Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Before the start of the EOGRTS study according to OECD 443, two dose range finding studies were performed to determine suitable dose levels.

The aim of these studies was to obtain preliminary information on possible effects of the test item IPDA on reproduction and/or development according to OECD guideline 421. The test item IPDA was administered orally to rats via the drinking water at dose levels of 20, 60or 160/120 mg IPDA/kg b.w./day (LPT 2019).

Unfortunately, it was not possible to determine adequate dose levels for the OECD 443 study. The animals refused to consummate the drinking water because of the bad smell of the test substance (LPT 2018). As a consequence, the OECD 421 study had to be performed with another route of exposure. It was chosen to perform the OECD 422 study with rats and gavage as administration route. The study started in July 2019 and a first draft report is expected soon (LPT 2020).

The requested OECD 443 with rats and dose groups of 0, 25, 80, 240 mg/kg study started in November 2019 and first draft report is expected in December 2020.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
August 2018 - November 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
WI IGS/ Crl:WI
Details on species / strain selection:
For this study Wistar rats bred by Charles River Laboratories Germany GmbH were used. The healthy nulliparous adult animals were randomised and assigned to the treatment groups and cages. The body weight range did not exceed 20% of the mean weight for each sex at the time of selection. The rat is a commonly used rodent species for such studies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld
- Strain: Rat / Wistar WI IGS / Crl:WI
- Sex. male and female
- Age: Young adult rats, approx 10 weeks old (71 days) at the start of the treatment
- Weight at study initiation: Males: 378.9 g - 454.0 g, females: 207.5 g - 258.2 g
- Housing: With the exception of the mating period, the males and females (F0-Generation) were kept singly in MAKROLON cages
- Diet (e.g. ad libitum): ssniff® R/Z V1324, ssniff Spezialdiäten GmbH, 59494 Soest, Germany
- Water (e.g. ad libitum): tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 55 +/- 15 %
- Air changes (per hr): was at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours daily
Route of administration:
oral: drinking water
Vehicle:
water
Remarks:
tap water
Details on exposure:
Route of administration was oral, via the drinking water. The test item-drinking water mixture was prepared once weekly by mixing the respective amounts of the test item with the drinking water. The test item-concentration in the drinking water was adjusted to the relative drinking water consumption of the animals of each group and sex at the beginning of the respective test week.
Details on mating procedure:
Sexually mature male and female rats were paired monogamously. One male and 1 female animal were placed together in one cage during the mating period. The female was placed with the same male until pregnancy had occurred or 2 weeks had elapsed. Each morning the females were examined for the presence of sperm or a vaginal plug. The day of conception (day 0 of gestation or GD0) was considered to be the day on which sperm was found.
With the exception of one female that showed a mating period of 16 test days all other animals were mated within this 14 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration and stability of the test item-drinking water mixtures were determined for the first preparation at the start of treatment, at the end of the study after weaning of the first litters and at dose change (group 4 only) for the males and females.
For the analysis of the test item-drinking water mixtures, two aliquots of approximately 5 mL each were taken and stored at ≤-20°C ± 10% until analysis at LPT.

Analysis and reporting of the test item-drinking water mixtures were perhormed by LPT.
With one exception, the concentration of the test item in the test item-drinking water mixtures was between 91.5% and 102.4% of the nominal concentration (see text table 7-25 below). This demonstrated that the test item-drinking water mixtures were correctly prepared and stable for at least 7 days under laboratory conditions.

Duration of treatment / exposure:
The study animals were treated during the following periods:
Males were treated 2 weeks prior to mating (from test day 15 until test day 29), during the mating period (from test day 30 until test day 33 at maximum) and during the post-mating period until test day 55.
The females were treated 2 weeks prior to mating (from test day 15 until test day 29), during the mating period (from test day 30 until test day 33 at maximum) and during the lactation period until test day 65 to 69.

Frequency of treatment:
continuously via drinking water
Details on study schedule:
- Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study
- Prior to the start of treatment and once weekly, all animals were observed for signs of functional/behavioural toxicity. Oestrus cyclicity is evaluated followed by daily monitoring of vaginal smears from beginning of treatmrnt until evidence of mating
- On Day 15, all animals were paired on a 1 male:1 female basis within each dose group for a maximum of fourteen days.

- Pregnant females were allowed to give birth and maintain their offspring until Day 4 post partum. Evaluation of each litter size, litter weight, mean pup weight, clinical observations and landmark developmental signs were also performed during this period.
- At the end of the dosing period on day of necropsy 5 males and 5 femals were randomly selected for laboratory examinations (clinical chemistry and haematology)
- Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically on Day 43.
- At Day 5 post partum, all females and offspring were killed and examined macroscopically.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
control group
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Remarks:
low dose group
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Remarks:
intermediate dose group
Dose / conc.:
160 mg/kg bw/day (actual dose received)
Remarks:
high dose group, reduction on test day 31 to 120 mg/kg bw d
No. of animals per sex per dose:
10 males and 10 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
The dose levels were selected in agreement with the Sponsor based on the results of a 90-day repeated dose toxicity study according to OECD guideline 408.
Nominal dose levels of 20, 60 and 160 mg/kg were selected for this OECD 421 study, since the treatment period was up to 12 weeks.
Positive control:
no
Parental animals: Observations and examinations:
Clinical signs
Throughout the test period, each animal (parental animals and pups) was observed for clinical signs at least once daily. Behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity were recorded. Mortality was checked twice daily. Animals which died prematurely were necropsied as soon as possible after exitus. However, no premature death was noted.

Body weight
The adult animals were weighed on each day of dosing for dose adjustment and at sacrifice; the individual body weights were recorded.
The report included weekly values for the male animals (starting on test day 15) and the body weight on the day of sacrifice.
Days on which body weight is reported:
Pre-mating period: Test days 15, 22,29
Gestation period: Gestation days 0, 7, 14, 20
Lactation period: Lactation days 1, 4, 13

Food and drinking water consumption
Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group upon completion of a treatment week (pre-mating and gestation) or treatment period (lactation). From these data the relative food consumption (in g/kg b.w./day) was determined.

Days on which food consumption is reported:

Study period Males Females
Pre-mating period TD15 - TD 22 TD15 - TD 22
TD 22 - TD 29 TD 22 - TD 29
Mating period None None
Gestation period Not applicable GD7, GD14 and GD21
Lactation period Not applicable LD1, LD8 and LD12
Drinking water consumption was monitored daily by visual appraisal throughout the study.


Reproductive parameters

Pre-coital time and gestation length
-number of pregnant females
-duration of pre-coital time
-gestation length
(The duration of gestation was calculated from gestation day 0 (day of positive sperm detection) until (but not including) lactation day 1 (lactation day 1: morning after littering when no signs of littering were noted anymore)).

Implantation sites
-distribution in the uterine horns (left or right uterus horn)
-total number per dam
-mean number per dam (mean value of the individual values per dam in each group)
-total number per group

Laboratory examinations
Blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight on the day of necropsy.
All relevant hematological and clinical bichemical parameter were determined.

T4 Determination
Blood samples were taken under isoflurane anaesthesia from animals fasted overnight always at the same time of day (in the morning between 7.30 a.m. and 9.30 a.m. for the adult animals) as scheduled below.
Blood withdrawal was performed by randomization of the parental male and female animals. The male animals of all test groups were completely randomized in a one- step process, the female animals in different staggers according to their litter day.
Oestrous cyclicity (parental animals):
During the 14-day pre-exposure period (TD 1 to TD 14), the estrus cycle of the female animals was monitored to yield study groups of at least 10 animals each with a normal estrus cycle. Animals that failed to exhibit typical 4 to 5 day cycles were excluded from the randomization process on test day 14 that was performed to allocate the female animals to the test groups of the main study.
Sperm parameters (parental animals):
Detailed histopathologic examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis during histopathology.
Litter observations:
As soon as possible after delivery, each litter was examined to establish the number and sex of pups, stillbirths, live births, runts (pups were considered as runts if their weight was less than 70% of the mean litter weight) and the presence of gross abnormalities.
Number of pups (for both sexes and for male and female pups)
- at birth (alive and dead)
- after 4 and 13 days of life
The above mentioned number of pups is given as total per group, total per dam and mean number per dam (mean value of the individual values per dam in each group)

Number of stillbirths
- total per group
- total number per dam

Number of pups with malformations
- total per group
- per dam

The following examinations/observations were done for the offspring.
Counting
Live pups were counted, sexed and weighed on post-natal days 1, 4 and 13.

Ano-genital distance
On post-natal day 4 before litter adjustment the ano-genital distance (AGD) of all pups was determined using a scale. The AGD was normalized to the cube root of body weight.

Litter adjustment on PND 4
After counting on PND 4, the litters were adjusted to 10 pups per litter by eliminating (culling) surplus pups using a randomization scheme generated by Provantis®.

Blood sampling for thyroid hormone (T4) determination
On PND 4 and on PND 13 blood samples for T4 hormone level determination were taken from 2 selected pups per litter, if possible from one male and one female pup. On PND 4 the culled surplus pups were used for blood collection.

Male nipples counting
Nipples were counted in all male pups on PND 13 (shortly before scheduled sacrifice).

T4 Determination
Determination of the pups used for blood withdrawal:
On lactation day 4 (PND4) and lactation day 13 (PND13) the litter sequence of pup blood withdrawal was determined by randomization of the dams. The collection of the pups for blood withdrawal from the individual litters occurred in an ascending order (the male and female pups per dam with the lowest number were used, if possible).

Postmortem examinations (parental animals):
Gross necropsy
Vaginal smears were examined on the day of necropsy to determine the stage of the oestrus cycle and allow correlation with the histopathology of the female reproductive organs.
The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically (gross necropsy) at the following times:
Males On test day 55
Dams On lactation days 14 - 16

At the time of sacrifice or premature death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
During necropsy the number of implantation sites was recorded in the female animals.
Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI.
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition to the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and the caecum were incised and examined. The lungs were removed and all pleural surfaces were examined under suitable illumination.
The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

Organs weighed
The organs which were weighed before fixation are listed below. The organ weighing was performed for all adult male and female animals.

Epididymis, Thyroid including parathyroids, Kidney, as a whole: prostate, seminal vesicles, Liver with coagulating glands, Ovary, Uterus including cervix, Testicle
Thyroid weight was determined after fixation; paired organs were weighed individually and identified as left or right.

Histopathology
Histopathological examination was performed on the organs of the animals of the control group and the high dose group (groups 1 and 4).
The organs (labelled with study number, species, animal number, type of sample) were dispatched to AnaPath for histopathological processing.
The histotechnique was performed by AnaPath Services according to all relevant AnaPath SOPs.

Prepared and evaluated sections from the adult animals of group 1 and 4.
Epididymis
Ovary
Testicle
Kidney
Liver
Thyroid

Detailed histopathologic examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology or interstitial testicular structure) of all males of groups 1 and 4 following H-E and PAS staining.

The other preserved organs (group 2 and 3 animals) will be examined when necessary, i.e. when changes are seen in the highest dose group.


Postmortem examinations (offspring):
Histopathological examination was performed on the organs of the pups of the control group and the high dose group (groups 1 and 4).
Prepared and evaluated sections from the pups of group 1 and 4 (1 male and 1 female pup):
Kidney
Liver
Thyroid
Statistics:
The statistical evaluation of the parametrical values was done by Provantis (Provantis® integrated preclinical software, version 9.4.0.1, Instem LSS Ltd) using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILK’s test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).
Significantly different data are indicated in the summary tables of the result sections of the report.

Non-parametrical data

The statistical evaluation of non-parametrical values was done by comparison of the group values using the FISHER or the Chi2 test with the StatXact 4.0.1 software:
FISHER test:
- Fertility index and gestation index of the parental females 'Reproductive Outcomes and Indices per Group - Females'.

Chi2 test:
- Birth and live birth index, post-impantation loss and viability indices of the pups

However, for the birth and live birth index, the post-implantation loss as well as for the viability indices an ANOVA / DUNNETT test was additionally performed using the individual indices per dam. The results are given in the summary tables of the respective sections.
Reproductive indices:
Gestation Index [%] =Number of dams with live pups / Number of pregnant rats x 100


Female Fertility Index [%] = Number of pregnant females / Number of females used for pairing x 100


For each litter/dam and group the following indices were determined:

Birth Index [%]= Total number of pups born (alive + dead) / Number of implantation scars x 100

Live Birth Index [%] = Number of pups alive on day 0/1 of lactation / Total number of pups (alive + dead) x 100

Viability Index [%] pre-cull = Number of pups alive on day 4 (pre-cull) / Number of pups alive on day 0/1 x 100

Viability Index [%] post-cull = Number of pups alive on day 13 / Number of pups alive on day 4 (post-cull) x 100

Post-implantation loss [%] = Implantations - living fetuses / Implantations x 100





Offspring viability indices:
Birth and live birth index, post-impantation loss and viability indices of the pups
Clinical signs:
no effects observed
Description (incidence and severity):
No changes in behaviour, the external appearance or the appearance of the faeces were noted for the male and female animals of the control group and the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day).
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No animal of the control group and in the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day) died prematurely.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Males
No test item-related changes in body weight and body weight gain were noted for the male rats between the control group and the low and the intermediate dose group (20 or 60 mg test item/kg b.w./day).
A marked reduction in body weight (negative body weight gain) was noted at the high dose level (160 mg test item/kg b.w./day) during the first 2 weeks of treatment (test weeks 3 and 4; test days 15 to 29). This led to a body weight value on test day 29 that was 18.9% (p ≤ 0.01) below the value of the control group.
This reduction in body weight was caused by a severely reduced drinking water consumption as the animals refused to drink the test item-drinking water mixture, most probably due to a bad taste of the test item. After the decrease of the test item concentration in the drinking water on test day 31 to obtain a dose level of 120 mg test item/kg b.w./day, the drinking water consumption and the body weight of the high dosed animals increased. However, the body weight of the high dosed animals remained below the body weight of the the animals of the control group and the low and the intermediate dose group. On test day 54 (one day before sacrifice), the body weight of the high dosed animals was still 9.5% below the value of the control group (p ≤ 0.01).
However, the reduced body weight that was noted for the high dosed animals was due to the reduced drinking water consumption caused by the bad taste of the test item and not caused by a toxic effect of the test item. Hence, the reductions in body weight and body weight gain (see below on the following page) were not considered to be of toxicological relevance.
The percentage of body weight gain was decreased in the high dose group in comparison to the control group and the low and the intermediate dose group

Females
No test item-related differences in body weight and body weight gain were noted between the female rats of the control group and the female rats of the low and the intermediate dose group (20 or 60 mg test item/kg b.w./day) during the pre-mating, the gestation and the lactation period.
At the high dose level (160/120 mg test item/kg b.w./day) a slightly reduced body weight in comparison to the control group was noted during the gestation and the lactation period (between 4.0% and 8.8% below the value of the control group; statistically significant at p ≤ 0.05 on gestation day 0 and lactation day 13).
As for the male animals, the slight reduction in body weight is considered to be a secondary effect of the reduced drinking water consumption of the high dosed females (caused by the bad taste of the test item) and not considered to be of toxicological relevance.
The slight reductions in body weight that were noted for the high dosed animals during the gestation and the lactation period had no effect on the body weight gain of the high dosed females during these periods

The mean body weights of the female rats during the pre-mating period, the gestation and the lactation period are shown in Figures 2 to 4 on the following pages.

BODY WEIGHT AT AUTOPSY
Males
No test item-related differences were noted on the body weight at autopsy between the control group and the treatment groups (20 or 60 mg test item/kg b.w./day).
A slight but statistically not significant reduction was noted at the high dose level (160/120 mg test item/kg b.w./day) (5.9% below the value of the control group). This was in accordance with the last difference in live body weight on test day 55 (6.1% below the value of the control group) after the animals fasted overnight and was considered not to be of toxicological relevance.
Females
No test item-related differences were noted on the body weight at autopsy between the control group and the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day).

Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Males: Pre-mating period
During the pre-mating period (test days 15 to 28) no test item-related difference in food consumption was noted between the control group and the low dose group (20 mg test item/kg b.w./day). The statistically significant (p ≤ 0.05) reduction that was noted during test week 4 (test days 22 -29) was considered to be spontaneous, as it was only very small.
The statistically significant reductions that were noted at the intermediate and the high dose group (60 or 160/120 mg test item/kg b.w./day) were considered as a secondary effect on the reduced drinking water consumption of the animals and not considered to be of toxicological relevance.
No food intake of female animals was recorded during the mating period as both sexes were housed together.

Females: Pre-mating, gestation and lactation period
No differences of toxicological relevance were noted between the female rats of the control group and the female rats of the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day) during the pre-mating, the gestation and the lactation period.
However, slight but statistically significant reductions in food consumption were noted in test week 4 for the animals of the intermediate and the high dose group. These reductions were considered as a secondary effect of the reduced drinking water consumption. Hence, they were not of toxicological relevance.

Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
MALES
There was a clear dose dependent tendency of the male rats to refuse the drinking of the test item-drinking water mixtures. The refusal of the drinking water was caused by the properties of the added test item (e.g. bad taste and/or smell) and is not considered as an adverse toxicological effect on the animals of the treatment groups (20, 60 or 160/120 mg/kg b.w./day).
The intake of the test-item drinking water mixture during the course of the study is described in detail below:
At the low dose level (20 mg/kg b.w./day) a statistically significantly reduced drinking water consumption was only noted in test week 7 (13.0% below the value of the control group).
At the intermediate dose level (60 mg/kg b.w./day) similarly reduced levels of drinking water were noted in nearly all test weeks (between 16.5% and 25.6% below the value of the control group; p ≤ 0.05 or 0.01).
At the high dose level (160/120 mg/kg b.w./day) marked and statistically significant (p ≤ 0.01) reductions in drinking water consumption were noted for all test weeks. The maximum reductions in drinking water consumption were noted in test week 4 (test days 22 - 29) with a test item concentration in the drinking water of 3486 mg/L and the beginning of test week 5 (from test days 29 - 31), equivalent to a dose of 160 mg/kg b.w./day.
Test week 5 started with the beginning of the mating period (test day 29). During the mating period the male and female pairs of the high dose group received a test item concentration of 2778 mg/L in the drinking water, which was an average value between the calculated test item concentrations for the male (4324 mg/L) and the female (2362 mg/L) animals alone, to achieve a nominal dose level of 160 mg/kg b.w./day for both sexes.
Between test days 29 - 31 most of the animals were in the mating period and received the averaged test item-concentration of 2778 mg/L to achieve a nominal dose level of 160 mg(kg b.w./day. Though the test item concentration was lower as in test week 4, the drinking water consumption of the male animals was still markedly reduced between test day 29 -31.
Due to this markedly reduced drinking water consumption that caused a marked reduction in body weight the high dose level was reduced from 160 mg/kg b.w./day to 120 mg/kg b.w./day on test day 31.
Due to this dose reduction, the averaged test item concentration during the mating period was reduced from 2778 mg/mL to 2083 mg/mL for both sexes. The test item concentration in the drinking water for the male animals who were sitting alone in their cage again after the mating period was reduced from 4324 mg/mL to 3243 mg/mL. The reduction of the test item concentration in the drinking water led to an increase in the drinking water consumption for the male rats of the high dose group from test day 31 onwards. However, due to the increased drinking water consumption the concentrations used turned out to be too high and led to a too high test item intake in the second part of test week 5. Hence the concentration of the test item was further reduced for the following test weeks. However, from test day 31 onwards, the drinking water consumption that were noted for the male animals of the high dose group remained on a nearly constant level. This level was still below the control group but above the values that were measured for the male animals of the high dose group before the reduction of the dose level.
Though the drinking water consumption of the high dosed animals remained below the control group and the low and the intermediate dose group, the drinking water consumption was now sufficient to support a positive body weight gain for the male rats of the high dose group during the post-mating period.

FEMALES
As for the male rats, a dose-dependent tendency of the rats to refuse the drinking of the test item-drinking water mixtures was also noted for the female animals.
The reductions in drinking water consumption were in the range of those noted for the male animals, with the exception of test weeks 3 and 4, where the reduction in drinking water consumption was much more pronounced for the male animals of the high dose group as for the female animals of the high dose group.

RELATIVE DRINKING WATER CONSUMPTION
Males and females
The reductions in the relative drinking water consumption in comparison to the control group for the male and female rats of the treatment groups were similar to the reductions that were noted for the total drinking water consumption for the male and female animals.

For detailed information on test item uptake via drinking water see section `other effects`
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related differences were noted between the male and female animals of the control group and the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day) for the examined haematological parameters. Decreased Neutrophilic granulocytes and decreased large unstained cells were observed in the high dose group males, however, these findings are considered to be spontanious and not treatment related.

Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related differences were noted between the male and female animals of the control group and the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day) for the examined biochemical parameters. aP was increased on test day 55 in the high dosed males. However, these finding is considered to be spontanious and not treatment related.

T4 hormone determination
Males
No test item-related changes were noted for the T4 levels of the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day).
A slight but statistically significant reduction was noted at the high dose level. However, this slight reduction was considered to be spontaneous.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The histopathological evaluation of selected organs from the animals of the control and the high dose group (160/120 mg test item/kg b.w./day) was performed and reported by AnaPath GmbH, Switzerland.
No indication of toxicity was noted for the examined organs (epididymides, testes, ovaries, kidneys, liver and the thyroid gland) of the male and female animals of the high dose group (160/120 mg test item/kg b.w./day).
The sperm staging revealed no test item-related alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
TEST ITEM INTAKE
The concentration of the test item in the drinking water was adjusted weekly to the relative drinking water consumption of the male and female animals. The real test item intake of the animals was calculated using the total amount of drinking water and the body weight of the animals and designated as the actual dose level (mg test item/kg b.w./day).

Males and females
In most cases the calculated actual dose level was nearly in the range of the nominal dose level for the male and female animals of all dose group (20, 60 or 160/120 test item mg/kg b.w./day).
However, the following noticeable discrepancies were noted for the male and the female animals:
Males:
At the high dose level (160 test item mg/kg b.w./day), the actual dose levels were clearly below the nominal dose level of 160 mg/kg in test weeks 3 and 4 and the beginning of test week 5 (test days 29 - 31). This was due to the markedly reduced consumption of the test-item drinking water mixtures during this period.
After the dose level reduction to 120 mg test item/kg b.w./day the actual dose level was in the range of the reduced nominal dose level of 120 mg/kg in test weeks 6 to 8. Only between test days 31 and 36 the actual dose level was clearly above the nominal dose level as the test item-concentration in the drinking water turned out to be too high for the period between test days 31 and 36, due to the increased drinking water consumption after the dose level reduction.

Females:
Increased actual dose levels were noted for all dose groups (20, 60 or 160/120 mg test item/kg b.w./day) in test weeks 8 and 9 when most animals where in the lactation period. This was due to the increased absolute and relative drinking water consumption that was noted for all dose groups during the lactation period in comparison to the pre-mating/mating and the gestation period.
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
No differences were noted in the mean number of oestrus cycles per dam during the pre-mating and mating period between the female animals of the control group and the female animals of the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day).
However, one animal each of the control group (no. 11), the low dose group (no. 31) and the high dose group (no. 71) was noted with an abnormal cyclus in the form of an elongated diestrus period (between 6 and 12 consecutive days in the diestrus stage). This observation was considered to be spontaneous, moreover as all 3 animals became pregnant.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
FERTILITY
No test item-related influence on the fertility index of the female rats was noted for any of the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day).
All female rats that were used for pairing were successfully mated (confirmed by sperm detection). However, one female animal of the control group and one female animal of the high dose group did not become pregnant, leading to a fertility index of 90% for the female animals of the control group and the high dose group. The occurrence of one non-pregnant female at the high dose level was considered to be spontaneous, as one non-pregnant female was also noted at the control group.

GESTATION INDEX
No test item-related influence on the gestation index was noted for the female rats treated with 20, 60 or 160/120 mg test item/kg b.w./day. All pregnant animals of the control group and the treatment groups delivered live pups, leading to gestation indices of 100% for all groups.

PRE COITAL TIME
No test item-related differences were noted in the length of the pre-coital time between the control group and the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day). With the exception of female no. 11 of the control group with a pre-coital time of 16 days, the pre-coital time of all other females was between 1 and 4 days. However, live pups were also noted for animal no. 11.

GESTATION LENGTH
No test item-related differences were noted for the length of the gestation period between the rats of the control group and the rats of the treatment groups (20 or 60 or 160/120 mg test item/kg b.w./day).

Birth indices and post-implantation loss
No test item-related differences were noted for the mean number of implantation sites, the mean number of pups born (alive and dead) and the mean number of live born pups between the control group and the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day).
Correlating, the reproductive indices as the birth index, the live birth index and the percentage of post implantation loss revealed no test item-related differences between the control group and the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day).
No premature death and no changes in behaviour, the external appearance and the appearance of the faeces were noted.
Also the laboratory examinations and the examinations at necropsy (examination of haematological and biochemical parameters, determination of the T4 level of the male animals, determination of organ weights, the macroscopical and histopathological examination) revealed no test item-related changes.
However, most probably due to the properties of the test item in the drinking water a dose dependent refusal of intake of the test item-drinking water mixtures was noted for the male and female animals. This was most pronounced for the male animals of the high dose group (160/120 mg IPDA/kg b.w./day) during the first and the second treatment week and caused a reduction in body weight for the male animals. The body weight of the high dosed males recovered as the consumption of drinking water increased after the reduction of the test item concentration in the drinking water (dose level reduction from 160 to 120 mg IPDA/kg b.w./day).
The concentration of the test item in the test item-drinking water mixture was adjusted weekly to the relative drinking water consumption of the male and female animals. The actual dose levels of test item intake were calculated for the male and female animals using the total drinking water consumption and the body weight of the animals:
In most cases the actual dose levels were nearly in the range of the nominal dose levels for the male and female animals of all dose groups.
However, for the male animals of the high dose group (160/120 mg test item/kg b.w./day), the marked reduction in drinking water consumption before the reduction of the dose level revealed a decreased intake of test item via the drinking water. Hence, the calculated actual dose level was below the nominal dose level during the first and second treatment week. After the dose level reduction the actual test item-intake was in the range of the nominal dose level.
For the female animals increased actual dose levels were noted during the lactation period for all dose groups (20, 60 or 160/120 mg IPDA/kg b.w./day). This was due to increased levels of absolute and relative drinking water consumption during the lactation period in comparison to the pre-mating and gestation period that were noted for all dose groups. No influence was noted on the estrus cycles, the fertility, the gestation index, the pre-coital time and the gestation length.
Key result
Dose descriptor:
NOAEL
Effect level:
> 160 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects were observed up to the highest tested dose
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Number of live pups
No test item-related difference was noted between the number of live pups per dam of the control group and the number of pups per dam of the low and the intermediate dose groups (20 or 60 mg test item/kg b.w./day).
At the high dose level (160/120 mg test item/kg b.w./day) a reduced number of pups per dam (22.6% below the value of the control group for male and female pups together; p ≤ 0.05) was noted on lactation day 13. This was due to the increased number of prematurely deceased pups at the high dose level during the post-cull period. However, this was not considered as a toxic effect on pup development.

Viability index: Pre-cull period
No difference between the control group and the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day).

Viability index: Post-cull period
No test item-related difference was noted for the post cull viability index between the control group and the low and the intermediate dose group (20 or 60 mg test item/kg b.w./day).
A statistically significantly decreased viability index was noted at the high dose level (160/120 mg test item/kg b.w./day) for the post-cull period. In detail, 24 pups died prematurely between lactation day 5 and 13 in comparison to 6 prematurely deceased pups in the control group.
The increased number of prematurely deceased pups at the high dose level was considered to be a secondary effect of the reduced drinking water consumption of the dams and not as a toxic effect of the test item on the development of the pups. In detail, the decreased drinking water consumption may have resulted in a decreased milk production of the dams, so that weaker pups failed to receive an adequate amount of milk. However, as 21 of 24 of the prematurely deceased pups during the post-cull period were cannibalized, an examination of the milk status was not possible.
The surviving pups seem to have received an adequate amount of milk as no difference in body weight was noted between the surviving pups of the high dose group and the pups of the control group on lactation day 13.



Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
BODY WEIGHT
+No test item-related difference was noted between the mean body weight of the pups from the dams of the control group and the mean body weight of the pups from the dams of the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day) on lactation days 1, 4 and 13.
No runt was noted in the control group and in the treatment groups.

LITTER WEIGHT
No test item-related difference was noted between the litter weight of the dams of the control group and the litter weight of the dams of the low and the intermediate dose group (20 or 60 mg test item/kg b.w./day).
At the high dose level (160/120 mg test item/kg b.w./day) a reduced litter weight (22.3% below the value of the control group for male and female pups together; statistically not significant) was noted on lactation day 13. This was due to the increased number of prematurely deceased pups that was noted at the high dose level for the post-cull period, leading to a reduced number of live pups on lactation day 13. However, as the premature death of these pups was not considered as a toxic effect on pup development (secondary effect of the reduced consumption of drinking water and a possibly reduced milk production of the dams), the reduced litter weight that was noted at the high dose level was not considered to be of toxicological relevance.

Food consumption and compound intake (if feeding study):
not examined
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:
no effects observed
Description (incidence and severity):
No test item-related changes were noted for the T4 levels of the male and female pups of the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day).
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
No test item-related influence was noted on the absolute and the relative ano-genital distance of the male and the female pups of all treatment groups (20, 60 or 160/120 mg test item/kg b.w./day).
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No test item-related difference in the number of nipples was noted between the male pups of the control group and in the male pups of the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day).
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
External examination of the pups
No gross abnormalities (e.g. malformations or variations) were noted during the macroscopic external examination of the control pups and the pups from the dams treated with 20, 60 or 160/120 mg test item/kg b.w./day after terminal sacrifice on lactation day 13 or for the pups that died during the lactation period.


Histopathological findings:
no effects observed
Description (incidence and severity):
The histopathological examination of selected organs of pups from the dams of the control and the high dose group (160/120 mg test item/kg b.w./day) was performed and reported by AnaPath GmbH, Switzerland.
No indications of toxicity were noted for the examined organs (kidneys, liver and the thyroid gland) from 2 selected pups per litter from the dams of the control and the high dose group (160/120 mg test item/kg b.w./day).
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
No influence on the pre-natal development was noted (number of implantation sites, birth and live birth index, percentage of post-implantation loss).
During the post-natal development no influence was noted on body weight, the ano-genial distance, the number of nipples of the male pups and the T4 level.
No abnormalities (malformations or variations) were noted during the external macroscopic examination of the pups at necropsy.
A reduced viability index was noted during the post-cull period for the pups of the high dose group (160/120 mg IPDA/kg b.w./day). This was considered as a secondary effect of the reduced consumption of drinking water of the high dosed females in comparison to the females of the control and the low and the intermediate dose group. This lower drinking water consumption led to a reduced milk production and hence, an inadequate feeding of some of the weaker pups. Therefore, the reduced viability index was not considered to be of toxicological relevance.
Though there were no toxicological relevant findings, the maximum dose level for possible further rat studies was considered to be 120 mg IPDA/kg b.w./day for the administration via the drinking water. At this dose level and also at the lower tested dose levels of 20 and 60 mg IPDA/kg b.w./day the test item in the drinking water already evoked a reduced drinking water consumption, but without secondary effects in the form of body weight loss or a possibly reduced milk production of the dams.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 160 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects were seen up to the highest tested dose with regard to pre- and postnatal development
Key result
Reproductive effects observed:
no
Treatment related:
no

Text table 1: Groups and dose levels

Group

IPDA dose (nominal)

[mg/kg b.w./day]

Number and sex of animals

 

Animal number

1

0

(vehicle control)

10

10

m

f

1 - 10

11 - 20

11 - 15

26 - 30

2

20

(low dose)

10

10

m

f

21 - 30

31 - 40

None

3

60

(low dose)

10

10

m

f

41 - 50

51 - 60

None

4

160 / 120 #

(low dose)

10

10

m

f

61 - 70

71 - 80

None

m = male

f = female

# Dose reduction as of September 14, 2018 (TD 31) for all animals of group 4.

Text Table 2 :Reproductive outcome of the female animals per group.

Test item

Group 1

Control

Group 2

20 mg/kg

Group 3

60 mg/kg

Group 4

160/120 mg/kg

Females started dosing #1

10

10

10

10

Females used for pairing

10

10

10

10

Females mated

10

10

10

10

Females pregnant #2

9

10

10

9

Females not pregnant

1

0

0

1

Females with litters #3

9

10

10

9

Females with live born pups

9

10

10

9

#1

Number of animals at start of the pre-mating and mating period

#2

Number of animals at start of the gestation period

#3

Number of animals at start of the lactation period

Text table 3: Fertility indices of the female animals per group.

Group / Dose level

Fertility index

%

Pregnant females / females used for pairing

Group 1

(Control)

90

9 / 10

Group 2

(20 mg/kg)

100

10 / 10

Group 3

(60 mg/kg)

100

10 / 10

Group 4

(160/120 mg/kg)

90

9 / 10

*/**:

p ≤ 0.05 / p ≤ 0.01, Fisher test

Text table 4: Gestation index per group.

Group / Dose level

Gestation index

%

Dams with live pups / pregnant rats

Group 1

(Control)

100

9 / 9

Group 2

(20 mg/kg)

100

10 / 10

Group 3

(60 mg/kg)

100

10 / 10

Group 4

(160/120 mg/kg)

100

9 / 9

*/**:

p ≤ 0.05 / p ≤ 0.01, Fisher test

Text table 5: Overview of the reproductive parameters.

 

Reproductive data

Group 1

(control)

Group 2

Group 3

Group 4

Parametrical values

(mean number per dam, #1)

Implantation sites

16.9

15.6

17.0

14.9

Pups

(born alive and dead)

14.7

14.7

15.7

13.7

Pups born alive

14.4

14.6

15.5

13.7

Indices [%]

Birth Index

mean per dam

group

87.45

86.84

93.09

94.23*

93.09

92.35

91.86

91.79

Live birth Index

mean per dam

group

98.29

98.48

99.29

99.32

98.75

98.73

100.00

100.00

Post-implantation loss

mean per dam

group

13.66

14.47

7.50

6.41*

8.17

8.82

8.14

8.21

Resorptions and stillbirths

Difference between

no. of implantation sites and no. of pups born alive

mean per dam

 

 

sum per group

2.4

 

 

22

1.0

 

 

10

1.5

 

 

15

1.2

 

 

11

Number of stillbirths

2

1

2

0

#1:

Statistical calculation was performed by ANOVA / DUNNETT

(*/**: p ≤ 0.05 / p ≤ 0.01).

Text table 6:Viability indices and prematurely deceased pups during the post-cull period.

Post-cull period

Parameter #

Group 1

Group 2

Group 3

Group 4

Prematurely deceased pups between lactation days 5 and 13

/

Number of pups alive on lactation day 4 after culling

6 / 90

11 / 96

7 / 100

24 / 89

Number of dams with dead pups between lactation days 5 and 13

/

Number of dams with live pups

1 / 9

6 / 10

2 / 10

7 / 9

Between lactation days 5 and 13:

Dams with 1 dead pup

Dams with 2 dead pup

Dams with ≥ 3 dead pup

 

-

-

1

 

3

1

2

 

-

1

1

 

1

3

3

Viability index (group values)

93.33

88.54

93.00

73.03**

 

(*/**: p ≤ 0.05 / p ≤ 0.01) Chi2test (group values)

Conclusions:
The aim of the study was to obtain preliminary information on possible effects of the test item IPDA on reproduction and/or development according to OECD guideline 421. The test item IPDA was administered orally to rats via the drinking water at dose levels of 20, 60 or 160/120 mg IPDA/kg b.w./day. Unfortunately it was not possible to determine adequate dose levels for the OECD 443 study. The animals refused to consumate the drinking water because of the bad smell of the test substance. As a consequence the OECD 421 study had to be performed with another route of exposure. It was chosen to perform the OECD 422 study with rats and gavage as administration route.
Executive summary:

The aim of the study was to obtain preliminary information on possible effects of the test item IPDA on reproduction and/or development according to OECD guideline 421. The test item IPDA was administered orally to rats via the drinking water at dose levels of 20, 60 or 160/120 mg IPDA/kg b.w./day.

 

Parental male and female animals

No premature death and no changes in behaviour, the external appearance and the appearance of the faeces were noted.

Also the laboratory examinations and the examinations at necropsy (examination of haematological and biochemical parameters, determination of the T4 level of the male animals, determination of organ weights, the macroscopical and histopathological examination) revealed no test item-related changes.

However, most probably due to the properties of the test item in the drinking water a dose dependent refusal of intake of the test item-drinking water mixtures was noted for the male and female animals. This was most pronounced for the male animals of the high dose group (160/120 mg IPDA/kg b.w./day) during the first and the second treatment week and caused a reduction in body weight for the male animals. The body weight of the high dosed males recovered as the consumption of drinking water increased after the reduction of the test item concentration in the drinking water (dose level reduction from 160 to 120 mg IPDA/kg b.w./day).

The concentration of the test item in the test item-drinking water mixture was adjusted weekly to the relative drinking water consumption of the male and female animals. The actual dose levels of test item intake were calculated for the male and female animals using the total drinking water consumption and the body weight of the animals:

In most cases the actual dose levels were nearly in the range of the nominal dose levels for the male and female animals of all dose groups.

However, for the male animals of the high dose group (160/120 mg test item/kg b.w./day), the marked reduction in drinking water consumption before the reduction of the dose level revealed a decreased intake of test item via the drinking water. Hence, the calculated actual dose level was below the nominal dose level during the first and second treatment week. After the dose level reduction the actual test item-intake was in the range of the nominal dose level.

For the female animals increased actual dose levels were noted during the lactation period for all dose groups (20, 60or160/120 mg IPDA/kg b.w./day). This was due to increased levels of absolute and relative drinking water consumption during the lactation period in comparison to the pre-mating and gestation period that were noted for all dose groups.

Reproductive toxicity

Parental females

No influence was noted on the estrus cycles, the fertility, the gestation index, the pre-coital time and the gestation length.

Pups

No influence on the pre-natal developmentwas noted (number of implantation sites, birth and live birth index, percentage of post-implantation loss).

During the post-natal development no influence was noted on body weight, the ano-genial distance, the number of nipples of the male pups and the T4 level.

No abnormalities (malformations or variations) were noted during the external macroscopic examination of the pups at necropsy.

A reduced viability index was noted during the post-cull period for the pups of the high dose group (160/120 mg IPDA/kg b.w./day). This was considered as a secondary effect of the reduced consumption of drinking water of the high dosed females in comparison to the females of the control and the low and the intermediate dose group. This lower drinking water consumption led to a reduced milk production and hence, an inadequate feeding of some of the weaker pups. Therefore, the reduced viability index was not considered to be of toxicological relevance.

 

Though there were no toxicological relevant findings, the maximum dose level for possible further rat studies was considered to be 120 mg IPDA/kg b.w./day for the administration via the drinking water. At this dose level and also at the lower tested dose levels of 20 and 60 mg IPDA/kg b.w./day the test item in the drinking water already evoked a reduced drinking water consumption, but without secondary effects in the form of body weight loss or a possibly reduced milk production of the dams.

Considering these secondary effects that were due to the refusal of intake of the test item-drinking water mixture by the animals and considering the fact that no systemic toxicological effects occured, the study has to be performed with another route of administration. Hence, an OECD 422 study via gavage was performed to be able to determine suitable dose levels for the requested OECD 443 study.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
other: study started in July 2019, first draft report is expected soon
Adequacy of study:
supporting study
Study period:
Study started on 24th of July 2019 and generation of draft report is in progress
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted July 29, 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
The study was performed as dose range finding study for the EOGRTS (OECD 443).
Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
Rat / CD® / Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Species / Strain / Stock: Rat / CD® / Crl:CD(SD)
Breeder: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
Body weight (at start of dosing): *
Age (at start of dosing): Approximately 11 to 12 weeks* (young adults; sexually mature)
Selection of species: The rat is a commonly used rodent species for such studies.
Number of animals: Pre-exposure period:At least 52 female animals will be evaluated pre-exposure for oestrus cyclicity to yield 40 females with a regular oestrus cycle for the study.
Main study: 80 animals (40 males and 40 females) A sufficient number in order to grant at least 8 pregnant females per group for evaluation of the F0 generation.

ENVIRONMENTAL CONDITIONS
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Route of administration: Oral, via gavage
Frequency of administration: Once daily; see section 2.6 for the treatment periods
Vehicle: Purified water
Application volume: 10 mL/kg b.w./day
Dosages: 0, 30, 100, 300 (240) mg/kg bw/d

The test item formulations will be freshly prepared every day.
The test item will be diluted in the vehicle to the appropriate concentrations and will be administered orally at a constant volume/kg b.w. once daily.
The amount of the test item will be adjusted to the animal's current body weight daily.
The vehicle control animals receive the vehicle at the same administration volume daily in the same way.
Details on mating procedure:
Sexually mature male and female rats are paired monogamously, i.e. 1 male and 1 female animal are placed in one cage during the dark period. The female is placed with the same male until evidence of mating is observed or two weeks have elapsed. The females are examined each morning for the presence of sperm. The day of conception (day 0 of gestation) is considered to be the day on which sperm is found. In case pairing is unsuccessful, re-mating of females with proven males of the same group can be considered after approximately 2 weeks of unsuccessful mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For each test item that is mixed with a vehicle, tests by appropriate analytical methods shall be conducted to determine the concentration, stability and homo¬geneity of the test item in the formulations.
For the analysis of the test item-vehicle mixtures, samples of 2 x 5 mL are taken as scheduled (see Table 2 1) and stored at -20°C±10% until analysis.
Duration of treatment / exposure:
The study animals will be treated during the following periods:
Males: 2 weeks prior to mating, during the mating period, and approximately 2 weeks post mating at least until the minimum total dosing period of 28 days has been completed (up to and including the day before sacrifice).
Females: 2 weeks prior to mating and continuing up to, and including, day 13 post-partum (up to and including the day before sacrifice).
In the case day 13 post-partum falls on a weekend, necropsy will be postponed to the next working day and dosing will be continued up to and including the day before sacrifice.
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control group
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
low dose group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
intermediate dose group
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
high dose group
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, concurrent vehicle
Positive control:
not needed
Parental animals: Observations and examinations:
CLINICAL SIGNS
Throughout the test period, each animal will be observed for clinical signs at least once daily. The frequency will be increased when signs of toxicitiy are observed. Behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity are recorded. Mortality is recorded twice daily. Animals which die or are sacrificed during the study are necropsied as soon as possible after exitus as described in section 4.
Individual animals will be observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness.
Additionally, once before the first exposure (to allow for within-subject comparisons) and once a week thereafter, detailed clinical observations will be made in all animals of the F0 generation animals outside the home cage in a standard arena and at the same time, each time preferably by observers unaware of the treatment.

VAGINAL SMEARS
Daily monitoring of vaginal smears will continue throughout the premating period until evidence of mating. A vaginal smear will also be taken in the morning of the day of scheduled necropsy.

NEUROLOGICAL SCREENING
Screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli) (based on Gad ), as well as the assessment of grip strength (Meyer ) and motor activity assessment will be conducted as described on the following pages in five males and five females randomly selected from each group. The screening will be conducted two hours after dosing and before any blood sampling for laboratory examinations:
5 male F0 animals per group: Shortly before scheduled sacrifice
5 female F0 animals per group: During lactation, shortly before scheduled sacrifice

Mortality
Further checks will be made early in the morning and again in the afternoon of each working day to look for dead or moribund period animals. This will allow post mortem examination to be carried out during the working period of that day. On Saturdays and Sundays a similar procedure will be followed except that the final check will be carried out at approximately 3:30 p.m.
Premortal symptoms will be recorded in detail; as soon as possible after exitus, a post mortem examination will be performed as described in section 4. In the case of prematurely sacrificed animals, laboratory examinations (see section 3.8) will be performed, if possible.

Body weight
The adult animals will be weighed on each day of dosing for dose adjustment and at sacrifice; the individual body weights will be recorded. The report will include weekly values as well as those determined on gestation days 0, 7, 14 and 20 and post partum days 1, 4, 8 and 13.

Food and water consumption
Food residue is weighed and recorded as follows:
Time points for food consumption
Pre-mating period Males: on Test day 8 and Test Day 15 Females on test day 8 and test day 15
Gestation period Females on Gestation day 7, 14, and 20
Lactation period Females on lactation day 1, 8, and 13


Reproductive performance
Evaluation/parameters

-Number of pregnant females
-Pre-coital time
-Gestation length calculated from day 0 of pregnancy
-Implantation sites (see also section 4)
- number per dam
- distribution in the uterine horns
- absolute number per group
- mean per group
- Number of pups absolute
- at birth (alive and dead)
- after 4 days of life (pre and post cull), after 13 days
- Number of pups per dam
- at birth
- after 4 days of life (pre and post cull), after 13 days
- Number of male and female pups
- at birth
- after 4 days of life (pre and post cull), after 13 days
- Number of stillbirths
- absolute
- per dam
- Number of pups with malformations (see appendix 1)
- absolute
- per dam

Haematology at sacrifice
Differential blood count (relative)
Differential blood count (absolute)
Erythrocytes (RBC)
Leucocytes (WBC)
Haematocrit value (HCT)
Haemoglobin content (HGB)
Platelets (PLT)
Reticulocytes (RET)
Mean corpuscular volume (MCV)
Mean corpuscular haemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)
Prothrombin time (PT)
Activated partial thromboplastin time (aPTT)

Clinical chemistry at sacrifice
Sodium
Potassium
Calcium
Chloride
Albumin
Total bilirubin
Total cholesterol
Glucose
Total protein
Blood urea (BUN)
Creatinine
Alanine amino-transferase (ALAT/GPT)
Alkaline phosphatase (aP)
Aspartate aminotransferase (ASAT/GOT)
Bile acids
Lactate dehydrogenase (LDH)
Globulin
Albumin/Globulin ratio




Oestrous cyclicity (parental animals):
During a 14-day pre-exposure period, the oestrus cycle of the female animals will be monitored to select 40 animals with regular oestrus cycles. Animals that fail to exhibit typical 4-5 day cycles will not be included in the study.
Daily monitoring of vaginal smears will continue throughout the premating period until evidence of mating.
A vaginal smear will also be taken in the morning of the day of scheduled necropsy.
Sperm parameters (parental animals):
Detailed histopathologic examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure will be performed on one testicle and one epididymis of the selected males of groups 1 and 4 following H-E and PAS staining.
Litter observations:
Each litter will be examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups are considered as runts if their weight is less than 70% of the mean litter weight) and the presence of gross abnormalities.
The pups will be examined as described below. Any abnormal behavior will be recorded.

Counting, sexing and weighing
Live pups will be counted and sexed. The litters will be weighed on post-natal days 1, 4, and 13 (PND 1, 4, and 13).

Ano-genital distance
On post-natal day 4 before litter adjustment the ano-genital distance (AGD) of all pups will be determined using a scale.

Litter adjustment
After counting on PND 4, the litters will be adjusted to 10 pups per litter (5 pups/sex/per litter) by eliminating surplus pups following a randomization scheme. Selective elimination of pups, e.g. based upon body weight is not appropriate. Whenever the number of male or female pups prevents having 5 pups/sex/per litter, partial adjustment (for example, 6 male and 4 female pups) is acceptable.

Blood sampling for thyroid hormone (T4) determination
On PND 4 and 13 blood samples for T4 hormone level determination will be taken from 2 selected pups, if possible from one male and one female pup. No pups will be eliminated when litter size will drop below the culling target (8 or 10 pups/litter). If there is only one pup available above the culling target, only one pup will be eliminated and used for blood collection for possible serum T4 assessments.

Nipples/areolae counting
Nipples/areolae will be counted in all male pups on PND 12 or 13.

Haematology at PND 4 and PND 13
Differential blood count (relative)
Differential blood count (absolute)
Erythrocytes (RBC)
Leucocytes (WBC)
Haematocrit value (HCT)
Haemoglobin content (HGB)
Platelets (PLT)
Reticulocytes (RET)
Mean corpuscular volume (MCV)
Mean corpuscular haemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)
Prothrombin time (PT)
Activated partial thromboplastin time (aPTT)

Clinical chemistry at PND 4 and PND 13
Sodium
Potassium
Calcium
Chloride
Albumin
Total bilirubin
Total cholesterol
Glucose
Total protein
Blood urea (BUN)
Creatinine
Alanine amino-transferase (ALAT/GPT)
Alkaline phosphatase (aP)
Aspartate aminotransferase (ASAT/GOT)
Bile acids
Lactate dehydrogenase (LDH)
Globulin
Albumin/Globulin ratio

Postmortem examinations (parental animals):
Gross necropsy
Vaginal smears prepared on the day of necropsy will be examined to determine the stage of the oestrus cycle and allow correlation with the histopathology of the female reproductive organs.
The animals will be euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis and weighed at the following times:
Males After a dosing period of at least 4 weeks
Dams On PND 13 or shortly thereafter

Dissection of all adult animals
At the time of sacrifice or death during the study, the adult animals will be dissected and examined macroscopically for any abnormalities or pathological changes. Special attention will be paid to the organs of the reproductive system.
The number of implantation sites will be recorded.
All superficial tissues will be examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues will be examined. The condition of the thoracic viscera will be noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera will be examined before and after removal; the urinary bladder will be examined externally and by palpation. The gastro-intestinal tract will be examined as a whole and the stomach and caecum will be incised and examined. The lungs will be removed and all pleural surfaces examined under suitable illumination.
The liver and the kidneys will be examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs will be recorded.

The weights of the following organs of all animals will be determined before fixation, where applicable. Thyroid weight will be determined after fixation. Paired organs will be weighed individually and identified as left or right.
Adrenal gland (2)
Brain
Epididymis (2)
Heart
Kidney (2)
Liver
Ovary (2)
Spleen
Testicle (2)
Thyroid
Thymus
As a whole: prostate, seminal vesicles with coagulating glands
Uterus including cervix

The following organs or parts thereof of all adult male and female animals will be preserved in an appropriate fixative:
Epididymis
Testicle
Gross lesions observed
Ovary and oviduct (2)
Prostate
Seminal vesicles with coagulating glands
Thyroid including parathyroids
Uterus with cervix
Vagina

Dissection of selected adult animals
The following organs or parts thereof of the animal groups listed below will be preserved in a suitable fixative.
• All deceased or prematurely sacrificed animals
• 5 males and 5 females randomly selected from each group

Fixative: Davidson’s solution
Eye with optic nerve (2)
Fixative: modified Davidson’s solution
Epididymis (2)
Fixative: 7% neutral buffered formalin
Adrenal gland (2)
Bone
Bone marrow (os femoris)
Brain (cerebrum, cerebellum, pons)
Gross lesions observed
Heart (3 levels: right and left ventricle,septum)
Intestine, small (duodenum, jejunum, ileum, including Peyer’s patches, Swiss roll method)
Intestine, large (colon, rectum)
Kidney and ureter (2)
Liver
Lungs (with mainstem bronchi and bronchioles), preserved by inflation with fixative and then immersion
Lymph node (1, cervical)
Lymph node (1, mesenteric)
Mammary gland
Muscle (skeletal)
Testicle (2)
Nerve (sciatic)
Oesophagus
Ovary and oviduct (2)
Pituitary
Prostate
Seminal vesicles with coagulating glands
Spinal cord (3 sections)
Spleen
Stomach
Thyroid (including parathyroids)
Thymus
Trachea (including larynx)
Urinary bladder
Uterus (including cervix)
Vagina
Any other organs displaying macroscopic changes will also be preserved.

Histopathology
Full histopathology will be performed on the preserved organs of the selected parental animals of groups 1 and 4 and all deceased or prematurely sacrificed animals, as well as the thyroid of the selected pups.
The organs listed above are examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining. Parathyroids cannot always be identified macroscopically. They are examined microscopically if in the plane of section and in all cases where they are noted as grossly enlarged.
In addition, frozen sections of the heart, liver and one kidney are prepared, stained with Oil Red O, and examined.
Detailed histopathologic examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure will be performed on one testicle and one epididymis of the selected males of groups 1 and 4 following H-E and PAS staining.
In case of test item-related changes in group 4, the Sponsor will be given sufficient notice before the corresponding organs of the animals of the intermediate/low dose level groups (group 2 and 3) are sectioned and examined histopathologically.





Postmortem examinations (offspring):
Dead pups and pups sacrificed on day 13 post-partum will be carefully examined externally for gross abnormalities. The external reproductive genitals will be examined for signs of altered development.
At day 13 post-partum, the thyroid of 1 male and 1 female pup from each litter will be preserved in 7% neutral buffered formalin, preferably from those animals used for T4 ELISA sampling (see section 3.8.4).
Statistics:
Toxicology data will be captured, whenever possible, using the departmental computerized systems (Provantis® Integrated preclinical software, Instem LSS Ltd.). Raw data not fully compatible with the computerized systems will be maintained on paper according to appropriate SOPs.

Parametrical data:
The statistical evaluation of the parametrical values will be done by Provantis (see flow chart of decision tree on the next page) using the following settings:
Homogeneity of variances and normality of distribution will be tested using the BARTLETT’s and SHAPIRO-WILK test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values will be performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group will be made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

Non-parametrical data:
The statistical evaluation of non-parametrical values will be done using the FISHER or Chi2 test:
FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01)
or
Chi2 test, n ≥ 100 (p ≤ 0.05 and p ≤ 0.01)

The respective calculations for the FISHER and Chi2 test will be performed using Provantis (maternal macroscopic findings at necropsy or findings during the external macroscopic examination of the fetuses) or an internal computer program (e.g. findings during the fetal skeletal or soft tissue examination).

Reproductive indices:
Gestation index, Fertility index, Birth index, Live Birth index, Viability index, Post-Implantation loss [%]
Dose descriptor:
other: no dose descriptor derived yet
Remarks on result:
other: study not yet completed
Critical effects observed:
not specified
Dose descriptor:
other: no dose descriptor derived yet
Remarks on result:
other: study not yet completed
Critical effects observed:
not specified
Reproductive effects observed:
not specified
Conclusions:
Up to now, there are no results of the study available.
Executive summary:

The OECD 422 study in rats started in July 2019 and the first draft report is expected to be available soon.

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
other: Study started in November 2019
Adequacy of study:
key study
Study period:
Study started in November 2019. Draft report will be available in December 2020.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
adopted 28 July 2011
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
The study design is based on final decision on acompliance check from ECHA dated 5th of April 2017. An Extended one-generation reproductive toxicity study (Annex X, Section 8.7.3.; test method: EU B.56./OECD TG 443) in rats, oral route with the registered substance specified as follows, was requested:
- Ten weeks premating exposure duration for the parental (P0) generation;
- Dose level setting shall aim to induce some toxicity at the highest dose level;
- Cohort 1A (Reproductive toxicity);
- Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation;
Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
Rat / CD® / Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
Breeder: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
Body weight (at start of dosing): *
Age (at start of mating): Approximately 20 weeks* (young adults; sexually mature)

Selection of species: The rat is a commonly used rodent species for such studies and required by the guideline.

Number of parental animals:
Pre-exposure period: At least 120 female animals will be evaluated pre-exposure for oestrus cyclicity to yield 96 females with a regular oestrus cycle for the study.
Main study: 192 animals (96 males and 96 females)

A sufficient number in order to grant at least 20 pregnant females per group for evaluation of the F0 generation.

Adaptation period: At least 5 days


ENVIRONMENTAL CONDITIONS

DIET Commercial diet ssniff R/M-Z V1324, offered ad libitum
Drinking water Tap water is offered daily ad libitum
Housing Singly in MAKROLON cages (type III plus)
Temperature 22°C ± 2 °C (maximum range)
Humidity 55% ± 10% (maximum range)
The rooms are alternately lit (about 150 lux at approx. 1.50 m room height) and darkened in a 12 hours dark/12 hours light cycle.
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
purified water
Details on exposure:
Route of administration: Oral, via gavage
Frequency of administration: Once daily; see section 2.9 for the treatment periods
Vehicle: Purified water
Administration volume: 10 mL/kg b.w.
Dosages: 0, 25, 80, 240 (160) mg/kg bw/d
Selection of route of administration: According to international guidelines.

Dose reduction since 9th of January 2020
Duration of treatment / exposure:
F0 ANIMALS
Males: 10 weeks prior to mating, during the mating period, and at least until weaning of the F1 generation (up to and including the day before sacrifice).
Females: 10 weeks prior to mating, during the mating, gestation and lactation period and until termination after weaning of their litters (up to and including the day before sacrifice).

F1 ANIMALS
Until weaning, F1 animals are indirectly exposed to the test item through the breast milk. After weaning, dosing will continue in the same way as for the parental generation
Cohort 1A: Until a dosing period of 10 weeks has been completed (up to and including the day before sacrifice, i.e. around PND 91)
Cohort 1B: Until a dosing period of 11 weeks has been completed (up to and including the day before sacrifice, i.e. around PND 98)
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control group
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
low dose group
Dose / conc.:
80 mg/kg bw/day (nominal)
Remarks:
intermediate dose group
Dose / conc.:
240 mg/kg bw/day (nominal)
Remarks:
high dose group; dose reduced on 9th of November to 160 mg/kg bw/d
No. of animals per sex per dose:
20 males and females in P generation and 20 males and females in Cohort 1A and Cohort 1B
Control animals:
yes, concurrent vehicle
Positive control:
no
Parental animals: Observations and examinations:
CLINICAL SIGNS
All animals
Throughout the test period, each animal will be observed for clinical signs at least once daily. The frequency will be increased when signs of toxicitiy are observed. Behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity are recorded.

FOOD CONSUMPTION
Food consumption is recorded daily during pre-mating period in males and females and during gestation period on GD 7, 14, 21 and during lactation period on PND 1, 7, 14, 21 in females. In males food consumption ois further recorded during post mating period on a weekly basis.

Individual animals will be observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment, or illness.
Cageside observations will include skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed will be recorded.
In addition, animals will be checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals will be checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m.
Dated and signed records of appearance, change, and disappearance of clinical signs will be maintained on clinical history sheets for individual animals.
F0 animals and F1 animals after weaning
Additionally, a more detailed examination of all F0 and F1 Cohort 1B animals is conducted on a weekly basis. F0 animals will be examined once before the first exposure (to allow for within-subject comparisons) and weekly thereafter until termination. F1 animals will be examined weekly after weaning until termination.
Detailed clinical observations will be made in all animals outside the home cage in a standard arena, approximately at the same time of day, each time preferably by observers unaware of the treatment. Signs noted will include changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) will also be recorded.

MORTALITY
Further checks will be made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This will allow post mortem examination to be carried out during the working period of that day. On Saturdays and Sundays a similar procedure will be followed except that the final check will be carried out at approximately 3:30 p.m.

BODY WEIGHT
The animals will be weighed and the weights recorded as scheduled daily during pre-mating, mating period. Females will be weighted daily during gestation (reported for GD 0, 7, 14, 21), lactation period (reported for PND 4, 7, 14, 21) and males during post mating period.

Water Water consumption is monitored by visual appraisal daily throughout the study.

HAEMATOLOGY
10 males and 10 females randomly selected from each F0 group.

Differential blood count (relative)
Differential blood count (absolute)
Erythrocytes (RBC)
Leucocytes (WBC)
Haematocrit value (HCT)
Haemoglobin content (HGB)
Platelets (PLT)
Reticulocytes (RET)
Mean corpuscular volume (MCV)
Mean corpuscular haemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY
Sodium
Potassium
Calcium
Chloride
Albumin
Total bilirubin
Total cholesterol
Glucose
Total protein
Blood urea (BUN)
Creatinine
Alanine amino-transferase (ALAT/GPT)
Alkaline phosphatase (aP)
Aspartate aminotransferase (ASAT/GOT)
Bile acids
Lactate dehydrogenase (LDH)
Sodium/Potassium ratio
Globulin
Albumin/globulin ratio
BUN/creatinine ratio

T4 and TSH Determination
From 10 males and females of F0 at sacrifice

Urinanalysis
At the end of the F0 dosing period

Parameters: Volume, pH and specific gravity
Protein, Glucose, Bilirubin, Urobilinogen, Ketones, Haemoglobin, Nitrite
The deposit will be examined for the presence of the following parameters:
- Epithelial cells
- Leucocytes
- Erythrocytes
- Organisms
- Further constituents (i.e. sperm, casts)
- Crystalluria
Oestrous cyclicity (parental animals):
Vaginal smears will be taken and the oestrus cycle stages will be determined at the following time points:

F0 animals 14-day pre-exposure period to select 96 animals with regular oestrus cycles (4-5 days) and during 10 weeks of premating until evidence of mating.
F1 animals, cohort 1A Start after onset of vaginal patency until first appearance of cornified cells and two weeks starting around PND 75.
F0 and F1 animals On the day of sacrifice, Shortly before necropsy.
Sperm parameters (parental animals):
Sperm viability and morphology (spermiogram)
• All F0 males
• All F1 Cohort 1A males
One epididymis and one testicle will be used for the sperm count. The sperm viability is determined and the sperm morphology is examined according to the method described by I. Chahoud and R. Franz (1993) as well as by S. Plassmann and H. Urwyler (2001).
Litter observations:
Littering
Each litter will be examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts4 and the presence of gross abnormalities. Based on these parameters, the reproductive performance of the dams will be evaluated.
The pups will be examined as described below. Any abnormal behavior will be recorded.

Counting, sexing and weighing
Live pups will be counted and sexed. Live pups are weighed on PND 1 and regularly thereafter.

Ano-genital distance
On post-natal day 4 before litter adjustment the ano-genital distance (AGD) of all pups will be determined using a scale.

Litter adjustment
After counting on PND 4, the litters will be adjusted to 10 pups per litter (5 pups/sex/per litter) by eliminating surplus pups following a randomisation scheme. Selective elimination of pups, e.g. based upon body weight is not appropriate. In case of unequal gender distribution, partial litter size adjustment is acceptable (e.g. 6 male and 4 female pups).

Nipples/areolae counting
Nipples/areolae will be counted in all male pups on PND 13.

Sexual maturation
Animals (all selected) are evaluated daily for balano-preputial separation or vaginal patency before expected achievement of these endpoints to detect if sexual maturation occurs early. Any abnormalities of the genitals are recorded. Sexual maturity of the animals is compared to physical development (i.e. body weight and age at balano-preputial separation or vaginal opening).
not yet available
Dose descriptor:
other: no Dose descriptor derived yet
Remarks on result:
other: study not yet completed
Critical effects observed:
not specified
not yet avialable
Dose descriptor:
other: no dose descriptor derived yet
Remarks on result:
other: study not completed
Reproductive effects observed:
not specified
Conclusions:
Up to now, there are no results of the study available.
Executive summary:

The OECD 443 study started in November 2019 and the first draft report is expected to be available in December 2020.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Species:
rat
Quality of whole database:
Klimisch 1
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Studies in animals from public sources

Partly cited from SIAR for SIAM 18 (Paris, April 2004): "No studies have been performed to explicitly address the question of reproductive effects in animals caused by 3-aminomethyl-3,5,5-trimethylcyclohexylamine. Histopathological results of a subchronic 90-day investigation on rats according to OECD TG 408 showed no effects regarding the reproductive organs (epididymides, mammary gland, ovaries, seminal vesicles, testes and uterus) in concentrations up to 160 mg/kg bw/day. Testes weights were also not affected (RCC Research and Consulting Company, 1986)."

The toxicological information regarding effects on Developmental toxicity ( no abortion or total resorption, no treatment releated effects on the pre- or post-implantation loss, no treatment releated effects on sex-ratio and on the fetal weight; CIT, 2002; see section 7.8.3 of IUCLID) and the fact that Isophorone diamine do not cause adverse effects on the examined reproductive organs in the 90 day subchronic study (RCC, 1986; see section 7.5 of IUCLID) leading to the conclusion that that effects on fertility of the substance Isophorone diamine at doses, which do not cause parental toxicity, are rather unlikely.

EOGRTS study according to OECD 443

However, in the Final decision on a compliance check (from 5thof April 2017) ECHA requested to perform the OECD 443 study in rats, oral route with the registered substance specified as follows:

 - Ten weeks premating exposure duration for the parental (P0) generation;

- Dose level setting shall aim to induce some toxicity at the highest dose level;

- Cohort 1A (Reproductive toxicity);

- Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation.

Before the start of the EOGRTS study according to OECD 443, two dose range finding studies were performed to determine suitable dose levels.

 The aim of the studies was to obtain preliminary information on possible effects of the test item IPDA on reproduction and/or development according to OECD guideline 421. The test item IPDA was administered orally to rats via the drinking water at dose levels of 20, 60 or 160/120 mg IPDA/kg b.w./day (LPT 2019). No premature death and no changes in behaviour, the external appearance and the appearance of the faeces were noted.

Also the laboratory examinations and the examinations at necropsy (examination of haematological and biochemical parameters, determination of the T4 level of the male animals, determination of organ weights, the macroscopical and histopathological examination) revealed no test item-related changes.

However, most probably due to the properties of the test item in the drinking water a dose dependent refusal of intake of the test item-drinking water mixtures was noted for the male and female animals. This was most pronounced for the male animals of the high dose group (160/120 mg IPDA/kg b.w./day) during the first and the second treatment week and caused a reduction in body weight for the male animals. The body weight of the high dosed males recovered as the consumption of drinking water increased after the reduction of the test item concentration in the drinking water (dose level reduction from 160 to 120 mg IPDA/kg b.w./day).

However, for the male animals of the high dose group (160/120 mg test item/kg b.w./day), the marked reduction in drinking water consumption before the reduction of the dose level revealed a decreased intake of test item via the drinking water. Hence, the calculated actual dose level was below the nominal dose level during the first and second treatment week. After the dose level reduction, the actual test item-intake was in the range of the nominal dose level.

For the female animals increased actual dose levels were noted during the lactation period for all dose groups (20, 60 or 160/120 mg IPDA/kg b.w./day). This was due to increased levels of absolute and relative drinking water consumption during the lactation period in comparison to the pre-mating and gestation period that were noted for all dose groups.

In the parental no influence was noted on the estrus cycles, the fertility, the gestation index, the pre-coital time and the gestation length. 

A reduced viability index was noted during the post-cull period for the pups of the high dose group (160/120 mg IPDA/kg b.w./day). This was considered as a secondary effect of the reduced consumption of drinking water of the high dosed females in comparison to the females of the control and the low and the intermediate dose group. This lower drinking water consumption led to a reduced milk production and hence, an inadequate feeding of some of the weaker pups. Therefore, the reduced viability index was not considered to be of toxicological relevance. No other effects could be observed.

 Unfortunately, it was not possible to determine adequate dose levels for the OECD 443 study. The animals refused to consummate the drinking water because of the bad smell of the test substance (LPT 2018). As a consequence, the OECD 421 study had to be performed with another route of exposure. It was chosen to perform the OECD 422 study with rats and gavage as administration route at the dose levels of 0, 30, 100 and 300 (240) mg/kg bw/d. The study started in July 2019 and a first draft report is expected soon (LPT 2020).

The requested OECD 443 study started in November 2019 and the draft report will be available in December 2020.

 

 

Effects on developmental toxicity

Description of key information

Partly cited from SIAR for SIAM 18 (Paris, April 2004): "3-Aminomethyl-3,5,5-trimethylcyclohexylamine did not show any teratogenic or embryofetotoxic effects in the gavage study with rats performed in accordance with OECD TG 414 (2001) up to and including the highest tested dose level of 250 mg/kg bw/day." The NOEL for maternal toxicity was 50 mg/kg bw/day, effects at 250 mg/kg bw/day were reduced food consumption and reduced body weight gain. The NOAEL for developmental toxicity is >=250 mg/kg bw/day (CIT, 2002). 

To determine suitable dose levels for the OECD 414 study with the second species, a dose range finding study was performed. In this prenatal developmental toxicity study, the test item was administered orally to female rabbits at dose levels of 5, 25 or 50 mg/kg b.w./day from the 6th to 28th day of pregnancy. Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was above 50 mg test item/kg b.w./day for the dams asno effects could be observed during the study.

In a supporting 14 days tolerance study with rabbits the test item was administered orally to two groups with 3 female rabbits once daily at dose levels of 75 mg/kg bw test item and 150 mg/kg bw. test item. Group 1 dosing was extended to 21 test day, group 2 dosing was terminated on test day 7 because of humane sacrifice. A reddened gastric mucosa was seen all dosed animals and a pale liver was noted in 2 of 3 animals of the high dose group. Furthermore, a reduction in food consumption was noted for the low dose animals. No or only minimal food intake was noted for the high dose animals from start of dosing onwards until premature sacrifice on test day 8 due to animal welfare reasons (LPT 2018).

In the following main OECD 414 study with the second species rabbit, the test substance was administered orally to female rabbits at dose levels of 10, 25 or 75 mg/kg b.w./day from the 6th to 28th day of pregnancy. Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 25 mg IPDA/kg b.w./day for the dams. The no-observed-adverse effect level (NOAEL) was above 75 mg IPDA/kg b.w./day for the fetuses and 25 mg IPDA/kg b.w./day for the rate of early resorptions/post implantation loss (LPT 2020).

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2018-04-12 to 2018-06-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted January 22, 2001
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
May 30, 2008
GLP compliance:
no
Remarks:
The study was performed based on Good Laboratory Practice' Regulations of the EC enacted in Germany in the 'Chemikaliengesetz' [Chemicals Act], current edition; and -OECD Principles of Good Laboratory Practice' Document Nos. 1, 8 and 13
Limit test:
no
Specific details on test material used for the study:
The test item was diluted in the vehicle to the appropriate concentration.
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ORGANISMS: 
- Species: Rabbit
- Source: Manfred Bauer Kaninchen, Lohe 7/1, 74632 Neuenstein, Germany
- Strain: New Zealand White
- Age: 5 months
- body weight: 3.55 - 4.30 kg
- Diet: ad libitum, Commercial ssniff K-Z V2323 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany,
- Water: ad libitum
- Adaptation period: 20 days
-Housing: Except during the mating period, the dams were kept separately in breeding cages with wire floors
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 °C +/- 3° C
- Humidity (%): 55% +/- 15 %
- Illumination: 12 hours artifical fluorescent light and 12 hours dark
- Ventilation rate: between fifteen to twenty air changes per hour.
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Purified water (aqua ad injectabilia)
Details on exposure:
ADMINISTRATION: 
- Frequency: once daily, day 6 to 28 of gestation
- Dose volume: 2 ml/kg b.w.
- Dose: 0, 5, 25, 50 mg/kg/bw
- Animals: 16 females in groups 1 to 4
Test item preparation:
The test item formulations were freshly prepared every day.
The test item was diluted in the vehicle to the appropriate concentration and was administered orally at a constant volume (2 mL/kg b.w.) once daily from the 6th to the 28th day of gestation.
The amount of the test item was daily adjusted to the current body weight of the animal. The control animals received the vehicle at the same administration volume daily in the same way.

Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
For each test or reference item that is mixed with a vehicle, test by appropriate analytical methods will be carried out for the main study to determine concentration, homogeneity and, if needed, stability of the test item in the formulations.

Details on mating procedure:
Sexually mature ('proved') male rabbits of the same breed served as partners. The female breeding partners were randomly chosen.
Mating was monogamous; 1 male and 1 female animal were placed in one cage in the forenoon. Successful copulation was ascertained by observation.The day of copulation was considered as day 0 of pregnancy. Only animals with positive signs of copulation were included in the study.
Duration of treatment / exposure:
23 administration days from gestation day 6 to 28
Frequency of treatment:
Once daily
Duration of test:
Laparotomy on gestation day 29
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Remarks:
Low dose
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Remarks:
Intermediate dose
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
High dose
No. of animals per sex per dose:
4 female rabbits/dose
Evaluated litters: at least 12 pregnant females, 3 litters per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Principle
Pregnant rabbits were treated with the test item starting from the 6th and lasting until the 28th day of pregnancy (the 'critical' phase of organogenesis and the fetal development). The development of the pregnant rabbits was observed during the gestation period (day of mating is day 0 of pregnancy). One day before the calculated date of birth (29 days after mating) the dams were laparotomised and examined for corpora lutea, implantation sites, resorptions in the uterus and for the condition of the fetuses.

Dose selection rationale:
The dose levels were selected in agreement with the Sponsor based on available toxicological data.
Maternal examinations:
Dated and signed records of all activities relating to the day to day running and maintenance of the study within the animal units, as well as to the group observations and examinations outlined in the Study Plan, were recorded in the appropriate documentation. In addition, observations relating to the individual animals made throughout the study were recorded.

The following observations were made during the course of the study:
Clinical signs
Individual animals were observed daily for any signs of behavioural changes, reaction to treatment, or illness.
Immediately after administration, any signs of illness or reaction to treatment were rec-orded. In case of changes, the animals were observed until thesymptoms disappeared. In addition, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m.
On Saturdays and Sundays, the animals were checked regularly starting from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately
3.30 p.m.
Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.


Viability
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This allowed post mortem examinations to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed except that the final check was carried out at approximately midday.
Animals showing signs of abortion or premature delivery would have been sacrificed on the same day. Fetuses obtained this way were examined for abnormal development, whenever possible. No abortion occurred in the study.
No abortion or premature delivery occurred during the study.

Body weight
The weight of each rabbit was recorded on the day of delivery (used for randomization), followed by daily weighings starting on gestation day 6 - always at the same time of the day. These measurements were also used for calculating the daily amount of test item to be administered.
The body weight gain was calculated in intervals (i.e. gestation day 6-9, 9 12, 12-15, 15-18, 18-21, 21-24, 24-27, 27-29) and for the period after the start of treatment until necropsy (gestation day 6-29).

Furthermore the carcass weight and the net weight change from day 6 are given.
Carcass weight: Carcass weight = Terminal body weight minus uterine weight

Net weight change from day 6 = Carcass weight minus body weight on day 6

These values are stated in the report.


Food and drinking water consumption
The quantity of food consumed by each rabbit was recorded daily. Food intake per rabbit (g/rabbit/day) was calculated using the total amount of food given to and left by each rabbit in each group on completion of a treatment day.

The relative food consumption (g/kg b.w./day) was calculated using the following formula:
Daily food consumption [g/kg b.w./day]= Total food intake in g / Body weight in kg

Daily monitoring by visual appraisal of the drinking water bottles was maintained throughout the study.

EXAMINATIONS (NECROPSY), Examination of the dams
Dissection technique and evaluation of the animals:
One day before the calculated parturition, i.e. on gestation day 29, all surviving rabbits were sacrificed by lethal intravenous injection of 300 mg Pentobarbital/kg b.w. and exsanguinated.
In order to check for possible drug effects, a dissection with macroscopic examination of the internal organs and placentae of the dams was carried out on the day of sacrifice or on the day on which the animals were found dead.
After ventral midline incision and skin reflection, the ovaries and uteri were removed; the gravid uteri (in toto) were weighed. A macroscopic examination of all subcutaneous tissues and internal organs of the dams was carried out. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal, the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intraabdominal lymph nodes and accessory reproductive organs were recorded.
In case of macroscopical findings, the affected maternal tissues were preserved in 10% buffered formalin for possible future histopathological examinations.


Ovaries and uterine content:
Corpora lutea
- number per dam
- absolute number per group
- mean per group

Implantations
- number per dam
- distributions in the uterine horns
- absolute number per group
- mean per group

Resorptions
- number per dam, % per litter
- distributions in the uterine horns
- absolute number per group
- mean per group
- mean % per group
- early resorptions < 2 g
- late resorptions > 2 g

Weight of placentae
- individual weight per fetus (alive and dead)
- mean placental weight per dam (placentae from male and female fetuses alone or combined)
- mean placental weight per dam and group (litter mean per group)

Weight of fetuses
- individual weight per fetus (alive and dead)
- mean fetal weight per dam (male and female fetuses alone or combined)
- mean fetal weight per dam and group (litter mean per group)

Fetuses
- absolute number per dam and group (alive and dead, male and female fetuses alone or combined)
- mean number of fetuses per dam and group (alive and dead, male and female fetuses alone or combined)
- sex ratio per litter
- distribution in the uterine horns

Runts
- number per dam
- mean per group

Malformed fetuses
- individual data per fetus
- type of malformation
- total number and incidence (%) of affected fetuses and litters per group

Total malformation rate [%] = malformed fetuses per group / fetuses per group x 100

Fetuses with variations
- individual data per fetus
- type of variation
- total number and incidence (%) of affected fetuses and litters per group8

Total variation rate [%] = fetuses per group with variations / fetuses per group x 100

Indices of pre-implantation loss and post-implantation loss:
Calculation of group indices

Pre-implantation loss [%] = corpora lutea (per group) - implantations (per group) / corpora lutea (per group) x 100


Post-implantation loss [%] = implantations (per group) - living fetuses (per group) / implantations (per group) x 100


Calculation of mean indices per litter

Pre implantation loss [%] = Sum of pre-implantation losses per litter in a group [%] / Number of litters in a group


Post implantation loss [%] = Sum of post-implantation losses per litter in a group [%] / Number of litters in a group




Fetal examinations:
The fetuses were removed and the following examinations performed:
(a) Macroscopic inspection (gross evaluation) of the placentae for example for focal indurations.
(b) The number of fetuses (alive and dead) and placentae (location in the uterus and the assignment of the fetuses) was determined.
(c) Sex and viability of fetuses were determined. Animals are said to be viable when they are found alive (spontaneous breathing, spontaneous movement).
(d) Number and size of resorptions were determined.
(e) Corpora lutea in the ovaries, implantations and location of fetuses in the uterus were determined.
(f) The gravid uterus weight was determined.
(g) Weights of fetuses and weights of the placentae were determined (fetuses were considered as runts if their weight was less than 70% of the mean litter weight).
(h) All fetuses (dead and alive) were inspected externally for damages, especially for malformations .
(i) The fetuses were sacrificed by a lethal intraperitoneal injection of 60 mg Pentobarbi-tal/kg b.w..

Each fetus was dissected:
The thorax and peritoneal cavity (without damage to ribs and sternum) were opened. Location, size and condition of the internal organs were determined and examined for abnormalities (e.g. liver, discolouration, situs inversus, alterations of urinary bladder, brain, lungs, cleft palate) of soft tissue.
The sex was determined.
The kidneys were removed and incised to check for damages (e.g. dilatation of the renal pelvis).
The abdominal organs were removed.
The diaphragm was carefully removed to check the position of the heart (left - right).
The thoracic organs were removed using surgical forceps; the heart was incised to check for damages.

Statistics:
The statistical evaluation of the parametrical values was done by Provantis using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), inter-group comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).
Non-parametrical data
The statistical evaluation of non-parametrical values was done using the FISHER or Chi2 test:
FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01)
or
Chi2 test, n ≥ 100 (p ≤ 0.05 and p ≤ 0.01)

The respective calculations for the FISHER and Chi2 test were performed using Provantis (maternal macroscopic findings at necropsy or findings during the external macro-scopic examination of the fetuses).
Note:
The statistical evaluation of the pre- and post-implantation index (per group) using the number of corpora lutea, implantation sites and/ or fetuses per group was done using StatXact 4.0.1 software.

Clinical signs:
no effects observed
Description (incidence and severity):
No changes in behaviour, the external appearance or faeces were noted for the control group or the test item-treated groups (5, 25 or 50 mg test item/kg b.w./day).
Mortality:
no mortality observed
Description (incidence):
No premature death was noted for the control group and the treatment groups (5, 25 or 50 mg test item/kg b.w./day).
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes in body weight or body weight gain were noted between the dams of the control group and the dams of the treatment groups (5, 25 or 50 mg test item/kg b.w./day).

Body weight gain:
The changes in body weight gain after the start of treatment on gestation day 6 until necropsy on gestation day 29 are given in the table below:

Body weight gain (mean %) # Group 1 Control Group2, 25 mg/kg Group 3, 25 mg/kg Group 4, 50 mg/kg
Gestation day 6 to gestation day 29 4.5% 7.8% 7.4% 7.4%
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes in food consumption were noted between the dams of the control group and the dams treated with 5, 25 or 50 mg test item/kg b.w./day.
However, a statistically significantly increased food consumption in comparison to the control group was noted on several test days between gestation day 19 and gestation day 24 for the dams of the treatment groups (5, 25 or 50 mg test item/kg b.w./day; at maximum 241.5% above the value of the control group at the intermediate dose level between gestation days 23 and 24). This was due to a low food intake of the control dams on several test days. Hence, the statistically significantly increased food consumption that was noted for the treatment groups was considered to be spontaneous.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test item-related changes in the consumption of drinking water were noted for the dams treated with 5, 25 or 50 mg test item/kg b.w./day by visual appraisal.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Gravid uterus and carcass weight
No test item-related changes in the gravid uterus weight and the carcass weight in comparison to the control group were noted for the dams of the treatment groups (5, 25 or 50 mg test item/kg b.w./day).

Net body weight gain from day 6
No test item-related changes between the control group and the treatment groups (5, 25 or 50 mg test item/kg b.w./day) were noted for the net body weight gain (without gravid uterus) between gestation day 6 and gestation day 29.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related pathological findings were noted during macroscopic inspection of the dams of the control group and dams treated with 5, 25 or 50 mg test item/kg b.w./day.
One animal of the high dose group revealed changes in the liver and the stomach. However, as only one animal was affected, these changes were considered to be spontaneous and not test item-related

Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
General toxicity maternal animals: no effects
Number of abortions:
no effects observed
Description (incidence and severity):
No test item-related influences on the number of resorptions and the number of live fetuses were noted for the dams of the treatment groups (5, 25 or 50 mg test item/kg b.w./day).

Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No test item-related influences on the number of resorptions and the number of live fetuses were noted for the dams of the treatment groups (5, 25 or 50 mg test item/kg b.w./day).
The values of pre- and post-implantation loss were in the range of normal variability.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No test item-related influences on the number of resorptions and the number of live fetuses were noted for the dams of the treatment groups (5, 25 or 50 mg test item/kg b.w./day).
Early or late resorptions:
no effects observed
Description (incidence and severity):
No test item-related influences on the number of resorptions and the number of live fetuses were noted for the dams of the treatment groups (5, 25 or 50 mg test item/kg b.w./day).
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead fetus was noted in the control group and in the treatment groups (5, 25 or 50 mg test item/kg b.w./day).
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
No effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:no effects
Key result
Dose descriptor:
NOAEL
Effect level:
> 50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no maternal toxicity
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related influence was noted on the fetal weight after administration of 5, 25 or 50 mg test item/kg b.w./day.
One runt (no. 15-7) was noted in the high dose group (50 mg/kg b.w./day). This was in the normal range of variability and considered to be spontaneous.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No dead fetus was noted in the control group and in the treatment groups (5, 25 or 50 mg test item/kg b.w./day).
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in the control group and in the treatment groups (5, 25 or 50 mg test item/kg b.w./day) was inside the range of normal biological variability.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No test item-related influences on the number of resorptions and the number of live fetuses were noted for the dams of the treatment groups (5, 25 or 50 mg test item/kg b.w./day).
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No external malformation was noted for the fetuses of the control group and the treat-ment groups (5, 25 or 50 mg test item/kg b.w./day).
External variations in the form of a hyperflexion of the extremities were noted for 3 fetuses from 2 dams of the low dose group (5 mg test item/kg b.w./day). As no extremities with a hyperflexion were noted for the fetuses of the intermediate and the high dose group (25 or 50 mg test itemkg b.w./day) the observations at the low dose level were considered to be spontaneous.
Skeletal malformations:
not examined
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Internal gross alterations
A macroscopic internal examination was performed to detect gross alterations of the internal organs. No internal malformation or variation was noted for the fetuses of the treatment groups (5, 25 or 50 mg test item/kg b.w./day).
At the control group a spontaneous variation in the form of a malpositioned left kidney was noted for fetus no. 4-8.
Other effects:
no effects observed
Description (incidence and severity):
One runt each was noted in the intermediate dose group (300 mg test item/kg b.w./day) and in the control group.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
No dead fetuses were noted in the litters of the control group and the treatment groups (treated orally with 5, 25 or 50 mg /kg b.w./day).
No test item-related malformations or variations were noted during the macroscopic external examination and the macroscopic gross inspection of the internal organs at laparotomy for the treatment groups (5, 25 or 50 mg test item/kg b.w./day).

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no developmental toxicity
Key result
Developmental effects observed:
no

Summary of animals examined

Test item

Group 1

Control

Group 2

5 mg/kg

Group 3

25 mg/kg

Group 4

50 mg/kg

Treated females

4

4

4

4

Not examined females

(excluded/spare animals)

none

none

none

1

Non-pregnant females

1

1

1

none

Deceased animals

none

none

none

none

Evaluated pregnant females

3

3

3

3

Dams without viable fetuses

(total post implantation loss)

none

none

none

none

Dams with abortion

none

none

none

none

Evaluated litters with

viable fetuses

3

3

3

3

Reproduction data of the dams

Parameter

Group 1

Control

(n=3)

Group 2

5 mg/kg

(n=3)

Group 3

25 mg/kg

(n=3)

Group 4

50 mg/kg

(n=3)

Corpora lutea

total

mean per dam

 

31

10.3

33

11.0

29

9.7

39

13.0

Implantation sites

total

mean per dam

 

31

10.3

30

10.0

27

9.0

35

11.7

Total resorptions

total

mean per dam

 

1

0.3

1

0.3

2

0.7

0

0.0

Early resorptions

total

mean per dam

 

1

0.3

1

0.3

2

0.7

0

0.0

Late resorptions

total

mean per dam

 

0

0.0

0

0.0

0

0.0

0

0.0

Fetuses (alive + dead)

total

mean per dam

 

30

10.0

29

9.7

25

8.3

35

11.7

Live fetuses

total

mean per dam

 

30

10.0

29

9.7

25

8.3

35

11.7

Dead fetuses

total

mean per dam

 

0

0.0

0

0.0

0

0.0

0

0.0

Pre-implantation loss

per group

 

0.0

9.1

6.9

10.3

[%]

mean per dam

 

0.0

7.1

6.7

8.9

Post-implantation loss

per group

 

3.2

3.3

7.4

0.0

[%]

mean per dam

 

3.3

3.0

7.2

0.0

* / **

Significantly different from the controls at p ≤ 0.05 / p ≤ 0.01, Chi2test, only performed for the group values of the pre- and post-implantation loss

* / **

Significantly different from the controls at p ≤ 0.05 / p ≤ 0.01, Dunnett test, performed for the mean values per group

Body weight gain

Body weight gain

(mean %)#

Group 1

Control

Group 2

5 mg/kg

Group 3

25 mg/kg

Group 4

50 mg/kg

Gestation day 6 to

gestation day 29

4.5%

7.8%

7.4%

7.4%

Sex distribution of fetuses

Parameter

Sex ratio

observed in this study

(male / female)

Sex ratio of

fetuses

(male/female)

Group 1:

0.76

Group 2:

0.71

Group 3:

0.92

Group 4:

1.33

Conclusions:
Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was above 50 mg test item/kg b.w./day for the dams. The no-observed-adverse effect level (NOAEL) for the fetal organism was also above 50 mg test item/kg b.w./day.

Based on the data obtained in this dose-range-finding study, it is not possible to derive dose levels for main study. Therefore, an additionally tolerance test was conducted (7.5.1 LPT_DRF_21 days_Rabbits_2018, LPT Study No. 36299).
Based on data obtained in both dose range finding studies, the following dose levels are suggested for LPT Study No. 35503 (Prenatal developmental toxicity study in rabbits):
Group 1: Control
Group 2: 10 mg test item/kg b.w./day, p.o
Group 3: 25 mg test item/kg b.w./day, p.o
Group 4: 75 mg test item/kg b.w./day, p.o
Executive summary:

The aim of this dose-range-finding study was to determine the dose levels for a prenatal developmental toxicity study of the test item in pregnant rabbits when administered orally during the critical period of organogenesis and the fetal development (6th to 28th day of gestation).

 

In this prenatal developmental toxicity study, the test item was administered orally to female rabbits at dose levels of 5, 25 or 50 mg/kg b.w./day from the 6th to 28th day of pregnancy.

Findings:

Examination of the dams

 

Mortality

No premature death was noted for the control group and the treatment groups (5, 25 or 50 mg test item/kg b.w./day).

Clinical signs

No changes in behavior, the external appearence and the consistency of the faeces were noted in the control group and the test item-treated groups.

Body weight and

body weight gain

 

No test item-related difference was noted for the body weight and the body weight gain.

Food consumption

No test item-related difference was noted for the food consumption.

Drinking water consumption

No test item-related influence was noted for the drinking water consumption at any of the tested dose levels (visual assessment).

Necropsy findings

No test item-related observations were noted for the treatment groups during necropsy.

Uterus and carcass weights

No test item-related influence on the uterus and carcass weight was noted for the treatment groups.

 

 

Examination of the fetuses

No test item-related influence was detected on the prenatal fetal development at 5, 25 or 50 mg test item/kg b.w./day with respect to the incidence of resorptions, number of live fetuses and the values calculated for the pre- and post-implantation loss.

 

Nodead fetuswas noted in the test item groups or the control group.


 

Sex distribution

 

No test item-related differences were noted.

 

Fetal weights

 

No test item-related influence on the fetal weights was noted for the treatment groups.

 

Placental weights

 

No test item-related influence was noted.

 

 

 

 

Fetal alterations

 

Malformations

No malformation was noted for the fetuses treated with 5, 25 or 50 mg test item/kg b.w./day during the inspection at laparotomy, including a macroscopic external examination and a macroscopic gross inspection of the internal organs.

 

 

Variations

The macroscopic inspection at laparotomy revealed no test item-related external variation for the fetuses treated with 5, 25 or 50 mg test item/kg b.w./day. No variation was noted during the gross inspection of the internal organs.

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was above 50 mg test item/kg b.w./day for the dams.

None of the animals died prematurely.

No changes were noted in behaviour, the external appearance and the consistency of the faeces.

No test item-related effect was noted for the body weight, body weight gain or food consumption.

Necropsy revealed no test item-related pathological findings.

The no-observed-adverse effect level (NOAEL) for the fetal organism was also above 50 mg test item/kg b.w./day.

No test item-related effects were noted for the reproduction parameters (number of implantation sites, number of fetuses, number of resorptions) in any of the treatment groups.

No test item-related influence was noted on the body weight of the fetuses in the treatment groups.

No dead fetuses, no malformation and no test item-related variations were noted.

Based on the data obtained in this dose-range-finding study, it is not possible to derive dose levels for main study. Therefore, an additionally tolerance test was conducted (LPTStudy No. 36299). Based on data obtained in both dose range finding studies, the following dose levels are suggested forLPTStudy No.35503 (Prenatal developmental toxicity study in rabbits):

Group 1:

Control

Group 2:

10 mg test item/kg b.w./day, p.o

Group 3:

25 mg test item/kg b.w./day, p.o

Group 4:

75 mg test item/kg b.w./day, p.o

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2018 - October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2018
The 2018 update is to include rat-specific requirements in the TG 414 only; thus applies to rats and not to rabbits (for details see overall remarks/attachments below)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Test species / Strain Rabbit / New Zealand White
Breeder Manfred Bauer Kaninchen, Lohe 7/1, 74632 Neuenstein, Germany
Age (on day 6 of gestation) 5 months

Body weight (on day 6 of gestation) 3.75 kg to 4.68 kg
Number of animals 128 treated females in groups 1 to 4:

Mated and treated animals:
Groups 1 + 2: 30 females per group
Groups 3 + 4: 34 females per group

Evaluated females with viable fetuses:
Group 1: 20 dams
Group 2: 20 dams
Group 3: 20 dams
Group 4: 20 dams

ENVIRONMENTAL CONDITIONS
DIET: ad libitum, Commercial diet ssniff® K-Z V2323 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
WATER: ad libitum, tap water in bottles, Samples of drinking water are taken periodically by the Wasserwerk Wankendorf (24601 Wankendorf, Germany)

ENVIRONMENTAL CONDITIONS
Room temperature: 20°C ± 3°C (maximum range)
Relative humidity: 55% ± 10% (maximum range)
Illumination: 12-hour light/12-hour dark cycle
Ventilation rate: 15 - 20 air changes/hour
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
purified water (aqua ad injectabilia)
Details on exposure:
ADMINISTRATION
Frequency of administration Once daily, via gavage
Treatment period Day 6 to 28 of gestation
Vehicle Purified water
Administration volume 2 mL/kg b.w./day
Selection of route of administration According to OECD guideline 414

DOSAGE PREPARATION
The test item formulations were freshly prepared every day.
The test item was diluted in the vehicle to the appropriate concentrations and was administered orally at a constant volume (2 mL/kg b.w.) once daily from the 6th to the 28th day of gestation.
The amount of the test item was daily adjusted to the current body weight of the animal.
The control animals received the vehicle at the same administration volume daily in the same way.
In addition, the stability, homogeneity, and concentration of the test item mixture was monitored


Analytical verification of doses or concentrations:
yes
Remarks:
For the analysis of the application formulations of groups 2 to 4, samples of approximately 2 x 5 mL each were taken at the following times and stored at 20°C ± 10% until analysis
Details on analytical verification of doses or concentrations:

For the analysis of the application formulations of groups 2 to 4, samples of approximately 2 x 5 mL each were taken at the following times and stored at 20°C ± 10% until analysis:

At start of dosing

Analysis of stability and concentration
Immediately after preparation of the formula-tion as well as after 8 and 24 hours storage of the test item preparations at room temperature.
(3 samples / test item group; groups 2 - 4).
Number of samples: 3 x 3 = 9
(Date of sampling: 26 June 2018)

Homogeneity
At start of administration, during (middle) administration and before administration to the last animal of the dose group.
(3 samples / test item group; groups 2 - 4).
Number of samples: 3 x 3 = 9
(Date of sampling: 26 June 2018)

At end of the dosing period (at a time when the majority of animals was dosed)

Analysis of concentration
During treatment with the test item always before administration to the last animal of the group.
(1 sample / test item group; groups 2 - 4).
Number of samples: 1 x 3 = 3
(Date of sampling: 23 July 2018)

The samples were labelled with the study number, species, type of sample, concentration, sampling time and date.

The method of analysis was validated in LPT Study No. 36470. The following parameters were determined:
- Linearity
- Accuracy
- Precision
- Sensitivity
- Specificity
- Stability
The validation of the method revealed that the method employed was suitable for the determination and quantification of IPDA in the test item formulation samples.
Details on mating procedure:
For this experiment, sexually mature, purebred female artificially inseminated rabbits (New Zealand White) were used.
Duration of treatment / exposure:
Day 6 to 28 of gestation
Frequency of treatment:
Once daily
Duration of test:
On gestation day 29 (one day before the calculated date of parturition) the dams were laparotomised.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control group
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
low dose group
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
intermediate dose group
Dose / conc.:
75 mg/kg bw/day (nominal)
Remarks:
high dose group
No. of animals per sex per dose:
20 pregnant females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were selected in agreement with the Sponsor based on the results of two dose-range-finding studies which were conducted in pregnant rabbits with IPDA dosed at 5, 25 and 50 mg/kg b.w./day (LPT Study No. 35502) and in non-pregnant rabbits with IPDA dosed at 75 and 150 mg/kg b.w./day (LPT Study No. 36299).
LPT Report No. 35502 revealed no test item-related influences on any of the treatment groups.
In LPT Study No. 36299 the high dose group (150 mg IPDA/kg b.w./day) was noted with a distinctly reduced food consumption and body weight and therefore the animals were prematurely sacrificed on TD8. Necropsy revealed a severely reddened gastric mucosa in 3 of 3 examined animals.
The animals of the low dose group (75 mg IPDA/kg b.w./day) were noted with a reduced food consumption but no test item-related change for the body weight. A reddened gastric mucosa and ulcers were noted for 2 of 3 low dose animals during necropsy.
Based on complete lethality at 150 mg IPDA/kg b.w./day, 75 mg IPDA/kg b.w./day
were considered as the maximal tolerated dose (MTD).

The oral route was selected since this has been proven to be suitable for the detection of a toxicological hazard.
Maternal examinations:
Dated and signed records of all activities relating to the day to day running and maintenance of the study within the animal units as well as to the group observations and examinations outlined in the Study Plan were recorded in the appropriate documentation. In addition, observations relating to the individual animals made throughout the study were recorded.

Clinical signs
Animals were individually observed at least once daily for any signs of behavioural changes, reaction to treatment or illness.
Immediately after administration any signs of illness or reaction to treatment were recorded. In case of changes the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m.
On Saturdays and Sundays, animals were checked regularly from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m.
Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.

Viability
Further checks were made early in each working day and again in the afternoon to look for dead or moribund animals. This would have allowed post mortem exami-nations to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed except that the final check was carried out at approximately midday.
Animals showing signs of abortion or premature delivery were sacrificed on the same day. Fetuses obtained this way were examined for abnormal development whenever possible.

Body weight
The weight of each rabbit was recorded on the day of delivery (used for randomiza-tion), followed by daily weighings starting on GD 6 - always at the same time of the day. The body weight gain was also calculated in intervals (i.e. day 6-9, 9 12, 12-15, 15-18, 18-21, 21-24, 24-27, 27-29). Furthermore, the carcass weight and the net weight gain from day 6 are given.

Food and drinking water consumption
The quantity of food consumed by each rabbit was recorded. Food intake per rabbit (g/rabbit/day) was calculated using the total amount of food given to and left by each rabbit in each group on completion of a treatment day.

Necropsy
One day before the calculated parturition, i.e. on gestation day 29, all surviving rabbits were sacrificed by lethal intravenous injection of 300 mg Pentobarbi-tal/kg b.w. and exsanguinated.
In order to check for possible drug effects, a dissection with macroscopic examina-tion of the internal organs and placentae of the dams was carried out on the day of sacrifice.
After ventral midline incision and skin reflection, the ovaries and uteri were removed; the gravid uteri (in toto) were weighed. A macroscopic examination of all subcutaneous tissues and internal organs of the dams was carried out. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal, the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumi-nation. The liver and the kidneys were examined. Any abnormalities in the appear-ance and size of the gonads, adrenal glands, uterus, intraabdominal lymph nodes and accessory reproductive organs were recorded.
In case of macroscopical findings, the affected maternal tissues were preserved in 10% buffered formalin for possible future histopathological examinations.
Ovaries and uterine content:
Corpora lutea
- number per dam
- absolute number per group
- mean per group

Implantations
- number per dam
- distributions in the uterine horns
- absolute number per group
- mean per group

Resorptions
- number per dam
- distributions in the uterine horns
- absolute number per group
- mean per group
- mean % per group
- number of early resorptions (< 2 g)
- number of late resorptions (> 2 g)

Weight of placentae
- individual data per fetus
- mean per litter
- mean per sex and litter
- litter mean per group
- litter mean per sex and group

Weight of fetuses
- individual data per fetus (alive and dead)
- mean per litter
- mean per sex and litter
- litter mean per group
- litter mean per sex and group

Fetuses
- number per dam (alive)
- number per dam (dead)
- number of fetuses (alive and dead) per sex and dam
- sex ratio per litter/group
- distribution in the uterine horns
- absolute number of fetuses alive per group
- mean number of fetuses alive/dead per group

Runts
- number per dam/group


Malformed fetuses
- individual data per fetus
- type of malformation: number and incidence (%) per group and litter
- number of affected fetuses per group
Total malformation rate [%] = malformed fetuses per group x 100
fetuses per group



Fetuses with variations7
- individual data per fetus
- type of variation: number and incidence (%) per group and litter
- number of affected fetuses per group8
Total variation rate [%] = fetuses per group with variations x 100
fetuses per group



Fetuses with retardations7
- individual data per fetus
- type of retardation: number and incidence (%) per group and litter
- number of affected fetuses per group8
Total retardation rate [%] = fetuses per group with retardations x 100
fetuses per group




Indices of pre-implantation loss and post-implantation loss:

Calculation of group indices (see table 7-1)
Pre implantation
loss [%] = Corpora lutea (per group) - implantations (per group) x 100
Corpora lutea (per group)

Post implantation
loss [%] = Implantations (per group) - living fetuses (per group) x 100
Implantations (per group)

Calculation of mean indices per litter (see table 7-2 and A7)
Pre implantation
loss [%] = Sum of pre-implantation losses per litter in a group [%]
Number of litters in a group

Post implantation
loss [%] = Sum of post-implantation losses per litter in a group [%]
Number of litters in a group



Fetal examinations:
The fetuses were removed and the following examinations performed:
(a) Macroscopic inspection (gross evaluation) of the placentae for example for focal indurations.
(b) The number of fetuses (alive and dead) and placentae (location in the uterus and the assignment of the fetuses) was determined.
(c) Sex and viability of fetuses were determined. Animals are said to be viable when they are found alive (spontaneous breathing, spontaneous movement).
(d) Number and size of resorptions were determined.
(e) Corpora lutea in the ovaries, implantations and location of fetuses in the uterus were determined.
(f) The gravid uterus weight was determined.
(g) Weights of fetuses and weights of the placentae were determined (fetuses were considered as runts if their weight was less than 70% of the mean litter weight).
(h) All fetuses (dead and alive) were inspected externally for damages, especially for malformations .
(i) The fetuses were sacrificed with pentobarbital (60 mg/fetus, i.p.).

Each fetus was dissected:
The thorax and peritoneal cavity (without damage to ribs and sternum) were opened. Location, size and condition of the internal organs were determined and examined for abnormalities (e.g. liver, discolouration, situs inversus, alterations of urinary bladder, brain, lungs, cleft palate) of soft tissue.
The sex was determined.
The kidneys were removed and incised to check for damages (e.g. dilatation of the renal pelvis).
The abdominal organs were removed.
The diaphragm was carefully removed to check the position of the heart (left - right).
The thoracic organs were removed using surgical forceps; the heart was incised to check for damages.

The head was removed from 50% of the fetuses and fixed in BOUIN'S solution. An examination according to WILSON was carried out, inspecting the internal head structures (e.g. eyes).
The cranium was opened for the remaining 50% of the fetuses and the brain was removed for external inspection in toto.
The eviscerated fetuses (intact and headless bodies) were dehydrated in ethanol and cleared in potassium hydroxide solution. The skeleton was stained with Alizarin red (according to DAWSON). The skeletal system was examined (determi-nation of the number and type of retardations, variations as well as malfor-mations).
Statistics:
The statistical evaluation of the parametrical values was done by Provantis using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT and SHAPIRO-WILK test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT test (p ≤ 0.01 and p ≤ 0.05).
Non-parametrical data:
The statistical evaluation of non-parametrical values was done using the FISHER or Chi2 test:
FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01)
or
Chi2 test, n ≥ 100 (p ≤ 0.05 and p ≤ 0.01)

The respective calculations for the FISHER and Chi2 test were performed using Provantis (maternal macroscopic findings at necropsy or findings during the external macroscopic examination of the fetuses) or an internal computer program (e.g. findings during the fetal skeletal or soft tissue examination).
Note:
The statistical evaluation of the pre- and post-implantation index (per group) using the number of corpora lutea, implantation sites and/ or fetuses per group (see table 7-1 'Reproduction Data - Summary - Values per Group') was done using StatXact 4.0.1 software, as such a calculation is not possible in Provantis.
Indices:
Malformation rate, total variation rate, pre implantation loss index, post implantation loss index, viability index, retardations index
Historical control data:
LPT Background Data
Values of control groups (n = 18) and test groups (n = 55) not significantly influenced by any test compound; data taken from the years 2013 to September 2018
Clinical signs:
no effects observed
Description (incidence and severity):
No changes in behaviour, the external appearance or faeces were noted for the control group or the test item-treated groups (10, 25 or 75 mg IPDA/kg b.w./day).
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No premature deaths were noted for the control group and the treatment groups (10, 25 or 75 mg IPDA/kg b.w./day).
However, one dam of the control group, two dams of the intermediate dose group and two dams of the high dose group were prematurely sacrificed after abortion. Macroscopic post mortem examination revealed no test item-related findings.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
BODY WEIGHT
No test item-related changes in body weight were noted in the dams after oral treatment with 10 mg IPDA/kg b.w./day.
In the intermediate dose group (25 mg IPDA/kg b.w./day), a not statistically significant but constantly lower body weight compared to the control group was noted from GD 9 until study termination on GD 29 (at maximum 2.4% below the value of the control group). Due to this slight reduction, the effect was considered to be not test item-related and within the normal range of variation.
At 75 mg IPDA/kg b.w./day, a more pronounced and constant reduction in body weight was noted from GD 9 until study termination on GD 29 (statistically signifi-cant between GD 9 and GD 23 at p ≤ 0.05 or 0.01) in comparison to the control group (at maximum 5.5% below the value of the control group on GD 16, p ≤ 0.05). The effect is considered a combination of two statistically non-significant effects, namely the reduced gravid uterus weight and the slightly reduced carcass weight. However, this distinctly and constantly lower body weight was dose dependent and therefore considered to be test item-related.

BODY WEIGHT GAIN
No test item-related differences between the control group and the low dose group (10 mg IPDA/kg b.w./day) was noted for the body weight gain.
A slightly but not statistically significantly lower body weight gain was noted for the intermediate dose group (25 mg IPDA/kg b.w./day). However, as the body weight, this small reduction was considered to be within the normal range of variation.
According to the lower body weight of the high dose group (75 mg IPDA/kg b.w./day), also the body weight gain was slightly lower but not statistically significantly different from the control group.
However, the distinctly and constantly lower body weight gain was dose dependent and therefore considered to be test item-related and adverse.

NET BODY WEIGHT GAIN FROM DAY 6
No differences of toxicological relevance between the control group and the treatment groups (10, 25 or 75 mg IPDA/kg b.w./day) were noted for the net body weight gain (without gravid uterus) between GD6 and GD29.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No test item-related changes in food consumption were noted between the dams of the control group and the dams treated with 10 or 25 mg IPDA/kg b.w./day.
At 75 mg IPDA/kg b.w./day, a decreased food consumption was noted between GD8 and GD17 (at maximum 22.9% below the value of the control group on GD14 to GD15, p ≤ 0.05). This distinctly lower food consumption was considered to be test item-related and adverse (results see figure below).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test item-related changes in the consumption of drinking water were noted for the dams treated with 10, 25 or 75 mg IPDA/kg b.w./day by visual appraisal.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Behaviour, external appearance, faeces
No changes in behaviour, the external appearance or faeces were noted for the control group or the test item-treated groups (10, 25 or 75 mg IPDA/kg b.w./day).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Gravid uterus weight
No test item-related changes in the gravid uterus weight in comparison to the control group were noted for the dams of the low and the intermediate dose group (10 or 25 mg IPDA/kg b.w./day).
A slightly decreased mean value of the gravid uterus weight was noted at the high dose level (75 mg IPDA/kg b.w./day) (8.7% below the control value, statistically not significant). This finding was mainly due to the low uterine weight of animal no. 82 with a total implantation loss and was considered to be test item-related.

Carcass weight
No test item-related changes in the carcass weight in comparison to the control group were noted for the dams of all treatment groups (10, 25 or 75 mg IPDA/kg b.w./day).
A slightly reduced carcass weight was noted at the high dose level (3.3% below the value of the control group). The slight reduction was related to the reduction in body weight and considered to be test item-related.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related pathological findings were noted during macroscopic inspection of the dams treated with 10, 25 or 75 mg IPDA/kg b.w./day at necropsy.
In the low dose group there was one female with blck foci in the placenta and in the high dose group marked red discoloration in the stomach of one female was observed.
These single findings were considered to be spontaneous and not test item-related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related abortions were noted for the treatment groups (10, 25 or 75 mg IPDA/kg b.w./day).
However, one control dam, two intermediate dose dams and two high dose dams were noted with spontaneous abortions. As abortions are known to occur spontaneously in rabbits of this strain and age and an abortion was also noted in the control group, the abortions noted in the intermediate and high dose group were considered to be not test item-related. Furthermore, the number of abortions is still in the range of LPT background data (see table below).

Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Animal no. 82 of the high dose group showed a total post-implantation loss.
Control 8.9 % showed pre-implantation loss & 2.9 % animals showed post implantation loss
Low dose 2.0% showed pre-implantation loss & 1% showed post implantation loss
Medium dose 4.6 % showed pre-implantation loss & 3.8% showed post implantation loss
High dose 2.6% showed pre-implantation loss & 13.8% showed post implantation loss

More details in attached table.
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
The treatment with the test substance at 75 mg IPDA/kg b.w./day revealed increased incidences of early and total resorptions, and in consequence an increased percentage of post-implantation loss, which were statistically significantly different from the control.
A treatment related effect for the statistically significantly increased rate of early resorptions and the statistically significantly increased percentage of post-implantation loss, cannot totally be excluded, since both values were at the upper end of the LPT historical control data.
Although treatment related and adverse effects cannot totally be excluded, the main reason for those increases is animal no. 82 that showed a total post-implantation loss. When assessing the number of live fetuses the high dose group showed no remarkable difference to the control group (199 vs. 194 out of 20 animals with viable fetuses each). A fetotoxic effect of the test substance can be excluded, since no test item-related malformations, variations or retardations were evident.
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
No test item-related influence was noted on the number of resorptions for the dams of the low and intermediate dose groups (10 or 25 mg IPDA/kg b.w./day).
The treatment with the test substance at 75 mg IPDA/kg b.w./day revealed increased incidences of early and total resorptions, and in consequence an increased percentage of post-implantation loss, which were statistically significantly different from the control.
A treatment related effect for the statistically significantly increased rate of early resorptions and the statistically significantly increased percentage of post-implantation loss, cannot totally be excluded, since both values were at the upper end of the LPT historical control data.
Although treatment related and adverse effects cannot totally be excluded, the main reason for those increases is animal no. 82 that showed a total post-implantation loss. When assessing the number of live fetuses the high dose group showed no remarkable difference to the control group (199 vs. 194 out of 20 animals with viable fetuses each). A fetotoxic effect of the test substance can be excluded, since no test item-related malformations, variations or retardations were evident.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
No dead fetus was noted in the control group and in the intermediate and high dose groups (25 or 75 mg IPDA/kg b.w./day).
At 10 mg IPDA/kg b.w./day, dam no. 30 was noted with two dead fetuses (nos. 30-8 and 30-9). As the two dead fetuses were only noted for one dam and no dose response-relationship was present, the two dead fetuses were considered to be spontaneous and not test item-related. Furthermore, the incidence of 2 dead fetuses in one of the treatment groups is in the range of the LPT background data (see table below).
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
Control: From 30 mated and treated females in the control group 6 animals became not pregnant and 21 animals were pregnant. 3 animals were excluded and not examined.
Low dose group. From 30 animals 5 became not not pregnant and 20 animals were pregnant, 5 Animals were excluded from further examinations.
Medium dose group. From 34 mated and treated animals, 8 animals not became pregnatn and 22 animals were pregnant.
High dose group. From 34 mated and treated naimals 7 animals became not pregnant and 23 animals were pregnant.
Altogether in all dose groups including the control group, a poor pregnancy rate was observed for seasonal reasons. For details see also attached table.
Other effects:
no effects observed
Description (incidence and severity):
No test item-related differences for the placental weights were noted for any of the dosing groups (10, 25 or 75 mg IPDA/kg b.w./day) in comparison to the control group. The placental weights were within the normal variability.
Details on maternal toxic effects:
Mortality: No test item-related premature death was noted for the control group and the treatment groups (10, 25 or 75 mg IPDA/kg b.w./day).
Abortions: No test item-related abortions were noted for the treatment groups.
Clinical signs: No changes in behaviour and the external appearance were noted in the control group and the test item-treated groups.
Body weight and body weight gain: A test item-related reduced body weight was noted for the high dose dams treated with 75 mg IPDA/kg b.w./day from GD9 until study termination on GD29 (at maximum 5.5% below the value of the control group on GD16, p ≤ 0.05). Accordingly a lower body weight gain was noted for the high dose group (12.6% body weight gain for the high dose group compared to 14.1% in the control group).
Food and drinking water consumption: A test item-related decrease in food consumption was noted for the high dose group (75 mg IPDA/kg b.w./day) from GD8 to GD17 (at maximum 22.9% below the value of the control group on GD14 to GD15, p ≤ 0.05).
Necropsy findings: No test item-related observations were noted for the treatment groups during necropsy.
Uterus weight, carcass weight and net body weight gain:
At 75 mg IPDA/kg b.w./day a slight reduction in the gravid uterus weight was noted (8.7% below the control value, statistically not significant).
A slight reduction was noted for the carcass weight at the high dose level (3.3% below the control value, statistically not significant).

Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity see below
Key result
Dose descriptor:
LOAEL
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
pre and post implantation loss
early or late resorptions
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related influence was noted on the mean fetal weights after administration of 10, 25 or 75 mg IPDA/kg b.w./day.
No test item-related effect was noted for the number of runts. In total, 12 runts were noted at laparotomy: 5 runts were noted in the control group, 4 runts in the low dose group (10 mg IPDA/kg b.w./day), one runt in the intermediate dose group (25 mg IPDA/kg b.w./day) and two runts were noted in the high dose group (75 mg IPDA/kg b.w./day).
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
No dead fetus was noted in the control group and in the intermediate and high dose groups (25 or 75 mg IPDA/kg b.w./day).
At 10 mg IPDA/kg b.w./day, dam no. 30 was noted with two dead fetuses (nos. 30-8 and 30-9). As the two dead fetuses were only noted for one dam and no dose response-relationship was present, the two dead fetuses were considered to be spontaneous and not test item-related. Furthermore, the incidence of 2 dead fetuses in one of the treatment groups is in the range of the LPT background data.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in the dose groups (10, 25 or 75 mg IPDA/kg b.w./day) was comparable to the control fetuses. Slight differences observed were within the biological variability.
Sex ratio in study groups(male/female):
Control 0.88
Low dose 0.76
Medium dose 1.06
High dose 1.06
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No visible gross alteration (malformation or variation) was noted for fetuses of the low and intermediate dose groups (10 or 25 mg IPDA/kg b.w./day).
In the high dose group (75 mg IPDA/kg b.w./day), the fetus no. 84-9 was noted with multiple malformations in form of an absent upper jaw and cranial roof as well as a small brain and a raschisis in the neck region. This malformation complex confirmed the external observation and was considered to be not test item-related but spontaneous according to type and incidence.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No alterations in the form of malformations were noted during skeletal examina-tions of the fetuses according to DAWSON at the low and intermediate dose level (10 or 25 mg IPDA/kg b.w./day).
At the high dose level (75 mg IPDA/kg b.w./day), the fetus no. 84-9 was noted with a malformation of the skull in form of an absent cranium and maxilla as well as a partly ossified temporal bone and cervical vertebral arches being partly reduced in size and misaligned. However this single occurrence was considered to be sponta-neous and not test item-related.

The skeletal variations observed during examination according to DAWSON were related to the caudal vertebral bodies (fused), to the lumbar vertebral bodies (misaligned), to the thoracic vertebral arches (fused), to the sternum (sternebra(e) bipartite, fused, misaligned, misshapen) or the rib(s) (accessory 13th ribs (uni- or bilateral), short, less than 12 ribs ossified).
No test item-related influences were noted on the incidence of the above men-tioned skeletal variations in the treatment groups (10, 25 or 75 mg IPDA/kg b.w./day).

The retardations observed during skeletal examination (according to DAWSON) were related to the skull (incomplete ossification), the lumbar vertebral arches (reduced in size), the lumbar vertebral bodies (reduced in size), the sternum (sternebra(e) unossified, incompletely ossified or reduced in size), the talus (unossified), the thoracic vertebral arches (unossified) and the thoracic vertebral bodies (reduced in size or unossified).
No test item-related influences were noted on the incidence of the above men-tioned skeletal retardations in the treatment groups (10, 25 or 75 mg IPDA/kg b.w./day).
However, statistically significant increases were noted for the incidences of the incomplete ossification of the skull in the intermediate dose group (p ≤ 0.05) as well as for the incidences of unossified sternebra(e) and the total retardations in the low dose group (for both p ≤ 0.01).
As no dose response relationship was noted and the values were within the range of the LPT background data ; these 3 statistically significantly increased incidences were considered to be not test item-related.

For details see attached table.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A macroscopic internal examination was performed to detect gross alterations of the internal organs. No malformations or variations were noted during the internal examination of the fetuses of the control group and the dose groups (10, 25 or 75 mg IPDA/kg b.w./day). The gross inspection of the brain in 50% of the fetuses revealed no changes for any of the fetuses after opening of the cranium and removal of the brain.

No alterations in the form of malformations were noted during soft tissue examina-tions of the fetal head according to WILSON at any tested dose level (10, 25 or 75 mg IPDA/kg b.w./day).
No test item-related influence was noted in the number of soft tissue variations of the fetal head compared to the control at any tested dose level (10, 25 or 75 mg IPDA/kg b.w./day).
The observed and classified soft tissue variations that were noted during the examination of the fetal head according to WILSON were in the form of dilatations of the 4th cerebral ventricle, subdural haemorrhages in the meninges and haemor-rhages in the cerebrum as well as cystic or semicircular cystic areas in the cerebral hemisphere. No test item-related differences in the incidences of the observed variations of the fetal head were noted between the control group and the test groups.
The observed cystic or semicircular cystic areas in the cerebrum noted in altogeth-er 6 fetuses of the test item groups are known to be fixation-induced artefacts .
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
One abortion was noted for the control group (no. 1) and two abortions each were noted for the intermediate dose group (nos. 58 and 63) and the high dose group (nos. 76 and 90) treated with 25 or 75 mg IPDA/kg b.w./day). As abortions are known to occur spontaneously in rabbits of this strain and age, these single incidences were considered to be an incidental finding and hence, are judged to be not test item-related.
Two dead fetuses were noted for dam no. 30 (nos. 30-8 and 30-9) treated with 10 mg IPDA/kg b.w./day. As no dose dependence-relationship was noted and because of the low incidence, the occurrence of one dam with two dead fetuses was considered to be not test item-related.
No test item-related increase was noted for the incidence of runts at any tested dose level.
No test item-related malformations or variations were noted during the macro-scopic external examination and the macroscopic gross inspection of the internal organs at laparotomy and the skeletal examination according to DAWSON or the soft tissue examination of the fetal heads according to WILSON for the treatment groups (10, 25 or 75 mg IPDA/kg b.w./day).
Furthermore, the skeletal examination according to DAWSON revealed no test item-related retardations.
Key result
Dose descriptor:
NOAEL
Effect level:
> 75 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no prenatal developmental toxicity effects observed up to the highest tested dose
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table: Overview on abortions of the study animals

Group

Dam no.

Day of abortion

(GD)

Time of abortion

Day of sacrifice

Abortion rate

(Aborted animals / evaluated pregnant females; row 5 in text table 4-1)

1

Control

1

22

during the day

22

1 / 21 = 4.8%

2

10 mg/kg

-

-

-

-

0 / 20 = 0.0%

3

25 mg/kg

58

20

during the night

21

2 / 22 = 9.1%

63

26

during the night

27

4

75 mg/kg

76

26

during the night

27

2 / 23 = 8.7%

90

21

during the night

22

 

Table: Comparison of the abortion rate with theLPTbackground data.


Abortions

Abortion rates

in this study

(LPT Report 35503)

 

LPT Background Data#1-

Values of control groups (n = 18)

and test groups (n = 55)

not significantly influenced by any test compound;

data taken from the years

2013 to September 2018

 

[Mean value from the mean values of the groups used]

[Range of mean values of the individual groups used]

Abortion rate (%)

Control:

4.8

4.13 ± 3.81

0.0 - 9.5

control groups

Group 2:

0.0

Group 3:

9.1

3.32 ± 3.65

0.0 - 10.0

test groups

Group 4:

8.7

#1:

not audited by QAU

Table:Summary of the mean body weight gain from GD6 to GD29

Mean Body Weight Gain

Group

Time interval

Gestation day 6 - 29

(whole study period)

 

Gain in kg

Gain in %

Difference

to control %

Control

0.60

14.1

n.a.

Group 2 (10 mg/kg)

0.64

14.7

+6.6

Group 3 (25 mg/kg)

0.53

12.8

-10.8

Group 4 (75 mg/kg)

0.52

12.6

-12.8

 

Table:Comparison of the resorption rates and the post-implantation loss with the LPT background data.


Parameter

Values observed

in this study

(LPT Report 35503)

 

[fetal incidence in %

per group]

LPT Background Data#1-

Values of control groups (n = 18)

and test groups (n = 55)

not significantly influenced by any test compound;

data taken from the years

2013 to September 2018

 

[Mean value from the mean values of the groups used]

[Range of mean values of the individual groups used]

Total resorptions

mean % per dam

Control:

0.3

0.70 ± 0.39

0.1 - 1.3

control groups

Group 2:

0.0

Group 3:

0.4

0.79 ± 0.41

0.0 - 1.6

test groups

Group 4:

1.5** #2

Early resorptions

mean % per dam

Control:

0.1

0.39 ± 0.30

0.0 - 1.0

control groups

Group 2:

0.0

Group 3:

0.3

0.45 ± 0.32

0.0 - 1.3

test groups

Group 4:

1.0** #2

Late resorptions

mean % per dam

Control:

0.3

0.33 ± 0.22

(0.0 - 0.7)

control groups

Group 2:

0.0

Group 3:

0.1

0.36 ± 0.23

(0.0 - 0.9)

test groups

Group 4:

0.4

Post-implantation loss

% per group

Control:

2.9

7.71 ± 4.66

(0.5 - 16.5)

control groups

Group 2:

1.0 #3

Group 3:

3.8

8.83 ± 4.36

(1.0 - 17.4)

test groups

Group 4:

13.8** #3

Post-implantation loss

mean % per dam

Control:

2.0

7.85 ± 4.92

(0.4 - 16.9)

control groups

Group 2:

1.0

Group 3:

3.9

8.71 ± 4.30

(1.0 - 15.7)

test groups

Group 4:

15.1** #2

#1:

not audited by QAU

#2*/**:

(p ≤ 0.05/0.01) Dunnett test

#3*/**:

(p ≤ 0.05/0.01) Chi2test

Table: :Summary of the reproduction data

Parameter

Group 1

Control

(n=20)

Group 2

10 mg/kg

(n=20)

Group 3

25 mg/kg

(n=20)

Group 4

75 mg/kg

(n=21) #1

 

Corpora lutea

total

mean per dam

 

225

11.3

199

10.0

195

9.8

231

11.0

 

Implantation sites

total

mean per dam

 

205

10.3

195

9.8

186

9.3

225

10.7

 

Total resorptions

total

mean per dam

 

6

0.3

0

0.0

7

0.4

31

1.5 ** #1

 

Early resorptions

total

mean per dam

 

1

0.1

0

0.0

5

0.3

22

1.0 ** #1

 

Late resorptions

total

mean per dam

 

5

0.3

0

0.0

2

0.1

9

0.4

 

Fetuses (alive + dead)

total

mean per dam

 

199

10.0

195

9.8

179

9.0

194

9.7 #²

 

Live fetuses

total

mean per dam

 

199

10.0

193

9.7

179

9.0

194

9.7 #²

 

Dead fetuses

total

mean per dam

 

0

0.0

2

0.1

0

0.0

0

0.0 #²

 

Post-implantation loss

per group

 

2.9

1.0

3.8

13.8 **

 

[%]

mean per dam

 

2.0

1.0

3.9

15.1 **

 

 

 

 

 

* / **

Significantly different from the controls at p ≤ 0.05 / p ≤ 0.01, Chi2test, only performed for the group values of the pre- and post-implantation loss (see table7-1).

 

* / **

Significantly different from the controls at p ≤ 0.05 / p ≤ 0.01, Dunnett test, performed for the mean values per group (see table7-2).

 

#1

A total post-implantation loss (9 early resorptions) was noted in 1 of 21 litters.

 

#2

The calculation was based on 20 litters with viable fetuses (see table7-2).

 

Table :Comparison of the number of dead fetuses with the LPT background data.


Parameter

Values observed

in this study

(LPT Report 35503)

 

 

LPT Background Data#1-

Values of control groups (n = 18)

and test groups (n = 55)

not significantly influenced by any test compound;

data taken from the years

2013 to September 2018

Dead fetuses

(total number per group)

Control:

0

0 - 1 #2

control groups

Group 2:

2

Group 3:

0

0 - 5

test groups

Group 4:

0

Dead fetuses

(mean per dam)

Control:

0.0

0.01 ± 0.02 #4

(0.0 - 0.1) #3

control groups

Group 2:

0.1 ± 0.4

Group 3:

0.0

0.06 ± 0.09

(0.0 - 0.4)

test groups

Group 4:

0.0

#1:

not audited by QAU

#2

Range of the total number of dead fetuses in the control groups.

#3

Range of the mean number of dead fetuses per dam in the cpntrol groups.

#4

Mean value of the mean numbers of dead fetuses per dam in the control groups.

Table: :Overview of the runts per group

IPDA

Group 1: Control

Group 2:

10 mg/kg

Group 3:

25 mg/kg

Group 4:

75 mg/kg

Dam no.

Runt

Fetus no.

Dam no.

Runt

Fetus no.

Dam no.

Runt

Fetus no.

Dam no.

Runt

Fetus no.

7

6 m

40

5 m

56

10 f

81

7 m

7 f

7 f

-

-

120

5 f

12

5 f

44

5 f

-

-

-

-

18

2 f

8 f

-

-

-

-

7 f

-

-

-

-

-

-

Table :Background data of statistically significant incidences of skeletal retardations


Skeletal retardations

Values observed

in this study

(LPT Report 35503)

 

[fetal incidence in %

per group]

LPT Background Data#1-

Values of control groups (n = 18)

and test groups (n = 55)

not significantly influenced by any test compound;

data taken from the years

2013 to September 2018

 

[fetal incidence in % per group]

[Mean value ±SD (range)]

Sternebra(e)

unossified

Control:

24.1

12.8 ± 7.8

2.2 - 25.7

control groups

Group 2:

38.5 ** #²

Group 3:

19.0

13.5 ± 7.8

4.0 - 39.0

test groups

Group 4:

25.3

Skull

incompletely ossified

Control:

0.0

0.37 ± 0.85

0.0 - 3.4

control groups

Group 2:

1.0

Group 3:

1.7 * #²

0.53 ± 0.84

0.0 - 2.9

test groups

Group 4:

1.5

Total fetal skeletal

retardations

Control:

68.3

56.4 ± 14.9

(20.7 - 77.1)

control groups

Group 2:

80.0 ** #2

Group 3:

66.5

53.6 ± 13.9

(25.2 - 80.5)

test groups

Group 4:

73.7

#1:

not audited by QAU

#2:

Considered to be not test item-related as withinLPTbackground data range.

*/**:

(p ≤ 0.05/0.01) Fisher or Chi2- test

Conclusions:
Under the conditions of the study, 3-Aminomethyl-3,5,5-trimethylcyclo-hexanamine did not show any teratogenic potential.
Executive summary:

Under the conditions of the study, IPDA did not show any teratogenic potential.In this prenatal developmental toxicity study according to OECD 414 (version 2001), the test item 3-Aminomethyl-3,5,5-trimethylcyclo-hexanamine was administered orally to 128 inseminated female rabbits at dose levels of 0, 10, 25 or 75 mg/kg b.w./day from the 6th to 28th day of pregnancy. One group served as control group, in which the animals received the vehicle (purified water) without test substance. The body weight of the 5-month-old animals at day 6 of gestation was 3.75 kg to 4.68 kg. 20 dams per dose group were examined and evaluated at the end of the study.

 

Examination of the dams         

Mortality:                       No test item-related premature death was noted for the control group and the treatment groups (10, 25 or 75 mg IPDA/kg b.w./day).

Abortions:                     No test item-related abortions were noted for the treatment groups.

Clinical signs:                  No changes in behaviour and the external appearance were noted in the control group and the test item-treated groups.

Body weight:                 A test item-related reduced body weight was noted for the high dose dams treated with 75 mg IPDA/kg b.w./day from GD9 until study termination on GD29 (at maximum 5.5% below the value of the control group on GD16, p ≤ 0.05). Accordingly, a lower body weight gain was noted for the high dose group (12.6% body weight gain for the high dose group compared to 14.1% in the control group).

Food and DW

Consumption:                A test item-related decrease in food consumption was noted for the high dose group (75 mg IPDA/kg b.w./day) from GD8 to GD17 (at maximum 22.9% below the value of the control group on GD14 to GD15, p ≤ 0.05).

 

Necropsy findings:        No test item-related observations were noted for the treatment groups during necropsy.

Uterus weight, carcass weight and net body weight gain:           

At 75 mg IPDA/kg b.w./day a slight reduction in the gravid uterus weight was noted (8.7% below the control value, statistically not significant).

A slight reduction was noted for the carcass weight at the high dose level (3.3% below the control value, statistically not significant).

Summary table:Overview of the animals (total numbers)

IPDA

Group 1

Control

Group 2

10 mg/kg

Group 3

25 mg/kg

Group 4

75 mg/kg

1

Mated and treated

females

30 #

30 #

34 #

34 #

2

Not examined females (excluded/spare animals)

3

5

4

4

3

Non-pregnant females

6

5

8

7

4

Deceased animals

none

none

none

none

5

Evaluated pregnant

females

21

20

22

23

6

Evaluated dams without viable fetuses (total post implantation loss)

none

none

none

1

7

Evaluated dams with abortion

1

none

2

2

8

Evaluated litters with

viable fetuses

20

20

20

20

 

#      Further animals were included to yield 20 evaluable litters with viable fetuses.

        A poor pregnancy rate was observed for seasonal reasons.

 

Examination of the fetuses      

In the high dose group (75 mg IPDA/kg b.w./day), a test item-related increase was noted for the number of early resorptions (22 early resorptions in the high dose group in comparison to 1 early resorption in the control group). Accordingly, also the post-implantation loss was increased in the high dose group (13.8% in the high dose group compared to 2.9% in the control group).

No test item-related deaths of fetuses were noted.

No test item-related increase was noted for the incidence of runts in the test item groups in comparison to the control group.

Sex distribution:             No test item-related differences were noted.

Fetal weights:                No test item-related influence on the fetal weights was noted for the treatment groups.

Placental weights:         No test item-related differences were noted.

Fetal alterations            

Malformations:             No test item-related malformation was noted during the external examination at laparotomy, the gross inspection of the internal organs, the skeletal examination according to DAWSON and the soft tissue examination of the fetal head according to WILSON.

Variations:                      The external examination at laparotomy, the gross inspection of the internal organs, the skeletal examination according to DAWSON and the soft tissue examination of the fetal head according to WILSON revealed no test item-related variations.

Retardations:                 Examination according to DAWSON revealed no test item-related delays in the ossification.

 

 

Analysis of test item formulations

The measured actual concentrations of the test item in the test item vehicle-mixtures were between 102.7% and 106.5% of the nominal concentrations, indicating correctly prepared, stable and homogeneous formulations.

Conclusion

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 25 mg IPDA/kg b.w./day for the dams:

No test item-related premature death was noted for any of the dose groups.

For the high dose group (75 mg IPDA/kg b.w./day), a reduction was noted for the body weight or the body weight gain. Also a decrease was noted in food consumption at 75 mg IPDA/kg b.w./day.

No changes in behaviour, external appearance or faeces were noted for the treatment groups.

No test item-related pathological findings were noted during necropsy for the treatment groups.

At 75 mg IPDA/kg b.w./day, a slight decrease was noted for the gravid uterus and the carcass weight.

 The no-observed-adverse effect level (NOAEL) was above 75 mg IPDA/kg b.w./day for the fetuses and 25 mg IPDA/kg b.w./day for the rate of early resorptions/postimplantation loss:

At the maternotoxic dose level of 75 mg IPDA/kg b.w./day (reduced body weight, reduced body weight gain, reduced carcass weight and reduced food consumption), an increased number of early resorptions and accordingly an increased post-implantation loss were noted. Due to the increased number of early resorptions, the total resorption rate was slightly above the LPT historical control data (up to 1.3). The early resorption rate and the percentages of post-implantation loss were at the upper range but still within the LPT historical control data.

No test item-related influence was noted on the mortality or body weight of the fetuses in the treatment groups.

No test item-related deaths of the fetuses and no test item-related malformations, variations or retardations were noted.

 Under the conditions of the study, IPDA did not show any teratogenic potential.

 

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-06-05 - 2001-06-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
1981
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
- Source: Charles River Laboratories, L'Arbresle (France)
- Age: 10-11 weeks
- Weight at study initiation: 206-301, mean 245 g. Mean weights in the four groups were similar.
- Housing: single
- Diet: Pelleted maintenance diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 50 +/- 20 %
- Air changes (per hr): 12 cycles/hour
- Photoperiod (hrs dark / hrs light): 12h/12h
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
- Vehicle: Water purified by reverse osmosis
- Concentration in vehicle: 1, 5, or 25 g/l
- Total volume applied: 10 ml/kg bw/treatment
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analyses demonstrated satisfactory stability and agreement between nominal and actual concentrations of the test material.
Details on mating procedure:
Females were mated at the breeder's facilities. The day of confirmed mating (detection of a vaginal plug) was designated as day 0 post-coitum.
Duration of treatment / exposure:
day 6 to day 19 post-coitum inclusive
Frequency of treatment:
daily
Duration of test:
until day 20 post-coitum
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control group
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
low dose group
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
intermediate dose group
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
high dose group
No. of animals per sex per dose:
24 (females only); only the first 20 pregnant females were taken into consideration for fetal examinations
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were determined based on the results of a preliminary study
Maternal examinations:
PARAMETERS ASSESSED DURING STUDY:
- Mortality: daily (twice during treatment period)
- Clinical signs: daily (twice during treatment period)
- Body weight gain: Days 2, 6, 9, 12, 15, 18, 20
- Food consumption: Cumulative for days 2-6; 6-9; 9-12; 12-15; 15-18; 18-20
ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): Principal thoracic and abdominal organs, gross evaluation of placentas
Ovaries and uterine content:
- Examination of uterine content: Weight of gravid uterus, number or corpora lutea, number and distrubution of implantation sites (or uterine scars), number and distrubution of early and late resorptions, number and distribution of dead and live fetuses
Fetal examinations:
- Examination of fetuses: weight, sex, detailed external examination (all); soft tissue including all organs and structures of head, neck, thorax, and
abdomen (one half); skeleton including bone structures and cartilage of head, spine, rib cage, pelvis, limbs (other half)
Statistics:
- Group mean values +/- standard deviation (one-way analysis of variance and Dunnett test): Maternal body weight and food consumption, fetal body weight and number of corpora lutea, implantations, fetuses and resorptions
- Proportions (Fisher exact probability test): Pre-implantation loss, post-implantation loss, fetal findings
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The only treatment-related clinical sign was ptyalism in most females of the 250 mg/kg bw/day group from day 11, 12, 13, or 14 until hysterectomy. Loud breathing and presence of material in the mouth, probably due to hold-up in the esophagus, were recorded in 4 females. These observations might be the consequence of the corrosive properties of the test item (pH between 10 and 12).
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Mortality and day of death: One female of the high-dose group was found dead on day 16. This death was attributed to an effect of hold-up in the esophagus following gavage. No other mortalities were observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Not affected in low and mid dose groups. A significant decrease (-35 %) was observed in the high-dose group after the first three days of treatment. Thereafter, the body weight was similar to that of the controls. The net body weight gain was also significantly lower (-25 %) at this dose level.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Not affected in low and mid dose groups; a slightly significant decrease was observed in the high dose group during the treatment period.
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):
No treatment related findings were observed at any dose level. An exception are whitish foci on the lung in the decedent female.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
No abortion in any group
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No treatment related findings were observed at any dose level.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
No total resorption in any group
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Number pregnant per dose level: Control 24; low- and high-dose 23; mid-dose 22
Other effects:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
- Mortality and day of death: One female of the high-dose group was found dead on day 16. This death was attributed to an effect of hold-up in the
esophagus following gavage. No other mortalities were observed.
- Clinical signs: The only treatment-related clinical sign was ptyalism in most females of the 250 mg/kg bw/day group from day 11, 12, 13, or 14 until hysterectomy. Loud breathing and presence of material in the mouth, probably due to hold-up in the esophagus, were recorded in 4 females. These observations might be the consequence of the corrosive properties of the test item (pH between 10 and 12).
- Number pregnant per dose level:
Control 24; low- and high-dose 23; mid-dose 22
- Number aborting: No abortion in any group
- Number of resorptions: No total resorption in any group
- Pre and post implantation loss: No treatment related findings were observed at any dose level.
- Body weight gain: Not affected in low and mid dose groups. A significant decrease (-35 %) was observed in the high-dose group after the first three days of treatment. Thereafter, the body weight was similar to that of the controls. The net body weight gain was also significantly lower (-25 %) at
this dose level.
- Food/water consumption: Not affected in low and mid dose groups; a slightly significant decrease was observed in the high dose group during the
treatment period.
- Gross pathology incidence and severity: No treatment related findings were observed at any dose level. An exception are whitish foci on the lung in
the decedent female.
Key result
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Fetal body weight changes:
no effects observed
Description (incidence and severity):
No treatment related findings were observed at any dose level.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No treatment related findings were observed at any dose level.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No treatment related findings were observed at any dose level.
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
No treatment related external malformations or variations were observed at any dose level.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No statistically significant treatment related skeletal malformations or variations were observed at any dose level.
There was a statistically insignificant increase in fetal incidence of incomplete ossification of the 5th sternebra in the 250 mg/kg bw/day group (106/134 fetuses = 79.1%, p<0.01 were affected vs. 88/130 = 67.7% in control group). In the same group there was a statistically nonsignificant increase in fetal incidence of incomplete ossification of the rib(s) (9/137 fetuses = 6.7 % vs. 2/130 = 1.5% in control group. When ossification was incomplete, cartilage was generally present, demonstrating that the skeletal variations recorded corresponded to slight fluctuations in the time of ossification rather than being a persistent alteration. In conclusion, these findings were considered to be incidental and of no toxicological significance.
Visceral malformations:
no effects observed
Description (incidence and severity):
No treatment related soft tissue malformations or variations were observed at any dose level.
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- Litter size and weights: No treatment related findings were observed at any dose level.
- Sex ratio: No treatment related findings were observed at any dose level.
- External abnormalities: No treatment related external malformations or variations were observed at any dose level.
- Soft tissue abnormalities: No treatment related soft tissue malformations or variations were observed at any dose level.
- Skeletal abnormalities: No statistically significant treatment related skeletal malformations or variations were observed at any dose level.
There was a statistically insignificant increase in fetal incidence of incomplete ossification of the 5th sternebra in the 250 mg/kg bw/day group
(106/134 fetuses = 79.1%, p<0.01 were affected vs. 88/130 = 67.7% in control group).
In the same group there was a statistically nonsignificant increase in fetal incidence of incomplete ossification of the
rib(s) (9/137 fetuses = 6.7 % vs. 2/130 = 1.5% in control group.
When ossification was incomplete, cartilage was generally present, demonstrating that the skeletal variations recorded corresponded to slight
fluctuations in the time of ossification rather than being a persistent alteration. In conclusion, these findings were considered to be incidental and of no toxicological significance.
Key result
Dose descriptor:
NOAEL
Effect level:
> 250 mg/kg bw/day
Basis for effect level:
other: embryotoxicity
Key result
Dose descriptor:
NOAEL
Effect level:
> 250 mg/kg bw/day
Basis for effect level:
other: fetotoxicity
Key result
Dose descriptor:
NOAEL
Effect level:
> 250 mg/kg bw/day
Basis for effect level:
other: teratogenicity
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
No teratogenic or embryofetotoxic effects were recorded at any dose level.
Conclusions:
Isophorone diamine did not show any teratogenic or embryofetotoxic effects in a gavage study with rats performed in accordance with OECD TG 414 (2001) up to and including the highest tested dose level of 250 mg/kg bw/day. The NOEL for maternal toxicity was 50 mg/kg bw/day, effects at 250 mg/kg bw/day were reduced food consumption and reduced body weight gain. The NOAEL for developmental toxicity is > 250 mg/kg bw/day
Executive summary:

Based on the results of a dose finding study, three groups of 24 mated female Sprague-Dawley rats received 3-aminomethyl-3,5,5-trimethylcyclohexylamine by daily oral administration (gavage) at 0 (water = control), 10, 50 and 250 mg/kg/day from day 6 to day 19 post-coitum inclusive. On day 20 post-coitum, the dams were sacrificed and subjected to macroscopic examination. The study was designed according to OECD TG 414.There was no treatment-related death in any of the dams. Clinical signs were not observed, except for ptyalism in most females of the 250 mg/kg/day group (from day 11, 12, 13 or 14 post-coitum until hysterectomy; effect not considered as adverse). Loud breathing and hold-up in the esophagus were recorded in 4 females of this group and a significantly lower body weight gain (-35%) was recorded after the first three days of treatment. Thereafter, the body weights were similar to that of the controls. Over the whole treatment period, the difference remained slight (-10 %, not statistically significant). The net body weight gain was also significantly lower at this dose-level (-25 %) when compared to the control group. In the 250 mg/kg/day group, a significant decrease in food consumption was recorded during the treatment period (-7%), with a more marked effect during the first three days of treatment (-21 %).Abortions or total resorptions were not observed in any of the groups, nor were there any macroscopic findings that were ascribed to treatment with the test item. No treatment related effects were observed on pre- or post-implantation loss, fetal weight or sex-ratio. With respect to the fetuses, no test item related external, soft tissue or skeletal malformations or variations were detected. There was an increase in fetal incidence of incomplete ossification of the 5th sternebra in the 250 mg/kg bw/day group (106/134 fetuses = 79.1 %, p < 0.01 were affected vs. 88/130 = 67.7 % in control

group, statistically insignificant on a fetus/litter basis). Because these findings are of low concern and occur only in the presence of maternal toxicity, they are considered to be secondary. In the same group there was a statistically nonsignificant increase in fetal incidence of incomplete ossification of the rib(s) (9/137 fetuses = 6.7 % vs. 2/130 = 1.5 % in control group. When ossification was incomplete, cartilage was generally present, demonstrating that the skeletal variations recorded corresponded to slight fluctuations in the time of ossification rather than being a persistent alteration. In conclusion, these findings were considered to be incidental and of no toxicological significance. The NOEL for maternotoxicity was 50 mg/kg/day and the NOAEL for embryonic development was >250 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
75 mg/kg bw/day
Species:
other: rat and rabbit
Quality of whole database:
Klimisch 1 (guideline studies)
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

Studies in animals

 

Partly cited from SIAR for SIAM 18 (Paris, April 2004):

"Based on the results of a dose finding study, three groups of 24 mated female Sprague-Dawley rats received 3-aminomethyl-3,5,5-trimethylcyclohexylamine by daily oral administration (gavage) at 0 (water = control), 10, 50 and 250 mg/kg/day from day 6 to day 19 post-coitum inclusive. On day 20 post-coitum, the dams were sacrificed and subjected to macroscopic examination. The study was designed according to OECD TG 414.There was no treatment-related death in any of the dams. Clinical signs were not observed, except for ptyalism in most females of the 250 mg/kg/day group (from day 11, 12, 13 or 14 post-coitum until hysterectomy; effect not considered as adverse). Loud

breathing and hold-up in the esophagus were recorded in 4 females of this group and a significantly lower body weight gain (-35%) was recorded after the first three days of treatment. Thereafter, the

body weights were similar to that of the controls. Over the whole treatment period, the difference remained slight (-10 %, not statistically significant). The net body weight gain was also

significantly lower at this dose-level (-25 %) when compared to the control group. In the 250 mg/kg/day group, a significant decrease in food consumption was recorded during the treatment

period (-7%), with a more marked effect during the first three days of treatment (-21 %). Abortions or total resorptions were not observed in any of the groups, nor were there any macroscopic

findings that were ascribed to treatment with the test item. No treatment related effects were observed on pre- or post-implantation loss, fetal weight or sex-ratio. With respect to the fetuses, no

test item related external, soft tissue or skeletal malformations or variations were detected. There was an increase in fetal incidence of incomplete ossification of the 5th sternebra in the 250 mg/kg

bw/day group (106/134 fetuses = 79.1 %, p < 0.01 were affected vs. 88/130 = 67.7 % in control group, statistically insignificant on a fetus/litter basis). Because these findings are of low concern

and occur only in the presence of maternal toxicity, they are considered to be secondary. In the same group there was a statistically nonsignificant increase in fetal incidence of incomplete

ossification of the rib(s) (9/137 fetuses = 6.7 % vs. 2/130 = 1.5 % in control group. When ossification was incomplete, cartilage was generally present, demonstrating that the skeletal variations recorded corresponded to slight fluctuations in the time of ossification rather than being a persistent alteration. In conclusion, these findings were considered to be incidental and of no toxicological significance."

The NOEL for maternotoxicity was 50 mg/kg/day and the NOAEL for embryonic development was >250 mg/kg/day (CIT, 2002).

 

ECHA requested to perform the OECD 414 study in a second species.

In this prenatal developmental toxicity study, the test item IPDA was administered orally to female rabbits at dose levels of 10, 25 or 75 mg/kg b.w./day from the 6th to 28th day of pregnancy.

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 25 mg IPDA/kg b.w./day for the dams:

No test item-related premature death was noted for any of the dose groups. For the high dose group (75 mg IPDA/kg b.w./day), a reduction was noted for the body weight or the body weight gain. Also a decrease was noted in food consumption at 75 mg IPDA/kg b.w./day. No changes were noted for the drinking water consumption. No changes in behaviour, external appearance or faeces were noted for the treatment groups. No test item-related pathological findings were noted during necropsy for the treatment groups. At 75 mg IPDA/kg b.w./day, a slight decrease was noted for the gravid uterus and the carcass weight.

The no-observed-adverse effect level (NOAEL) was above 75 mg IPDA/kg b.w./day for the fetuses and 25 mg IPDA/kg b.w./day for the rate of early resorptions/postimplantation loss: At the maternotoxic dose level of 75 mg IPDA/kg b.w./day (reduced body weight, reduced body weight gain, reduced carcass weight and reduced food consumption), an increased number of early resorptions and accordingly an increased postimplantation loss were noted. Due to the increased number of early resorptions, the total resorption rate was slightly above the LPT historical control data (up to 1.3). The early resorption rate and the percentages of post-implantation loss were at the upper range but still within the LPT historical control data. No test item-related influence was noted on the mortality or body weight of the fetuses in the treatment groups. No test item-related deaths of the fetuses and no test item-related malformations, variations or retardations were noted.

Under the conditions of the study, IPDA did not show any teratogenic potential.

The treatment with the test substance at 75 mg IPDA/kg b.w./day revealed increased incidences of early and total resorptions, and in consequence an increased percentage of post-implantation loss, which were statistically significantly different from the control. A treatment-related effect for the statistically significantly increased rate of early resorptions and the statistically significantly increased percentage of postimplantation loss cannot totally be excluded since both values were at the upper end of the LPT historical control data.

Although treatment-related and adverse effects cannot totally be excluded, the main reason for those increases is animal no. 82 that showed a total post-implantation loss. When assessing the number of live fetuses, the high dose group showed no remarkable difference to the control group (194 vs. 199 fetuses out of 20 dams with viable fetuses each). A feto-toxic effect of the test substance can be excluded, since no test item-related malformations, variations or retardations were evident.

 

 

Toxicity to reproduction: other studies

Description of key information

no other studies available

Justification for classification or non-classification

Because of the results of all available studies the substance isophorone diamine is not classified according to CLP regulation (1272/2008).