150556) are 125, 250 and 500 mg/kg.


Dose Range Finder Study (pregnant female New Zealand White rabbits) via oral gavage:
Method: The test substance and vehicle were administered to the appropriate animals by once daily oral gavage from Day 6 to Day 16 post-coitum, inclusive (instead of to Day 28 post-coitum). Treatment was discontinued due to severe toxicity in all groups, including control. All females were sacrificed in extremis on Days 14-17 post-coitum. A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures (Study No. 521128 was used for DCS). The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test substance. Dose levels were selected based on the results of a tolerability study in non-pregnant females (Test Facility Study No. 20150559). In this latter study, two groups of three females each were treated with either the vehicle alone (corn oil) or with 500 mg/kg/day of the test substance in formulation (dose volume: 2 mL/kg). In Group 1, the two different conditions were tested (treatment Period 1 or 2) separated by a treatment-free (wash-out) period of 24 days. In Group 2, only the condition test substance in formulation was tested (Period 1). Group 1/Period 1 (vehicle, 10 days): Reduced food consumption (up to severe in the first two days) was observed during the first six days of treatment (followed by a partial recovery afterwards). This resulted in a slight and transient body weight loss (up to 3% versus start of dosing) during the first 5-6 days of treatment. Persistent reduced faeces production was observed in 2/3 animals from Day 5 onwards (including two days during the wash-out period for one female), whereas the other animal was observed with reduced faeces production on Days 3 to 6 of treatment only. Group 2/Period 1 (500 mg/kg/day, 12-14 days): Treatment of one female (No. 4) had to be discontinued based on the severely reduced faecal production, low/absent food consumption and persistent body weight loss from Day 5 onwards. Clinical signs of the remaining two females (Nos. 5 and 6) included reduced faeces production (slight to moderate) for several days. In addition, Female No. 5 was noted with transient pale faeces on Day 11. Both females showed recurrent body weight loss (up to 3%) during the treatment period. Female No. 5 was noted with transient reduction (up to severe) in food consumption over various intervals, where the food consumption of Female No. 6 remained within normal values. No abnormalities were noted during necropsy. Group 1/Period 2 (500 mg/kg, 14 days): Clinical signs included reduced faeces production for six to nine days during the treatment period in all three females, being moderate to severe for five consecutive days in Female No. 2. Body weight loss was observed in all three females, ranging from 1-5% body weight loss compared to start of dosing. Slightly reduced food consumption was observed in Female No. 3 on Days 9-10, severely reduced food consumption was observed in Female No. 1 on Days 7-8. Female No. 2 was observed with a moderate reduction in food consumption on Days 1-2, with a recovery thereafter. However, a recurrent decrease (up to severe) was noted on Days 5-6 and Days 7-12, with a recovery over Days 12-15. Based on these results, and after consultation with the Sponsor, dose levels of 125, 250 and 500 mg/kg/day were selected for the current dose range finding study. The high-dose level should produce some maternal and/or developmental toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal toxic effects. The low-dose level should produce no observable indications of toxicity.

Results: In this dose range finding study, 1/6, 1/6, 3/6 and 3/6 females in the control, 125, 250 and 500 mg/kg/day groups, respectively, had to be euthanised for humane reasons on Days 14-16 post-coitum. Based on the poor general health of the remaining animals in all groups, including control, it was decided to sacrifice all residual animals on Day 17 post-coitum and thereby to terminate the dose range finding study. Maternal Findings: Clinical signs that were observed during the treatment period included piloerection in 1/6 females in the control group, in 5/6 females at 125 mg/kg/day, in 4/6 females at 250 mg/kg/day and in 3/6 females at 500 mg/kg/day. In addition, paleness was observed in 1/6 females at 125 mg/kg/day, in 2/6 females at 250 mg/kg/day and in 2/6 females at 500 mg/kg/day. Lethargy was observed in 2/6 females at 250 mg/kg/day and in 3/6 females at
500 mg/kg/day. Besides that, flat posture in 2/6 females at 250 mg/kg/day, and hunched posture in 1/6 females at 250 mg/kg/day and in 2/6 females at 500 mg/kg/day was observed. Lastly, lean appearance was observed in one female (500 mg/kg/day) only. Reduced faeces production (up to severe) were observed in all females among all groups, including control, in the absence of a dose response. In addition, pale faeces were observed in
4/6 females in the control group, in 5/6 females at 125 mg/kg/day, in 3/6 females at 250 mg/kg/day and in 5/6 females at 500 mg/kg/day. Incidental finding that were noted included, scabs, swelling of the flews or mouth, and scars. These findings occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance. Body weight loss (up to 7%) at the majority of the animals across all groups, including controls, were noted without a dose relationship. Severely reduced food consumption was observed for all animals, including controls, at the majority of the intervals, without a clear relationship to dose level. Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. One female (treated at 500 mg/kg/day) was observed with scab formation on the lip and nose region. Maternal Pregnancy Data: As no females survived until scheduled necropsy, the results described below should be considered as indicative only. Except for two females treated at 125 mg/kg/day all females were found to be pregnant with normal implantations in development at the time of preterm sacrifice. However, one female in the control group and two females at 125 mg/kg/day had litters with high numbers of early resorptions (5/9, 4/5 and 6/14 early resorptions of total implantations, respectively). Mean numbers of corpora lutea and implantation sites were within the normal range in all groups. No foetuses were available for foetal examination, as no females survived until the day of scheduled necropsy.

Conclusion: Based on the results from this dose range finder study it can be concluded that pregnant rabbits housed and treated under the conditions of the current study do not tolerate corn oil when administered at a dose volume of 2 mL/kg. Therefore, it was decided in consultation with the Sponsor not to continue with corn oil as vehicle in the planned main prenatal development study.

Tolerability study (non-pregnant female New Zealand White rabbits) via diet:
The objective of this study was to determine the highest tolerated dose of the test substance in non-pregnant rabbits, for a dose range finding study in pregnant rabbits and subsequent prenatal developmental toxicity study.
Results showed No mortality occurred during the study period. No test substance-related clinical signs were noted. Reduced faeces production was observed for one Female (Period 2; 25000 ppm) on Days 3 to 7 of treatment and occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, this was considered to be unrelated to treatment with the test substance. At 33500 ppm, no body weight gain was observed. Two out of three treated females were observed with a slight body weight loss (up to 2%) compared to the body weight at the initiation of treatment. Normal body weight (gain) was observed up to 25000 ppm. Normal food consumption was observed up to 33500 ppm. Notably, one female had a lower food consumption during both treatment periods (Period 1, 16750 ppm and Period 2, 25000 ppm), which was considered unrelated to treatment with the test substance as this was noted in one female only and no trend was apparent regarding dose. Macroscopic observations at necropsy did not reveal any alterations.

Conclusion: Based on the results of this tolerability study, selected dose levels for the Dose Range Finder in pregnant rabbits were 10050, 20100 and 33500 ppm.

Dose Range Finder (pregnant female New Zealand White rabbits) via diet:
The test substance was administered to the appropriate animals once daily by dietary administration from Day 7 to Day 29 post-coitum, inclusive. The dose levels were selected based on the results of a tolerability study with the test substance in rabbits via dietary exposure (Test Facility Study No. 20247985). The in-life procedures, Observations, and measurements in the DRF were identical as for the Main study. Terminal Procedures of the F0-generation were identical as for the Main study. Each viable foetus of animals surviving to planned necropsy was externally examined in detail and weighed. A gross external examination was performed for late resorption (control). No visceral (internal) or skeletal examination was performed. Foetuses and late resorptions without malformations were discarded.

Results showed no mortality occurred during the study period. No toxicologically significant clinical signs were noted up to treatment at 33500 ppm. Reduced faeces production was incidentally noted for all control animals, 5/6 animals treated at 10050 ppm and in general more frequently during the treatment period for all animals treated at 20100 and 5/6 animals treated at 33500 ppm. Incidental findings that were noted included dark faeces, focal erythema, alopecia and/or scabs. These findings occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance. Overall, body weights, body weight gain and (for uterus weight) corrected body weight gain at necropsy of treated animals remained in the same range as controls over the study period. At the start of treatment (Day 7-8 post-coitum), mean food consumption was decreased for animals treated at 20100 and 33500 ppm, which recovered from Days 8-9 post-coitum onwards. Food consumption and relative food consumption tended to be lower in females of all treatment groups when compared to controls (reaching statistical significance on a few occasions and often in absence of a clear dose-response). Food consumption was comparable between all groups over post-coitum Days 22-29. Macroscopic observations at necropsy did not reveal any alterations that were considered to be toxicologically relevant. One female treated at 10050 ppm (Animal No. 12) and two females treated at 20100 ppm (Animal Nos. 13 and 18) were non-pregnant. All other animals were found to be pregnant with viable foetuses. The numbers of corpora lutea and implantation sites, pre- and post-implantation loss and early and late resorptions in the control and test groups were similar and in the range of normal biological variation. There were no test item-related effects on litter size up to 33500 ppm. Mean litter sizes were 9.7, 10.8, 8.8 and 10.0 foetuses/litter for the control, 10050, 20100 and 33500 ppm groups, respectively. All foetuses were alive. Mean combined (male and female) foetal body weights were 39.4, 34.0, 37.2 and 36.5 for the groups treated at 10050, 20100 and 33500 ppm, respectively. Mean combined (male and female) foetal weights were slightly lower at 10050 ppm and 33500 ppm and below the range of historical control data (4/5 litters at 10050 ppm and 4/6 litters at 33500 ppm; HCD: mean: 39.1, P5-P95: 36.7-42.3). As no dose-related trend was noted, the lower foetal weight was considered not toxicologically relevant. External examination of the foetuses did not show any test item-related abnormalities.

Conclusion: Based on the results of the dose range finder, selected dose levels for the Main study were 3350, 10050 and 33500 ppm (corresponding to an actual test substance intake of 0, 99, 291, 904 mg/kg body weight/day, respectively)




Maternal examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily throughout the study
- Cage side observations checked: Mortality/moribundity, clinical observations (performed from day 7) and cage debris to detect premature births

BODY WEIGHT:
- Time schedule for examinations: Animals were weighed on Days 7, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum

FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: Food consumption of animals was measured daily from Day 3 post-coitum onwards.

WATER CONSUMPTION:
- Time schedule for examinations: Water consumption was monitored by visual inspection of the water bottles.

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 29 post-coitum or within 24 hours of early delivery
- All animals (including animals sacrificed before planned necropsy and females with that delivered on the day of scheduled necropsy) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of life/dead foetuses: Yes
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
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:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Numerical data collected on scheduled occasions were analysed according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever possible. In case deemed appropriate, values were also be expressed as a percentage of predose or control values.
Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric: Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution were compared using the Mann Whitney test. Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05). intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.
Incidence: An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test was significant. No statistics was applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and postimplantation loss.
Indices:
- Preimplantation loss (%): ((number of corpora lutea - number of implantation sites) / number of corpora lutea) x 100
- Postimplantation loss (%): ((number of implantation sites - number of live foetuses) / number of implantation sites ) x 100
- Viable fetuses affected/litter (%): (number of viable foetuses affected/litter / number of viable foetuses/litter) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Reduced faeces production was observed in 13/22 females of the control group, in 17/22 females treated at 3350 ppm, in 20/22 females treated at 10050 ppm and in 20/22 females treated at 33500 ppm, which corresponded with periods of reduced food consumption. In general, reduced faeces production was observed more frequently and at a higher severity during the treatment period of animals treated at 33500 ppm. One animal was noted with clinical observations of the right eye from post-coitum Day 27 onwards (i.e focal erythema, watery discharge, swelling of the conjunctiva, mucous membrane and/or a tilted head). This incidental finding was considered not related to treatment with the test substance. Other clinical signs noted during the treatment period included alopecia, fissures with bleedings on the bridge of the nose and scabs. These clinical signs occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test substance.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two females (33500 ppm) were sacrificed on post-coitum Days 20 and 19, respectively due to early abortions:
- For one female, one fetus was found on the manure tray on Day 20 post-coitum. On post-coitum Days 19 and 20, a decreased food consumption was noted and concurrent reduced faeces production (severe degree) was recorded. At necropsy, ectopic splenic tissue was observed. The uterus contained one implantation site only.
- For one female, blood and organic material were found on the manure tray on post-coitum Days 18 and 19, respectively. Furthermore, slightly reduced food consumption and reduced faeces production (moderate degree) were recorded during post-coitum Days 18 and 19. At necropsy, the uterus contained 4 early resorptions only.

One female (33500 ppm) was sacrificed in extremis for animal welfare reasons on Day 13 post-coitum, as absent food consumption was noted for 11 consecutive days (Days 3-14 post-coitum). This resulted in body weight loss (5%) on Day 12 post-coitum vs start of treatment on Day 7 post-coitum and reduced faeces production for 7 consecutive days with pale faeces on Days 12-13 post-coitum. Macroscopic findings included a dark-red discolored vagina with dark-red, watery content, and 10 normal implantations in development were present in the uterus. As a reduced food consumption was already noted during the acclimatisation period, this sacrifice was considered unrelated to treatmentwith the test substance. Another female (33500 ppm) was sacrificed on Day 29 post-coitum after it started to deliver its offspring before scheduled necropsy (8 pups already delivered and 8 fetuses inside the uterus). At necropsy, no findings were noted. As early deliveries at this term occur more often in rabbits and this is the only female with an early delivery, it was considered incidental and unrelated to treatment with the test substance.

Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Before start of treatment on post-coitum Day 7, mean body weights were slightly lower compared to the initial body weight on post-coitum Day 0 in all groups, most likely due to acclimatisation to the powder diet.
Slightly reduced body weight gain was noted at 3350, 10050 and 33500 ppm compared to concurrent controls over post-coitum Days 7-9, which recovered from post-coitum Day 12 onwards for females treated at 3350 and 10050 ppm. At 33500 ppm, body weight gain remained slightly lower during the entire treatment period (statistically significant on Day 15 post-coitum only). This resulted in a statistically significantly reduced mean body weight (6% lower compared to controls) at the end of the treatment period. Since the initial body weights of females treated at 33500 ppm were lower (-3%) than controls at the start of treatment, the test substance-related effect of these body weight changes were considered minimal.
At 33500 ppm, mean body weight gain corrected for gravid uterus was lower compared to control (-206.2 gram (-6.1%) vs -114.5 gram (-3.1%) in controls).
Body weights and body weight gain (corrected for gravid uterus) at 3350 and 10050 ppm were considered not affected by treatment with the test substance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
During the acclimatisation period (Day 3-7 post-coitum), individual food consumption was reduced for several females in all groups (including controls) due to acclimatization to the powder diet. For most of these females, food consumption recovered to similar values as observed for other animals of the same dose group. Therefore, it was considered that the low food consumption at the end of the acclimatisation period did not affect the outcome of the study. During treatment, mean absolute food consumption of females treated at 33500 ppm was lower than in concurrent controls over post-coitum Days 7-23 and over post-coitum Days 27- 28 (statistically significant on a few occasions). Relative food consumption was decreased from post-coitum Days 7-20 (not always statistically significant), which recovered to values comparable to concurrent controls from post-coitum Days 21-22 onwards. Overall, mean over mean absolute food consumption over the post-coitum period was 17% lower than concurrent controls.
At 3350 and 10050 ppm, mean food consumption (both absolute and relative) was slightly lower than concurrent controls over post-coitum Days 7-18, reaching statistical significance on a few occasions. Relative food consumption was similar to concurrent controls from postcoitum Days 17-18 (3350 ppm) or 20-21 (10050 ppm) onwards. Overall, mean over mean absolute food consumption over the post-coitum period was 7 and 6% lower in the groups treated at 3350 and 10050 ppm, respectively when compared to concurrent controls. Due to the minimal effect, these differences in food consumption were considered not to represent a change of toxicological significance
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine 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
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Description (incidence and severity):
As two cases of early abortions were noted in the high-dose group of this study and these early abortions occur very rarely, it was considered possibly related to treatment with the test substance. Although early abortion is defined as reproduction endpoint, which is not included in the objective for this type of study.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
For the two females at 33500 ppm that aborted on post-coitum Days 20 or 19, respectively, individual pre-implantation loss was higher when compared with mean concurrent control values (90% and 43% for the two females vs. 10.7% in the control group). As treatment was initiated after the implantation period was completed, this was considered to be unrelated to treatment with the test substance.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Five females in the control group, one female in the 3350 and 10050 ppm groups each, and two females in the 33500 ppm group were not pregnant at necropsy. The incidence of non-pregnancy was considered to be unrelated to treatment with the test substance as no dose-related response was observed. Given the four females at 33500 that were sacrificed in extremis, delivered early or aborted (prior to scheduled necropsy), in total 17, 21, 21 and 16 females (with their litters) were available for evaluation in the control, 3350, 10050 and 35000 ppm groups, respectively.
Key result
Dose descriptor:
NOAEL
Remarks:
General toxicity
Effect level:
>= 33 500 ppm
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
corresponding to an actual test substance intake of 904 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Remarks:
Maternal developmental toxicity
Effect level:
>= 33 500 ppm
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
corresponding to an actual test substance intake of 904 mg/kg bw/day
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean combined (male and female) foetal body weights were 38.3, 37.4, 37.0 and 36.8 g for the control, 3350, 10050 and 33500 ppm groups, respectively.
Mean male, female and combined (male and female) foetal weights were slightly lower at 33500 ppm (3-4% compared to control), mainly attributed to the high mean litter weight of a control female. At 33500 ppm, a relatively low mean litter weight was noted in two females, which was below the range of the mean litter weight in concurrent controls. However, as these differences were minor and mean values per litter remained within the range of historical control data, they were considered to be unrelated to treatment with the test substance.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on litter size up to 33500 ppm. Mean litter sizes were 9.1, 10.2, 9.0 and 9.5 fetuses/litter for the control, 3350, 10050 and 33500 ppm groups, respectively
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects on skeletal morphology following administration of the test substance up to 33500 ppm.
Skeletal malformations were noted in 1 (1), 1 (1), 2 (2) and 1 (1) foetuses (litters) of the control, 3350, 10050 and 33500 ppm groups, respectively.
The incidental occurrence of malformations does not indicate a test substance relationship. Moreover, all but one (sternoschisis) were seen previously in historical foetuses.
All skeletal variations occurred at low incidences, in the absence of a dose-related incidence trend and/or at frequencies that were within the range of available historical control data.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects on visceral morphology following administration of the test substance up to 33500 ppm. Visceral malformations occurred in one foetus of the control, one foetus at 3350 ppm and two foetuses at 33500 ppm. The various malformations all affected the cardiovascular system, and due to the isolated incidence and occurrence in historical control foetuses, these were not related to the test substance.
The visceral variation supernumerary spleen showed a remarkable group distribution as it was observed in 3.7% of foetuses per litter in the control group and not in the test substance groups, yielding statistical significance for these latter groups. However, the control incidence was higher than the historical maximum value and not having a supernumerary spleen is considered normal. Therefore, this remarkable group distribution of supernumerary spleen was considered to have occurred by chance.
All other visceral variations that were noted were considered unrelated to treatment with the test substance as they occurred in the absence of a dose-related trend, infrequently and/or in control foetuses only.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
The numbers of foetuses (litters) available for a full foetal morphological examination were 155 (17), 215 (21), 189 (21) and 152 (16) in the control, 3350, 10050 and 33500 ppm groups, respectively. At 33500 ppm, two females were euthanised before the scheduled day of necropsy and two other females had an early delivery. Results of the uterus contents from these dams are recorded separately and are not included in the summary. Except from hyperextended hindlimbs (no underlying skeletal abnormality) in a foetus of the control group, no other malformations occurred These results are considered to be non-treatment related.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 33 500 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Corresponding to an actual test substance intake of 904 mg/kg/day
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Results analytical study: accuracy and homogeneity:

Accuracy: The concentrations analyzed in the diets of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%). A small response at the retention time of the test item was observed in the chromatograms of the Group 1 diet. It was considered not to derive from the diet since a similar response was obtained in the matrix matched analytical blanks.

Homogeneity: The diets of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Table 1. Body weight (gram) summary females (F0 -generation)

 

Group 1: control

Group 2: 3350 ppm

Group 3: 10050 ppm

Group 4: 33500 ppm

Post- coitum
day 0

Mean

3679

3502

3578

3588

 

ST.DEV.

347.6

336

309.2

326.7

 

N

17

21

21

20

Day 7

Mean

3523

3381

3455

3419

 

ST.DEV.

344.1

252.1

249.6

239.6

 

N

17

21

21

20

Day 9

Mean

3596

3414

3489

3439

 

ST.DEV.

324.8

254

251.9

241.9

 

N

17

21

21

20

Day 12

Mean

3636

3476

3540

3482

 

ST.DEV.

309.3

259.1

250.7

259.3

 

N

17

21

21

20

Day 15

Mean

3740

3566

3623

3556

 

ST.DEV.

303.9

274.5

239.9

272.1

 

N

17

21

21

19

Day 18

Mean

3766

3608

3637

3589

 

ST.DEV.

323.8

266.6

253

277

 

N

17

21

21

19

Day 21

Mean

3754

3615

3646

3566

 

ST.DEV.

302

261

234.8

245.7

 

N

17

21

21

17

Day 24

Mean

3801

3661

3686

3603

 

ST.DEV.

303.2

261.3

219.4

255

 

N

17

21

21

17

Day 27

Mean

3849

3717

3717

3651

 

ST.DEV.

301.2

264.1

201.6

251.5

 

N

17

21

21

17

Day 29

Mean

3891

3766

3765

3674*

 

ST.DEV.

296.7

269.6

203

238

 

N

17

21

21

17

Table 2. Summary of maternal survival and pregnancy status

Dose group

1

2

3

4

No.

NO.

%

NO.

%

NO.

%

NO.

%

Females on study                               

 22

 

22

 

22

 

22

 

Females that aborted or delivered

0

0.0

0

0.0

0

0.0

2

9.1

Females that died

0

0.0

0

0.0

0

0.0

0

0.0

Females that aborted

0

0.0

0

0.0

0

0.0

0

0.0

Nongravid

0

0.0

0

0.0

0

0.0

0

0.0

Gravid

0

0.0

0

0.0

0

0.0

0

0.0

Females that were euthanised

0

0.0

0

0.0

0

0.0

2

9.1

Nongravid

0

0.0

0

0.0

0

0.0

0

0.0

Gravid

0

0.0

0

0.0

0

0.0

2

100.0

Females examined at scheduled necropsy

22

100.0

22

100.0

22

100.0

18

81.8

Nongravid

5

22.7

1

4.5

1

4.5

2

11.1

Gravid

17

77.3

21

95.5

21

95.5

16

88.9

With resorptions only

0

0.0

0

0.0

0

0.0

0

0.0

With viable fetuses

17

100.0

21

100.0

21

100.0

16

100.0

Total females gravid

17

77.3

21

95.5

21

95.5

20

90.9

Dose group: 1 - 0 PPM, 2 - 3350 PPM, 3 - 10050 PPM, 4- 33500 PPM

Table 3. Summary of foetal data at scheduled necropsy

 

 

Sex

Viable foetuses

Dead foetuses

Resorptions

 

Post implantation loss

Implantation sites

Corpora lutea

Pre implantation loss

Foetal weights in grams

No of gravid females

Group

 

M

F

 

 

Early

Late

 

 

 

 

 

 

1

Total

86

69

155

0

10

2

12

167

187

20

NA

17

 

Mean

5.1

4.1

9.1

0.0

0.6

0.1

0.7

9.8

11.0

1.2

38.3

 

 

S.D.

2.08

2.41

2.23

0.00

0.80

0.49

0.85

2.43

2.40

1.63

5.00

 

2

Total

110

105

215

0

7

0

7

222

243

21

NA

21

 

Mean

5.2

5.0

10.2

0.0

0.3

0.0

0.3

10.6

11.6

1.0

37.4

 

 

S.D.

1.95

1.48

1.89

0.00

0.58

0.00

0.58

1.94

2.01

1.05

3.70

 

3

Total

87

102

189

1

1

6

8

197

224

27

NA

21

 

Mean

4.1

4.9

9.0

0.0

0.0

0.3

0.4

9.4

10.7

1.3

37.0

 

 

S.D.

1.62

1.90

2.00

0.22

0.22

0.46

0.50

2.06

1.74

1.59

4.47

 

4

Total

73

79

152

0

4

1

5

157

171

14

NA

16

 

Mean

4.6

4.9

9.5

0.0

0.3

0.1

0.3

9.8

10.7

0.9

36.8

 

 

S.D.

1.75

2.43

2.10

0.00

0.58

0.25

0.70

2.29

1.66

1.15

5.49

 

None significantly different from control group

NA = NOT APPLICABLE

MEAN NUMBER OF VIABLE FOETUSES, MEAN NUMBER OF IMPLANTATION SITES, MEAN NUMBER OF CORPORA LUTEA, FOETAL WEIGHTS COMPARED USING DUNNETT'S TEST

Dose group: 1 - 0 PPM, 2 - 3350 PPM, 3 - 10050 PPM, 4- 33500 PPM

Table 4. Summary of litter proportions of malformations

% Per litter                                                

 

 

 

 

Day 29

Dose group:

 

1

2

3

4

Number of litters examined skeletally

 

17

21

21

16

Sternoschisis

Mean

0

0

0

0.8

 

S.D.

0

0

0

3.13

Vertebral centra anomaly

Mean

0

0

0.6

0

 

S.D.

0

0

2.73

0

Vertebral anomaly with or without associated rib anomaly

Mean

0.6

0.5

0

0

 

S.D.

2.43

2.18

0

0

Rib anomaly

Mean

0

0

0.4

0

 

S.D.

0

0

1.98

0

Dose group: 1 - 0 PPM, 2 - 3350 PPM, 3 - 10050 PPM, 4- 33500 PPM

None significantly different from control group

Table 5. Summary of foetuses and litters with variations [Absolute no.]

 

Foetuses

Litters

Dose group

1

2

3

4

1

2

3

4

Number examined externally

155

215

189

152

17

21

21

16

Number with findings

0

0

0

0

0

0

0

0

Number examined viscerally

155

215

189

152

17

21

21

16

Gallbladder- bilobed

0

1

0

0

0

1

0

0

Spleen- split

0

1

0

0

0

1

0

0

Spleen- supernumerary

4

0

0

0

4

0

0

0

Retrocaval ureter

1

8

6

1

1

5

5

1

Lung- absent accessory lobe

1

4

2

2

1

4

2

2

Liver- small supernumerary lobe(s)

2

1

5

0

2

1

2

0

Gallbladder- absent or small

1

1

3

5

1

1

1

2

Liver- cyst(s)

0

0

1

0

0

0

1

0

Left carotid- originating from brachiocephalic trunk

0

5

3

0

0

2

3

0

Aortic arch- supernumerary artery

0

1

1

0

0

1

1

0

Ovary- cyst(s)

0

0

1

0

0

0

1

0

Gallbladder- contents

0

0

1

0

0

0

1

0

Spleen- constricted

0

1

0

0

0

1

0

0

Renal papilla(e)- absent and/or small

0

2

0

0

0

1

0

0

Right subclavian- retroesophageal

2

0

0

0

1

0

0

0

Number examined skeletally

155

215

189

152

17

21

21

16

13th full rib(s)

69

117

103

91

16

20

18

16

13th rudimentary rib(s)

17

14

19

10

11

9

12

10

Sternebra(e) #5 and/or #6 unossified

30

47

43

41

10

14

12

8

Sternum- supernumerary ossification site

0

0

0

1

0

0

0

1

Pelvic girdle- caudal shift

32

50

66

35

9

15

17

12

Supernumerary sternebra

0

1

0

1

0

1

0

1

Vertebral centra- reduced ossification

1

1

0

0

1

1

0

0

Number examined skeletally

155

215

189

152

17

21

21

16

Sternebrae fused

1

3

0

0

1

3

0

0

Metacarpal(s) and/or metatarsal(s) unossified

11

6

11

14

7

3

9

5

Sternebra(e) malaligned

7

7

5

0

4

5

4

0

Hyoid body and/or arches unossified

1

2

3

3

1

1

3

3

7th cervical ossification site(s)

3

14

4

1

2

5

3

1

Sternebra(e)- branched

0

0

1

0

0

0

1

0

Caudal vertebral anomaly

0

0

2

0

0

0

2

0

Hyoid arch(es) bent

0

2

3

0

0

2

2

0

Tarsal(s) unossified

3

0

3

3

2

0

3

1

Skull- supernumerary site

0

2

2

1

0

1

1

1

Skull bone- unossified line

0

1

0

0

0

1

0

0

Pubis- unossified

0

1

3

0

0

1

2

0

Dose group: 1 - 0 PPM, 2 - 3350 PPM, 3 - 10050 PPM, 4- 33500 PPM

Conclusions:
Based on the results in this prenatal developmental toxicity study, the NOAEL for maternal toxicity and developmental toxicity is set at 33500 ppm (corresponding to an actual test substance intake of 904 mg/kg bw/day)
Executive summary:

The objectives of this study were to determine the potential the test substance to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterise maternal toxicity at the exposure levels tested when given via diet to time-mated female New Zealand White rabbits from Days 7 to 29 post-coitum, inclusive. This study was compliant to GLP conducted according to OECD TG 414. The dose levels in this study were selected to be 0, 3350, 10050, 33500 ppm (corresponding to an actual test substance intake of 0, 99, 291, 904 mg/kg body weight/day, respectively), based on the results of the Dose Range Finder study (No. 20247987). Chemical analyses of dietary preparations were conducted once during the study to assess accuracy and homogeneity. The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, test substance intake, macroscopic examination, uterine contents, corpora lutea, implantation sites, and pre- and post-implantation loss. The following parameters were determined for the F1-generation: the number of live and dead foetuses, foetal body weights, sex ratio, external, visceral and skeletal malformations and developmental variations. Analysis of diet preparations confirmed that the achieved dietary concentrations of the test substance were in agreement with the target concentrations and that the test substance was homogeneously distributed in the diet.

Results showed at 33500 ppm, two females were sacrificed before scheduled necropsy on post-coitum Days 19 or 20, as these females were observed with organic material or a foetus on the manure tray, indicating early abortion. Both females showed a reduced food consumption and reduced faeces production only on two days prior to sacrifice. As two cases of early abortions were noted in the high-dose group of this study and these early abortions occur very rarely, it was considered possibly related to treatment with the test substance. A lower mean food consumption was observed in females at 33500 ppm between Days 7-23, which appeared to recover to control levels from Day 21 post-coitum onwards. This was accompanied with a slightly reduced body weight gain in females treated at 33500 ppm during the entire treatment period. Additionally, body weight gain corrected for gravid uterus was lower compared to control (-206.2 gram versus -114.5 gram in controls). As food consumption of treated females recovered to levels similar as concurrent controls by the end of the treatment period and the changes in body weight or (corrected) body weight gain were minor, these changes were considered non-adverse. No test substance-related gross findings were observed at necropsy. The number of corpora lutea, implantation sites, viable or dead foetuses, early or late resorptions, pre- and post-implantation loss were considered not affected by treatment with the test substance up to 33500 ppm. No test substance-related changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, foetal body weights, external, visceral and skeletal malformations and developmental variations).

In conclusion, based on the results in this prenatal developmental toxicity study, the NOAEL for maternal toxicity and developmental toxicity is set at 33500 ppm (corresponding to an actual test substance intake of 904 mg/kg bw/day)

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Aug 2018 to 30 Aug 2018
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 January 2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Wistar Han
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld, Germany
- Age at study initiation: 10-14 weeks
- Weight at study initiation: 186 – 266 g
- Housing: individually in Macrolon plastic cages (MIII type, height 18 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) provided ad libitum throughout the study
- Water: Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 21
- Humidity (%): 49 to 78
- Air changes (per hr): ≥ 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
12 Aug 2018 to 30 Aug 2018
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 6 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. The dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: The use of corn oil was required due to the physicochemical properties of the test substance (low water solubility and stability)
- Concentration in vehicle: 25, 75 and 250 mg/mL
- Amount of vehicle: 4 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
CONCENTRATION ANALYSIS
Concentration was determined based on Zinc content since there was no better, feasible analytical method available for this type of molecule.
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration.

HOMOGENEITY ANALYSIS
Homogeneity was determined based on Zinc content since there was no better, feasible analytical method available for this type of molecule.
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was =< 10%.

STABILITY ANALYSIS
The stability of the test item in the vehicle was not determined in this study since the available analytical method was only capable of measuring the zinc content in the test substance and, therefore, unable to measure the test item itself. Since the formulations were prepared fresh shortly before administration to the test animals and there is no known or deemed instability of the test item in an oil suspension; stability was not considered an issue during the performance of the study and the integrity of the study or its results are not affected.
Details on mating procedure:
The females arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating).
Duration of treatment / exposure:
The test item and vehicle were administered 7 days a week from day 6 to day 20 post-coitum, inclusive.
Frequency of treatment:
Once daily
Duration of test:
Animals surviving until scheduled euthanasia were euthanised on day 21 post-coitum.
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
Group 1 to 4: 22 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of the dose range finder. In this dose range finder, doses up to 1000 mg/kg bw/day were tolerated by pregnant rats. Only transient test item-related clinical signs were noted during the first two days of treatment in all treated groups. No toxicologically relevant changes in mean body weight and food consumption were observed.
Based on the results of the dose range finder, selected dose levels for the main study were 100, 300 and 1000 mg/kg bw/day.
- Animal assignment: random
Maternal examinations:
CAGE SIDE OBSERVATIONS
- Time schedule: twice daily, in the morning and at the end of the working day
- Cage side observations included general health/mortality and moribundity.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: once daily, beginning on Day 2 post-coitum and lasting up to the day prior to necropsy.
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.
Cage debris was examined to detect premature birth.

BODY WEIGHT
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum

FOOD CONSUMPTION
- Time schedule for examinations: Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum
- Food consumption was determined for each animal and mean daily diet consumption calculated as g food/kg body weight/day.

POST-MORTEM EXAMINATIONS
- Sacrifice on gestation day 21 post-coitum
- Organs examined: All animals were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No organs (except for the uterus) were weighed.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination
Examinations included:
- Gravid uterus weight
- Number of corpora lutea
- Number of implantations
- Number of early resorptions
- Number of late resorptions
- Other: the number and distribution of live and dead fetuses; the sex of each fetus based on the ano-genital distance
Fetal examinations:
- External examinations: all viable fetuses per litter. Each viable foetus was sexed, examined in detail to detect macroscopic visible abnormalities and their weight was determined. For late resorptions, a gross external examination was performed.
- Soft tissue examinations: The sex of all fetuses was confirmed by internal examination and approximately one-half of the fetuses in each litter (all groups) were examined for visceral anomalies by dissection in the fresh (non-fixed) state. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development.
- Skeletal examinations: half per litter. In order to further examine an externally noted malformation, one fetus selected for visceral examination, was also subjected to skeletal examination.
- Head examinations: half per litter. This examination included serial sectioning of the head in at least seven even slices after removal of the lower jaw. For each slice, the front- and back side was examined. Multiple structures were distinguished, special attention was paid to nose/throat, eyes and brains.
Statistics:
STATISTICAL ANALYSIS
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

PARAMETRIC
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

NON-PARAMETRIC
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution were compared using the Mann Whitney test.
Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences.

INCIDENCE
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
MATERNAL VARIABLES
Body Weight Gains: Calculated against the body weight on Day 6 post-coitum
Corrected Body Weight Gains: Terminal body weight minus the body weight on Day 6 postcoitum and the weight of gravid uterus.
Relative Food Consumption: Calculated against the body weight for scheduled intervals.

REPRODUCTION AND DEVELOPMENTAL VARIABLES
For each group, the following calculations were performed:

Pre-implantation loss (%): [(number of corpora lutea - number of implantation sites) / number of corpora lutea] x 100

Post-implantation loss (%): [(number of implantation sites - number of live fetuses) / number of implantation sites] x 100

Viable fetuses affected per litter (%): [(number of viable fetuses affected/litter) / (number of viable fetuses/litter)] x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Piloerection was observed in one control female for 3 consecutive days during the pre-treatment period and in one animal at 1000 mg/kg bw/day during the last 3 days of treatment. This clinical sign was considered to be unrelated to treatment at the incidence observed (in one high dose female only) and as it was also noted in one control animal.
Another clinical sign noted during the treatment period was alopecia which occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, this was considered to be unrelated to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weight and body weight gain of animals treated up to 1000 mg/kg remained in the same range as controls over the treatment period.
Body weight gain corrected for gravid uterus was considered to be unaffected by treatment up to 1000 mg/kg.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes in food consumption before or after correction for body weight were observed over the study period.
Over Days 6-9 post-coitum, mean food consumption (before or after allowance for body weight) was significantly increased at 1000 mg/kg bw/day (relative food consumption 11% higher vs concurrent control mean). This significant increase in food consumption was considered to be due to a slightly low mean value of the concurrent control group. Mean values of all treated groups were well within the range of the historical control data.
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
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The numbers of pre-implantation loss in the control and test groups were similar and/or in the range of normal biological variation.
No toxicologically relevant changes in post-implantation loss were observed by treatment up to 1000 mg/kg.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes in the litter incidence of early and late resorptions were observed by treatment up to 1000 mg/kg.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
General toxicity
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
Maternal developmental
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on fetal body weights (both sexes) noted by treatment up to 1000 mg/kg.
Mean combined (male and female) fetal body weights were 5.3, 5.4, 5.4 and 5.3 gram for the control, 100, 300 and 1000 mg/kg groups, respectively.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratios were unaffected by treatment up to 1000 mg/kg.
Mean sex ratios (males:females) were 47:53, 48:52, 49:51 and 57:43 for the control, 100, 300 and 1000 mg/kg groups, respectively.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on litter size by treatment up to 1000 mg/kg.
Mean litter sizes were 10.8, 10.7, 11.2 and 11.1 fetuses/litter for the control, 100, 300 and 1000 mg/kg groups, respectively.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects on external morphology following treatment up to 1000 mg/kg.
Two externally malformed fetuses were observed in this study, each in the control and 100 mg/kg bw/day groups: the low dose fetus was noted with a small lower jaw and cleft palate and the control fetus had a malrotated hindlimb. Skeletal examination substantiated the jaw and palate findings, but there was no apparent skeletal origin for the limb finding. Due to the single occurrence of these external malformations and their occurrence only in the low dose and control groups, these malformations were considered to be of spontaneous origin.
No external variations were noted in any of the groups.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects on skeletal morphology following treatment up to 1000 mg/kg.
Aside from the underlying skull malformations of the low dose fetus that presented with a small lower jaw and cleft palate, bent limb bones were observed in another low dose fetus and a vertebral anomaly was found in a mid dose fetus. The low incidence and group distribution of these malformations did not suggest a treatment relationship and litter incidences were well within the historical control range. Therefore, the above malformations were considered to be spontaneous in origin and not test item related.
Skeletal variations occurred at an incidence of 70.6%, 82.5%, 75.4% and 70.0% per litter in the control, 100, 300 and 1000 mg/kg groups, respectively. All skeletal variations noted occurred in the absence of a dose-related incidence trend, infrequently and/or at frequencies that were within the range of available historical control data. Therefore, they were not considered treatment related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects on visceral morphology following treatment up to 1000 mg/kg. No remarkable observations were noted in the heads which were removed from one-half of the fetuses in each litter.
Only one visceral malformation (situs inversus) was observed and as it occurred in a single control fetus, it was as such considered to be of spontaneous origin.
Only one visceral variation (small supernumerary liver lobes) was observed in one and four fetuses at 100 and 300 mg/kg, respectively. This finding was considered to be unrelated to treatment in the absence of a dose-response relationship and at the low incidence observed (litter incidences were well within the range of the historical control data).
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

TABLE 1.6 BODY WEIGHTS (GRAM) SUMMARY

F0-GENERATION

 

 

GROUP 1

CONTROL

GROUP 2

100 MG/KG

GROUP 3

300 MG/KG

GROUP 4

1000 MG/KG

POST COITUM

 

 

 

 

 

DAY 2

MEAN

209

209

209

210

 

ST.DEV.

19.6

19.7

17.0

18.3

 

N

22

22

22

22

DAY 6

MEAN

224

225

225

227

 

ST.DEV.

20.0

19.9

17.7

18.5

 

N

22

22

22

22

DAY 9

MEAN

232

234

234

235

 

ST.DEV.

20.2

23.1

18.8

20.0

 

N

22

22

22

22

DAY 12

MEAN

246

248

249

251

 

ST.DEV.

21.3

24.5

18.1

21.0

 

N

22

22

22

22

DAY 15

MEAN

261

262

263

264

 

ST.DEV.

23.1

26.2

19.9

23.7

 

N

22

22

22

22

DAY 18

MEAN

292

294

296

296

 

ST.DEV.

25.0

31.1

23.6

26.0

 

N

22

22

22

22

DAY 21

MEAN

329

332

335

333

 

ST.DEV.

30.8

36.8

28.0

29.6

 

N

22

22

22

22

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

TABLE 1.7 BODY WEIGHT GAIN (%) SUMMARY

F0-GENERATION

 

 

GROUP 1

CONTROL

GROUP 2

100 MG/KG

GROUP 3

300 MG/KG

GROUP 4

1000 MG/KG

POST COITUM

 

 

 

 

 

DAY 2

MEAN

-7

-7

-7

-8

 

ST.DEV.

2.3

2.0

2.4

2.3

 

N

22

22

22

22

DAY 6

MEAN

0

0

0

0

 

ST.DEV.

0.0

0.0

0.0

0.0

 

N

22

22

22

22

DAY 9

MEAN

4

4

4

4

 

ST.DEV.

1.8

1.9

2.0

2.3

 

N

22

22

22

22

DAY 12

MEAN

10

10

11

10

 

ST.DEV.

2.3

3.0

2.8

3.1

 

N

22

22

22

22

DAY 15

MEAN

16

16

17

16

 

ST.DEV.

2.7

3.8

3.8

4.9

 

N

22

22

22

22

DAY 18

MEAN

30

30

32

31

 

ST.DEV.

3.7

5.2

4.4

6.0

 

N

22

22

22

22

DAY 21

MEAN

47

47

49

47

 

ST.DEV.

7.2

7.1

6.4

6.8

 

N

22

22

22

22

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

TABLE 1.8 FOOD CONSUMPTION (G/ANIMAL/DAY) SUMMARY

F0-GENERATION

 

 

GROUP 1

CONTROL

GROUP 2

100 MG/KG

GROUP 3

300 MG/KG

GROUP 4

1000 MG/KG

POST COITUM

 

 

 

 

 

DAYS 2-6

MEAN

22

22

21

21

 

ST.DEV.

3.5

2.6

2.6

2.2

 

N

22

22

22

22

DAYS 6-9

MEAN

16

18

18

18 *

 

ST.DEV.

3.0

2.8

2.1

2.9

 

N

22

22

22

22

DAYS 9-12

MEAN

20

20

20

21

 

ST.DEV.

2.7

2.8

2.0

2.7

 

N

22

22

22

22

DAYS 12-15

MEAN

19

19

19

20

 

ST.DEV.

2.4

3.1

2.4

2.1

 

N

22

22

22

22

DAYS 15-18

MEAN

24

24

24

25

 

ST.DEV.

2.4

2.8

2.5

3.1

 

N

22

22

22

22

DAYS 18-21

MEAN

23

23

23

24

 

ST.DEV.

2.8

3.5

2.8

2.4

 

N

22

22

22

22

MEAN OF MEANS

 

21

21

21

22

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

TABLE1.9 RELATIVE FOOD CONSUMPTION (G/KG BODY WEIGHT/DAY) SUMMARY

F0-GENERATION

 

 

GROUP 1

CONTROL

GROUP 2

100 MG/KG

GROUP 3

300 MG/KG

GROUP 4

1000 MG/KG

POST COITUM

 

 

 

 

 

DAYS 2-6

MEAN

98

97

95

93

 

ST.DEV.

14.8

9.3

11.6

7.8

 

N

22

22

22

22

DAYS 6-9

MEAN

70

76

76

78 *

 

ST.DEV.

12.5

7.5

7.0

9.7

 

N

22

22

22

22

DAYS 9-12

MEAN

79

82

81

84

 

ST.DEV.

8.7

8.4

7.5

7.5

 

N

22

22

22

22

DAYS 12-15

MEAN

74

74

73

77

 

ST.DEV.

6.3

7.1

7.4

6.0

 

N

22

22

22

22

DAYS 15-18

MEAN

83

83

82

85

 

ST.DEV.

7.2

5.7

7.4

7.9

 

N

22

22

22

22

DAYS 18-21

MEAN

71

70

70

73

 

ST.DEV.

4.4

8.1

7.2

8.0

 

N

22

22

22

22

MEAN OF MEANS

 

79

80

80

82

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

TABLE 1.10 MACROSCOPIC FINDINGS SUMMARY

 

GROUP 1

CONTROL

GROUP 2

100 MG/KG

GROUP 3

300 MG/KG

GROUP 4

1000 MG/KG

POST COITUM

 

 

 

 

Animals examined

22

22

22

22

Animals without findings

22

21

22

20

Animals affected

0

1

0

2

Liver
Focus/foci

0

0

0

1

Skin

 

 

 

 

Alopecia

0

1

0

1

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

TABLE 1.11 SUMMARY OF MATERNAL SURVIVAL AND PREGNANCY STATUS

DOSE GROUP :

1

 

2

 

3

 

4

 

 

NO.

%

NO.

%

NO.

%

NO.

%

FEMALES ON STUDY

22

 

22

 

22

 

22

 

 

 

 

 

 

 

 

 

 

FEMALES THAT ABORTED

 

 

 

 

 

 

 

 

OR DELIVERED

0

0.0

0

0.0

0

0.0

0

0.0

 

 

 

 

 

 

 

 

 

FEMALES THAT DIED

0

0.0

0

0.0

0

0.0

0

0.0

  FEMALES THAT ABORTED

0

0.0

0

0.0

0

0.0

0

0.0

  NONGRAVID

0

0.0

0

0.0

0

0.0

0

0.0

  GRAVID

0

0.0

0

0.0

0

0.0

0

0.0

 

 

 

 

 

 

 

 

 

FEMALES THAT WERE EUTHANIZED

0

0.0

0

0.0

0

0.0

0

0.0

  NONGRAVID

0

0.0

0

0.0

0

0.0

0

0.0

  GRAVID

0

0.0

0

0.0

0

0.0

0

0.0

 

 

 

 

 

 

 

 

 

FEMALES EXAMINED AT

 

 

 

 

 

 

 

 

SCHEDULED NECROPSY

22

100.0

22

100.0

22

100.0

22

100.0

  NONGRAVID

0

0.0

0

0.0

0

0.0

0

0.0

  GRAVID

22

100.0

22

100.0

22

100.0

22

100.0

     WITH RESORPTIONS ONLY

0

0.0

0

0.0

0

0.0

0

0.0

     WITH VIABLE FETUSES

22

100.0

22

100.0

22

100.0

22

100.0

 

 

 

 

 

 

 

 

 

TOTAL FEMALES GRAVID

22

100.0

22

100.0

22

100.0

22

100.0

 1-  0 MG/KG; 2-  100 MG/KG; 3 -  300 MG/KG; 4- 1000 MG/KG

1.12 SUMMARY OF FETAL DATA AT SCHEDULED NECROPSY

 

 

 

 

 

 

 

 

POST

 

 

PRE

FETAL

NO. OF

SEX

VIABLE

DEAD

RESORPTIONS

IMPLANTATION

IMPLANTATION

CORPORA

IMPLANTATION

WEIGHTS

GRAVID

GROUP

 

M

F

FETUSES

FETUSES

EARLY

LATE

LOSS

SITES

LUTEA

LOSS

IN GRAMS

FEMALES

1

TOTAL

112

125

237

0

11

0

11

248

274

26

NA

22

MEAN

5.1

5.7

10.8

0

0.5

0

0.5

11.3

12.5

1.2

5.3

S.D.

2.07

2.06

2.65

0

0.8

0

0.8

2.83

2.42

1.76

0.25

2

TOTAL

109

126

235

0

11

0

11

246

265

19

NA

22

MEAN

5.0

5.7

10.7

0

0.5

0

0.5

11.2

12

0.9

5.4

S.D.

2.01

2.55

2.5

0

0.96

0

0.96

2.38

1.91

1.21

0.26

3

TOTAL

120

127

247

0

7

0

7

254

273

19

NA

22

MEAN

5.5

5.8

11.2

0

0.3

0

0.3

11.5

12.4

0.9

5.4

S.D.

2.18

2.11

2.02

0

0.48

0

0.48

2.11

1.87

1.25

0.22

4

TOTAL

141

103

244

0

6

1

7

251

264

13

NA

22

MEAN

6.4

4.7

11.1

0

0.3

0

0.3

11.4

12

0.6

5.3

 

S.D.

1.71

1.25

2.04

0

0.55

0.21

0.65

2.24

1.75

0.85

0.23

 

None significantly different from control group

NA = NOT APPLICABLE

MEAN NUMBER OF VIABLE FETUSES, MEAN NUMBER OF IMPLANTATION SITES, MEAN NUMBER OF CORPORA LUTEA, FETAL WEIGHTS COMPARED USING DUNNETT'S TEST

1- 0 MG/KG; 2- 100 MG/KG; 3- 300 MG/KG; 4- 1000 MG/KG

TABLE 1.13 SUMMARY OF FETAL DATA AT SCHEDULED NECROPSY [% PER LITTER]

GROUP

0 MG/KG

100 MG/KG

300 MG/KG

1000 MG/KG

CORPORA LUTEA

 

 

 

 

MEAN

12.5

12.0

12.4

12.0

S.D.

2.42

1.91

1.87

1.75

N

22

22

22

22

 

 

 

 

 

IMPLANTATION SITES

 

 

 

 

MEAN

11.3

11.2

11.5

11.4

S.D.

2.83

2.38

2.11

2.24

N

22

22

22

22

 

 

 

 

 

VIABLE FETUSES (%)

 

 

 

 

MEAN

96.0

95.3

97.4

97.6

S.D.

6.27

8.65

4.01

4.84

N

22

22

22

22

 

 

 

 

 

DEAD FETUSES (%)

 

 

 

 

MEAN

0.0

0.0

0.0

0.0

S.D.

0.00

0.00

0.00

0.00

N

22

22

22

22

 

 

 

 

 

EARLY RESORPTIONS (%)

 

 

 

 

MEAN

4.0

4.7

2.6

2.1

S.D.

6.27

8.65

4.01

4.08

N

22

22

22

22

 

 

 

 

 

LATE RESORPTIONS (%)

 

 

 

 

MEAN

0.0

0.0

0.0

0.4

S.D.

0.00

0.00

0.00

1.64

N

22

22

22

22

 

 

 

 

 

TOTAL RESORPTIONS (%)

 

 

 

 

MEAN

4.0

4.7

2.6

2.4

S.D.

6.27

8.65

4.01

4.84

N

22

22

22

22

 

 

 

 

 

PRE-IMPLANTATION LOSS (%)

 

 

 

 

MEAN

10.0

7.6

6.8

5.6

S.D.

14.92

11.30

10.27

9.10

N

22

22

22

22

 

 

 

 

 

POST-IMPLANTATION LOSS (%)

 

 

 

 

MEAN

4.0

4.7

2.6

2.4

S.D.

6.27

8.65

4.01

4.84

N

22

22

22

22

 

 

 

 

 

MALES (%)

 

 

 

 

MEAN

47.1

47.7

48.5

57.0

S.D.

14.89

18.12

16.55

11.94

N

22

22

22

22

 

 

 

 

 

FEMALES (%)

 

 

 

 

MEAN

52.9

52.3

51.5

43.0

S.D.

14.89

18.12

16.55

11.94

N

22

22

22

22

 

 

 

 

 

MALE FETAL WEIGHTS (g)

 

 

 

 

MEAN

5.5

5.5

5.5

5.4

S.D.

0.27

0.27

0.30

0.24

N

22

22

22

22

 

 

 

 

 

FEMALE FETAL WEIGHTS (g)

 

 

 

 

MEAN

5.2

5.2

5.2

5.1

S.D.

0.25

0.24

0.23

0.24

N

22

22

22

22

 

 

 

 

 

COMBINED FETAL WEIGHTS (g)

 

 

 

 

MEAN

5.3

5.4

5.4

5.3

S.D.

0.25

0.26

0.22

0.23

N

22

22

22

22

PROPORTIONAL (%) DATA COMPARED USING THE MANN-WHITNEY TEST

CORPORA LUTEA, IMPLANTATION SITES AND FETAL WEIGHTS COMPARED USING DUNNETT'S TEST

None significantly different from control group

TABLE 1.14 SUMMARY OF FETUSES AND LITTERS WITH MALFORMATIONS [ABSOLUTE NO.]

 

DAY 21

F E T U S E S

L I T T E R S

DOSE GROUP:

1

2

3

4

1

2

3

4

NUMBER EXAMINED EXTERNALLY

237

235

247

244

22

22

22

22

LOWER JAW- ABSENT OR SMALL

0

1

0

0

0

1

0

0

LIMB(S)- MALROTATED

1

0

0

0

1

0

0

0

CLEFT PALATE

0

1

0

0

0

1

0

0

 

 

 

 

 

 

 

 

 

NUMBER EXAMINED VISCERALLY

120

118

122

123

22

22

22

22

SITUS INVERSUS

1

0

0

0

1

0

0

0

 

 

 

 

 

 

 

 

 

NUMBER EXAMINED SKELETALLY

118

117

125

121

22

22

22

22

VERTEBRAL ANOMALY WITH OR WITHOUT ASSOCIATED RIB ANOMALY

0

0

1

0

0

0

1

0

BENT LIMB BONE(S)

0

1

0

0

0

1

0

0

 

 

 

 

 

 

 

 

 

TOTAL NUMBER WITH MALFORMATIONS

 

 

 

 

 

 

 

 

EXTERNAL:

1

1

0

0

1

1

0

0

SOFT TISSUE :

1

0

0

0

1

0

0

0

SKELETAL :

0

1

1

0

0

1

1

0

 

 

 

 

 

 

 

 

 

COMBINED :

2

2

1

0

2

2

1

0

1-  0 MG/KG; 2-  100 MG/KG; 3-  300 MG/KG; 4-  1000 MG/KG

 

TABLE 1.15 SUMMARY OF LITTER PROPORTIONS OF MALFORMATIONS% PER LITTER

 

DAY 21

DOSE GROUP:

 

1

2

3

4

NUMBER OF LITTERS EXAMINED EXTERNALLY

 

22

22

22

22

LOWER JAW- ABSENT OR SMALL

MEAN

0.0

0.6

0.0

0.0

 

S.D.

0.00

2.67

0.00

0.00

LIMB(S)- MALROTATED

MEAN

0.4

0.0

0.0

0.0

 

S.D.

1.94

0.00

0.00

0.00

CLEFT PALATE

MEAN

0.0

0.6

0.0

0.0

 

S.D.

0.00

2.67

0.00

0.00

NUMBER OF LITTERS EXAMINED VISCERALLY

 

22

22

22

22

SITUS INVERSUS

MEAN

0.9

0.0

0.0

0.0

 

S.D.

4.26

0.00

0.00

0.00

NUMBER OF LITTERS EXAMINED SKELETALLY

 

22

22

22

22

VERTEBRAL ANOMALY WITH OR WITHOUT ASSOCIATED RIB ANOMALY

MEAN

0.0

0.0

0.8

0.0

 

S.D.

0.00

0.00

3.55

0.00

BENT LIMB BONE(S)

MEAN

0.0

0.9

0.0

0.0

 

S.D.

0.00

4.26

0.00

0.00

TOTAL MALFORMATIONS

 

 

 

 

 

PERCENT PER LITTER WITH EXTERNAL MALFORMATIONS

MEAN

0.4

0.6

0.0

0.0

 

S.D.

1.94

2.67

0.00

0.00

PERCENT PER LITTER WITH SOFT TISSUE MALFORMATIONS

MEAN

0.9

0.0

0.0

0.0

 

S.D.

4.26

0.00

0.00

0.00

PERCENT PER LITTER WITH SKELETAL MALFORMATIONS

MEAN

0.0

0.9

0.8

0.0

 

S.D.

0.00

4.26

3.55

0.00

TOTAL PERCENT PER LITTER WITH MALFORMATIONS

MEAN

1.3

1.5

0.8

0.0

 

S.D.

4.60

4.92

3.55

0.00

1-  0 MG/KG; 2-  100 MG/KG; 3-  300 MG/KG; 4-  1000 MG/KG

None significantly different from control group

TABLE 1.16 SUMMARY OF FETUSES AND LITTERS WITH VARIATIONS [ABSOLUTE NO.]

DAY 21

 

F E T U S E S

L I T T E R S

DOSE GROUP:

1

2

3

4

1

2

3

4

NUMBER EXAMINED EXTERNALLY

237

235

247

244

22

22

22

22

NUMBER WITH FINDINGS

0

0

0

0

0

0

0

0

NUMBER EXAMINED VISCERALLY

120

118

122

123

22

22

22

22

LIVER- SMALL SUPERNUMERARY LOBE(S)

0

1

4

0

0

1

3

0

NUMBER EXAMINED SKELETALLY

118

117

125

121

22

22

22

22

14TH RUDIMENTARY RIB(S)

58

70

72

64

19

21

20

21

14TH FULL RIB(S)

13

10

7

9

9

6

6

7

PELVIC GIRDLE- CAUDAL SHIFT

8

7

4

7

7

5

3

4

BENT RIB(S)

12

11

11

7

6

9

7

3

STERNEBRA(E) MALALIGNED

4

12

6

7

4

10

5

5

REDUCED OSSIFICATION OF THE SKULL

13

12

10

8

8

7

3

5

METACARPAL(S) AND/OR METATARSAL(S) UNOSSIFIED

2

4

1

2

2

2

1

2

VERTEBRAL CENTRA- REDUCED OSSIFICATION

4

2

0

1

3

2

0

1

7TH CERVICAL OSSIFICATION SITE(S)

7

6

7

7

6

5

4

4

SKULL- SUPERNUMERARY SITE

0

0

0

1

0

0

0

1

7TH CERVICAL FULL RIB(S)

0

0

1

0

0

0

1

0

STERNEBRA(E)- BRANCHED

0

2

0

0

0

2

0

0

STERNEBRA(E) #5 AND/OR #6 UNOSSIFIED

0

1

0

0

0

1

0

0

1-  0 MG/KG; 2-  100 MG/KG; 3-  300 MG/KG; 4-  1000 MG/KG

TABLE 1.17 SUMMARY OF LITTER PROPORTIONS OF VARIATIONS % PER LITTER

 

 

DAY 21

DOSE GROUP:

1

2

3

4

NUMBER OF LITTERS EXAMINED EXTERNALLY

22

22

22

22

NUMBER OF LITTERS WITH FINDINGS

0

0

0

0

1-  0 MG/KG; 2-  100 MG/KG; 3-  300 MG/KG; 4-  1000 MG/KG

None significantly different from control group

Conclusions:
In this GLP compliant OECD 414 study, time-mated female Wistar Han rats were treated with the test substance from day 6 to 20 post-coitum. Based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for the test substance was established as being at least 1000 mg/kg bw/day.
Executive summary:

In this GLP compliant OECD 414 study, the potential of the test substance to induce developmental toxicity after maternal exposure during the critical period of organogenesis was examined and maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female Wistar Han rats from Day 6 to 20 post-coitum, inclusive was characterized. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. The dose levels in this study were selected to be 0, 100, 300, 1000 mg/kg bw/day, based on the results of the dose range finder. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. The following parameters and endpoints were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents. In addition, the following parameters were determined for the F1-generation:the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, external, visceral and skeletal malformations and developmental variations.

Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously. No maternal and developmental toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups.

In conclusion, based on the results in this prenatal developmental toxicity study a maternal and developmental No Observed Adverse Effect Level (NOAEL) for the test substance of at least 1000 mg/kg bw/day was established.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
904 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
A guideline study (comparable to OECD 414) performed in compliance with GLP
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

Developmental toxicity test in rat, 2018 (Key):

In this GLP compliant OECD 414 study,the potential of the test substance to induce developmental toxicity after maternal exposure during the critical period of organogenesis was examined and maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female Wistar Han rats from Day 6 to 20 post-coitum, inclusive was characterized. In addition, theNo Observed Adverse Effect Levels(NOAELs) for maternal toxicity and developmental toxicity were evaluated.The dose levels in this study were selected to be 0, 100, 300, 1000 mg/kg bw/day, based on the results of the dose range finder.Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity.The following parameters and endpoints were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents.In addition, the following parameters were determined for the F1-generation:the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, external, visceral and skeletal malformations and developmental variations.

Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously.No maternal and developmental toxicity was observedin the 100, 300 and 1000 mg/kg bw/day groups.

In conclusion, based on the results in this prenatal developmental toxicity study a maternal and developmental No Observed Adverse Effect Level (NOAEL) for the test substance of at least 1000 mg/kg bw/day was established.

Developmental toxicity test in rabbit, 2021 (Key):

Justification of route and dose levels: The oral route was selected because this is a possible route of human exposure during manufacture, handling or use of the test substance. For the test substance, a pilot developmental toxicity study in rabbits was previously performed via oral gavage with corn oil as a vehicle (Test Facility Study No. 20150564). During the pilot for the developmental toxicity study, all rabbits (including controls) had to be sacrificed between post coitum Days 14 and 17 due to poor health conditions (low or absent food consumption, low bodyweights and reduced faeces production) and the study had to be terminated. It was concluded that pregnant rabbits housed and treated under the conditions of this study did not tolerate corn oil as vehicle when administered at a dose volume of 2 mL/kg. This was confirmed by a dedicated pilot study, in which the tolerability of corn oil (2 mL/kg) was tested after maternal exposure during the critical period of organogenesis when given orally by gavage in rabbits after optimisation of several husbandry conditions (Test Facility Study No. 20227635). Due to the physicochemical properties of this group of substances (hydrolytical instability), the use of aqueous vehicles was not an option. As no other suitable vehicle was available for administration in rabbits, it was decided in consultation with the Sponsor to perform the current pilot and main developmental toxicity study with the test substance in rabbits via dietary administration. The dose levels were selected based on the results of the Dose Range Finder (Test Facility Reference No. 20247987), and in an attempt to produce graded responses to the test substance.

Executive summary: The objectives of this study were to determine the potential the test substance to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterise maternal toxicity at the exposure levels tested when given via diet to time-mated female New Zealand White rabbits from Days 7 to 29 post-coitum, inclusive. This study was compliant to GLP conducted according to OECD TG 414. The dose levels in this study were selected to be 0, 3350, 10050, 33500 ppm (corresponding to an actual test substance intake of 0, 99, 291, 904 mg/kg body weight/day,respectively), based on the results of the Dose Range Finder study (No. 20247987). Chemical analyses of dietary preparations were conducted once during the study to assess accuracy and homogeneity. The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, test substance intake, macroscopic examination, uterine contents, corpora lutea, implantation sites, and pre- and post-implantation loss. The following parameters were determined for the F1-generation: the number of live and dead foetuses, foetal body weights, sex ratio, external, visceral and skeletal malformations and developmental variations. Analysis of diet preparations confirmed that the achieved dietary concentrations of the test substance were in agreement with the target concentrations and that the test substance was homogeneously distributed in the diet.

Results showed at 33500 ppm, two females were sacrificed before scheduled necropsy on post-coitum Days 19 or 20, as these females were observed with organic material or a foetus on the manure tray, indicating early abortion. Both females showed a reduced food consumption and reduced faeces production only on two days prior to sacrifice. As two cases of early abortions were noted in the high-dose group of this study and these early abortions occur very rarely, it was considered possibly related to treatment with the test substance. A lower mean food consumption was observed in females at 33500 ppm between Days 7-23, which appeared to recover to control levels from Day 21 post-coitum onwards. This was accompanied with a slightly reduced body weight gain in females treated at 33500 ppm during the entire treatment period. Additionally, body weight gain corrected for gravid uterus was lower compared to control (-206.2 gram versus -114.5 gram in controls). As food consumption of treated females recovered to levels similar as concurrent controls by the end of the treatment period and the changes in body weight or (corrected) body weight gain were minor, these changes were considered non-adverse. No test substance-related gross findings were observed at necropsy. The number of corpora lutea, implantation sites, viable or dead foetuses, early or late resorptions, pre- and post-implantation loss were considered not affected by treatment with the test substance up to 33500 ppm. No test substance-related changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, foetal body weights, external, visceral and skeletal malformations and developmental variations).

In conclusion, based on the results in this prenatal developmental toxicity study, the NOAEL for maternal toxicity and developmental toxicity is set at 33500 ppm (corresponding to an actual test substance intake of 904 mg/kg bw/day)

Justification for classification or non-classification

Based on the available information the substance is not classified for reproductive toxicity in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.

Additional information