3-aminomethyl-3,5,5-trimethylcyclohexylamine

Developmental toxicity / teratogenicity

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-aminomethyl-3,5,5-trimethylcyclohexylamine

EC Number:

220-666-8

EC Name:

3-aminomethyl-3,5,5-trimethylcyclohexylamine

Cas Number:

2855-13-2

Molecular formula:

C10H22N2

IUPAC Name:

3-aminomethyl-3,5,5-trimethylcyclohexylamine

Test material form:

liquid

Details on test material:

3-aminomethyl-3,5,5-trimethylcyclohexylamine from Evonik, Batch: 18021307

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system 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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control group

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

low dose group

Doses / concentrations 3

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

intermediate dose group

Doses / concentrations 4

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.

Examinations

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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).

Effect levels (maternal animals)

1

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

2

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

early or late resorptions

food consumption and compound intake

organ weights and organ / body weight ratios

pre and post implantation loss

Results (fetuses)

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.

Effect levels (fetuses)

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

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

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) Chi^2test

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, Chi^2test, 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 Chi^2- test

Applicant's summary and conclusion

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.