Hexane, 1,6-diisocyanato-2,2,4(or 2,4,4)-trimethyl-

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-01-02 to 2017-02-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted January 22, 2001

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 30, 2008

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,2,4(or 2,4,4)-Trimethylhexane-1,6-diisocyanate

EC Number:

915-277-1

Cas Number:

32052-51-0

Molecular formula:

C11H18N2O2

IUPAC Name:

2,2,4(or 2,4,4)-Trimethylhexane-1,6-diisocyanate

Details on test material:

Trimethylhexandiisocyanat produced by Evonik, Batch No.: 1135051503

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ORGANISMS: 
- Species: Rat
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Strain: CD / Crl: CD(SD)
- Age: 59 days
- body weight: 203.3 - 271.0 g
- Diet: ad libitum, Commercial diet ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water: ad libitum
- Acclimatisation period: 5 days
-Housing: Except during the mating period, the dams were kept singly in MAKROLON cages
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °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.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

ADMINISTRATION: 
- Frequency: once daily, day 6 to 20 of gestation
- Dose volume: 2 ml/kg b.w.
- Dose: 0, 100, 300, 1000 mg/kg/bw
- Animals: 25 female rats/group, 20 litters per group
DOSAGE PREPARATION:
- The test item formulations were freshly prepared every day.
- The test item was suspended in the vehicle to the appropriate concentrations and was administered orally at a constant volume once daily from the 6th to the 20th 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.
- The male rats for mating remained untreated.

Analytical verification of doses or concentrations:

yes

Remarks:

The analytical method was successfully re-validated by LPT: Analytical Method for the Determination of test item in Formulations with subsequent HPLC UV Detection

Details on analytical verification of doses or concentrations:

For the analysis of the test item-vehicle formulations, samples of approximately 2 mL were taken at the following times and stored at -20°C or colder until analysis at LPT:
At start of dosing
- Analysis of stability and concentration: Immediately after preparation of the formula-tions as well as after 8 and 24 hours storage of formulations at room temperature. (3 samples/test item group), Number of samples: 3 x 3 = 9,
- Homogeneity: At the start of dosing, during (middle) admin-istration and before dosing to the last animal of the test item group. (3 samples/test item group), Number of samples: 3 x 3 = 9,

At the 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 dose level group. (1 sample/test item group), Number of samples: 1 x 3 = 3

Sum of all samples: 21

Details on mating procedure:

Sexually mature ('proved') male rats 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 together in one cage during the dark period. Each morning a vaginal smear was taken to check for the pres-ence of sperm. If findings were negative, mating was repeated with the same partner. The day on which sperm was found was considered as the day of conception (day 0 of pregnancy). This procedure was repeated until enough pregnant dams were available for all groups.
Rats which did not become pregnant were excluded from the analysis of the results and replaced by other animals.
A post-mortem negative staining according to SALEWSKI was carried out in the replaced animals in order to confirm the non-pregnancy status.

Duration of treatment / exposure:

From gestation day 6 until gestation day 20

Frequency of treatment:

Once daily

Duration of test:

On gestation day 21, all rats were euthanized by carbon dioxide (CO2) inhalation and laparotomised.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Low dose

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Intermediate dose

Doses / concentrations 4

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

HIgh dose

No. of animals per sex per dose:

25 female rats/dose , orally dosed with 0, 30, 100 or 300 mg test item/kg b.w.
Evaluated litters: 20 litters per 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 a dose-range-finding study for a prenatal developmental toxicity (LPT study no. 34043).
In this dose-range finding study, test item was administered to pregnant female rats at dose levels of 30, 100 or 300 mg/kg b.w./day orally, by gavage, once daily from gestation day 6 to 20.
No premature deaths were noted. Slight to marked adverse effects were noted at 300 mg test item/kg b.w./day in form of salivation, reduced body weight, reduced food consumption and macroscopic changes in the stomach (thickened mucosa or haemor-rhagic foci).
At 300 mg test item/kg b.w./day, one dam was noted with an increased post-implantation loss.
No embryotoxic properties (no dead fetuses, no malformations and no test item-related variations) were noted at any of the tested dose levels.

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.

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.


Body weight
The weight of each rat was recorded on day 0 of gestation (the day of detection of a positive mating sign), followed by daily weighing - always at the same time of the day.
The body weight gain was calculated in intervals (i.e. day 0-3, 3 6, 6-9, 9-12, 12-15, 15-18 and 18-21), for the whole study (gestation day 0 - 21) and for the period after the start of dosing (gestation day 6 to gestation day 21). Furthermore the carcass weight and the net weight gain from day 6 is given.
These values are stated in the report.
These measurements were also used for calculating the daily amount of test item to be administered.

Food and drinking water consumption
The quantity of food consumed by each rat was recorded daily. Food intake per rat (g/rat/day) was calculated using the total amount of food given to and left by each rat 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. Dehydration of the dams was avoided.

EXAMINATIONS (NECROPSY), Examination of the dams
Dissection technique and evaluation of the animals:
On gestation day 21, the rats were laparotomised under ether narcosis. The ovaries and the uteri of the dams were removed; the gravid uteri (in toto) were weighed. In order to check for possible test item 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. In case of macroscopical findings, the affected maternal tissues were preserved in 7% 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
- early resorptions < 2 mm
- Late resorptions > 2 mm

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

Weight of fetuses
- individual data per fetus (alive and dead)
- mean per 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
- distribution in the uterine horns
- absolute number of fetuses alive per group
- mean number of fetuses alive per group
- mean % of fetuses alive per group
- male/female ratio (alive and dead)

Runts
- number per dam
- mean per group

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

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

Fetuses with variations
- type of variation
- individual data per fetus
- number and incidence (%) per group and litter

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

Fetuses with retardations
- type of retardation
- individual data per fetus
- number and incidence (%) per group and litter

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)] / 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 (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 or abnormal appearance (e.g. size, colour, shape).
(b) The number of fetuses (alive and dead) and placentae (location in uterus and assignment to the fetus) 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) 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).
(g) All fetuses (dead and alive) were inspected externally for damages, especially for malformations .
(h) The fetuses were sacrificed by an ether atmosphere.
(i) Examination of fetuses and determination of number and kind of retardations, variations or malformations:
1) 50% of the number of fetuses in each litter were examined for skeletal anomalies. The thorax and peritoneal cavity (without damage to ribs and sternum) were opened and the location, size and condition of the internal organs were determined.
Then the skeleton was double-stained with Alcian blue for the examination of cartilage and with Alizarin red to reveal ossifications (according to DAWSON). The skeletal system was examined (determination of the number and type of retardations, variations as well as malformations).
2) The remaining 50% of the number of fetuses in each litter were examined for soft tissue anomalies. Body sections were made and examined according to WILSON.
The fetuses were allocated to the evaluation of DAWSON or WILSON on an alternating basis.

Statistics:

Parametrical data:
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 ≤ 0.01 (p ≤ 0.05 and p ≤ 0.01)

The respective calculations for the FISHER and Chi2 test were performed using Provan-tis (maternal macroscopic findings at necropsy or findings during the external macro-scopic 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 Re-production Data - Summary - Values per Group) was done using StatXact 4.0.1 software, as such a calculation is not possible in Provantis.

Significantly different data are indicated in the summary tables of the result sections of the report.

Indices:

Fertility Index, , Viability Index, Resorption Index, Pre-Implantation Loss Index, Post-Implantation Loss Index, Runts Index, Variation Index,
Number of litters having abnormalities, Number of abnormalities per litter

Historical control data:

LPT Background Data, see Appendix 4
Summarized results of the 59 last embryotoxicity studies in Sprague-Dawley rats (Charles River Deutschland GmbH) performed at LPT in the years 2000 to July 2016

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No test item-related changes in behaviour, the external appearance or the faeces that were considered to be of toxicological relevance were noted in any of the treatment groups (30, 100 or 300 mg test item/kg b.w./day).
Changes in behaviour and external appearance that were noted in the intermediate and high dose group (100 or 300 mg test item/kg b.w./day).
At 100 mg test item/kg b.w./day, a haemorrhagic nose/snout and breathing sounds were noted for dam 63 from GD 19 to 21. As only one dam was affected the observations of the intermediate dose group were considered to be incidental.
At 300 mg test item/kg b.w./day, slight to moderate salivation (5 of 20 animals) and haemorrhagic nose/snout (1 of 20) were noted for the dams that survived until study termination. As mentioned above, the prematurely deceased dam no. 90 was noted with extreme salivation and gasping on the day before it was found dead on GD 13.
Haemorrhagic nose/snout was noted only once and therefore was considered to be incidental.
Gasping was noted for the prematurely deceased dam no. 90 on the day before it was found dead. Therefore, gasping was considered to be a premortal symptom rather than an adverse effect of the test item.
Salivation was noted in 6 of 21 dams and therefore was considered to be test item-related.

Start and duration of reduced motility
All observations of salivation for the high dose group (300 mg test item/kg b.w./day) started immediately to 5 minutes post administration and disap-peared within 20 to 60 minutes after administration.

Mortality:

mortality observed, treatment-related

Description (incidence):

No premature deaths were noted in the control group and in the low and intermediate dose groups (30 or 100 mg test item/kg b.w./day).
Dam no. 90 of the high dose group (300 mg test item/kg b.w./day) was found dead in the morning of GD 13. Dam no. 90 was noted with gasping and severe salivation as pre-mortal symptom as well as with a reduced food consumption and a decreased body weight and body weight gain before death. Necropsy revealed changes of the stomach in form of a dilated, thin and translucent stomach with the mucosa in detachment and a few haemorrhagic foci.
The death was considered to be test item-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight
No test item-related differences in body weight were noted between the dams of the control group and the low and intermediate dose groups (30 or 100 mg test item/kg b.w./day).
At 300 mg test item/kg b.w./day, a constantly lower body weight compared to the control group was noted from GD 11 onwards until the end of the study. As a result, statistically significant decreased values for the body weight in comparison to the control group were noted on GD 13 and in the time interval from GD 16 until the end of the study on GD 21 (at maximum 7.4% below the value of the control group on GD 18, p ≤ 0.05).
As the body weight of the high dose group was constantly below the value of the control group, the reduced body weight was considered to be test item-related.

Body weight gain
No test item-related difference for the body weight gain compared to the control group was noted for the animals treated with 30 or 100 mg test item/kg b.w./day.
At 300 mg test item/kg b.w./day, a distinctly lower body weight gain compared to the control group was noted in the 3-day interval between GD 9 and GD 12 (63.5% be-low the value of the control group, p ≤ 0.01). This decrease was mainly due to 3 of 21 dams including the moribund dam no. 90. Furthermore, distinctly decreased values for body weight gain were noted in the 3-day intervals between GD 6 to 9 and GD 15 to 18 (at maximum 31.1% below the value of the control group, not significant). As a result, the body weight gain was statistically significant decreased in comparison to the control group for the treatment period (GD 6 - GD 21) and for the whole study period (GD 0 - GD 21). Therefore, the decreased body weight gain was considered to be test item-related.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related differences were noted between the control group and the low and intermediate dose groups (30 or 100 mg test item/kg b.w./day).
At 300 mg test item/kg b.w./day, a transient reduction in food consumption was noted during the time intervals between GD 9 and GD 12 (at maximum 17.5% below the value of the control group between GD 11 and GD 12, p ≤ 0.01). Furthermore, a lower food consumption was noted from GD 17 to GD 18 (18.0% below the value of the control group, not significant) and from GD 18 to GD 19 (17.7% below the value of the control group, p ≤ 0.01). This reduction in food consumption normalized until study termination.
As reductions in food consumption were only transient, the reduced food consumption was considered to be not adverse

Food efficiency:

not specified

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

no effects observed

Description (incidence and severity):

No test item-related changes in drinking water consumption were noted between the dams of the control group and the dams of the treatment groups 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:

effects observed, non-treatment-related

Description (incidence and severity):

Gravid uterus and carcass weight
No test item-related differences were noted between the gravid uterus weight and car-cass weight of the control dams and the dams of the low and intermediate dose group (30 or 100 mg test item/kg b.w./day).
At 300 mg test item/kg b.w./day, there was no difference in the gravid uterus weight compared to the control group but a reduction was noted for the carcass weight (8.8% below the value of the control group, p ≤ 0.01).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related observations were noted for the dams of the control group and the dams of the treatment groups (30, 100 or 300 mg test item/kg b.w./day) during the macroscopic inspection of the organs and tissues.
However, one finding each was noted in the control (no. 17) and the intermediate dose group (no. 63) that is listed in the table below. Furthermore, the prematurely deceased dam no. 90 of the high dose group (300 mg test item/kg b.w./day) revealed several findings related to the stomach (dilatation with a thin and translucent stomach wall, mucosa in detachment and a few haemorrhagic foci with approx. 1 mm in diameter).
Since in the different dams different tissues were affected, these observations were considered to be spontaneous and not test item-related or due to the moribund condition of dam no. 90.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Body weight gain from gestation day 6
No test item-related differences for the absolute body weight gain and the net body weight gain between the control group and the low and intermediate dose group (30 or 100 mg test item/kg b.w./day) were noted between gestation day 6 and gestation day 21.
As already stated in section 8.4 'Body weight and body weight gain', a decreased absolute body weight gain (18.9% below the value of the control group, p ≤ 0.01) was noted in the high dose group (300 mg test item/kg b.w./day), which resulted from a decreased net body weight gain (78.0% below the value of the control group, p ≤ 0.01).

Details on results:

no findings

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation) were noted between the dams of the control group and the dams of the treatment groups (30, 100 or 300 mg test item/kg b.w./day). see table "any other iformation on results"

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation) were noted between the dams of the control group and the dams of the treatment groups (30, 100 or 300 mg test item/kg b.w./day). see table "any other iformation on results"

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation) were noted between the dams of the control group and the dams of the treatment groups (30, 100 or 300 mg test item/kg b.w./day). see table "any other iformation on results"

Early or late resorptions:

no effects observed

Description (incidence and severity):

No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation) were noted between the dams of the control group and the dams of the treatment groups (30, 100 or 300 mg test item/kg b.w./day). see table "any other iformation on results"

Dead fetuses:

no effects observed

Description (incidence and severity):

No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation) were noted between the dams of the control group and the dams of the treatment groups (30, 100 or 300 mg test item/kg b.w./day). see table "any other iformation on results"

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation) were noted between the dams of the control group and the dams of the treatment groups (30, 100 or 300 mg test item/kg b.w./day). see table "any other iformation on results"

Details on maternal toxic effects:

No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation) were noted between the dams of the control group and the dams of the treatment groups (30, 100 or 300 mg test item/kg b.w./day). see table "any other iformation on results"

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The placental and fetal weights showed no test item-related differences between the control group and the treatment groups (30, 100 or 300 mg test item/kg b.w./day).
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): The placental and fetal weights showed no test item-related differences between the control group and the treatment groups (30, 100 or 300 mg test item/kg b.w./day).

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 test item treated groups (30, 100 or 300 mg test item/kg b.w./day).
One runt was noted in the control group (no. 13-10) and one runt (no. 48-3) was noted in the low dose group (30 mg test item/kg b.w./day). This was within the normal range of variation.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No test item-related differences between the ratio of male and female fetuses (range: 0.93 - 1.05) were noted between the control group and the treatment groups (30, 100 or 300 mg test item/kg b.w./day).

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No macroscopically visible external alterations (malformations or variations) were noted for the fetuses of the treatment groups (30, 100 or 300 mg test item/kg b.w./day) during the macroscopic inspection at laparotomy.
Dam no. 13 of the control group was noted with 2 live twins at implantation site 10. Both of the female twins were alive and one twin (no. 13-10) was a runt. The observation of twins was considered to be spontaneous.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No skeletal malformations were noted for the fetuses of the control group and the test item-treated groups (30, 100 or 300 mg test item/kg b.w./day) during the skeletal examination according to DAWSON.

Skeletal variations
Skeletal variations were noted for the ribs (less than 13 ribs ossified, short, wavy or fused to a slight degree) and the sternebrae (bipartite, dumbbell-shaped, misaligned or fused to a slight degree).
No test item-related increase in the incidence of the observed skeletal variations in com-parison to the control group was noted for the fetuses of the treatment groups (30, 100 or 300 mg test item/kg b.w./day).
Observed variations with statistically significant incidences in comparison to the control group that were considered to be not test item-related are shown in the table on the following page.

Skeletal retardations
Retardations (delayed ossifications) were related to the skull (incomplete ossification of frontal, parietal, interparietal and/or supraoccipital areas), the hyoid (unossified), the sternum (sternebra(e) incompletely ossified, reduced in size or unossified), the thoracic vertebral bodies (bipartite or dumbbell-shaped), the caudal vertebral bodies (no or only one body ossified), the sacral vertebral bodies (unossified) the os pubis and the os ischii (incompletely ossified or unossified) and the metacarpalia and metatarsalia (absence of ossification in metacarpalia 2 to 5 and metatarsalia 2 to 5).
No test item-related increase in the incidence of skeletal retardations at 30, 100 or 300 mg test item/kg b.w./day was noted during skeletal examination according to DAWSON.
Observed retardations with statistically significant incidences in comparison to the control group that were considered to be not test item-related are shown in the table on the following page.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The macroscopic inspection of the organs and tissues for gross alterations at laparotomy revealed no malformations or variations for the fetuses of the control group and the fetuses of the treatment groups (30, 100 or 300 mg test item/kg b.w./day).

Soft tissue examination according to WILSON
Malformations
No malformations were noted for the fetuses of the control group and the fetuses of the treatment groups (30, 100 or 300 mg test item/kg b.w./day) during the soft tissue examination according to WILSON.
Variations
During the examination of the organs and tissues according to WILSON variations were noted for the kidneys (adrenals darkly discoloured, uni- or bilateral dilatation of the renal pelvis or malpositioned) and the liver (haemorrhagic focus/foci).
No test item-related differences and no statistically significant differences in the incidences of the observed variations were noted between the control group and the test item-treated groups (30, 100 or 300 mg /kg b.w./day).

Unclassified observations
A thoracic cavity filled with blood was noted for one fetus (no. 69-9) of the intermediate dose group (100 mg test item/kg b.w./day). This observation is a preparation induced artefact and not considered as test item-related.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Number of runts
One runt was noted in the control group (no. 13-10) and one runt (no. 48-3) was noted in the low dose group (30 mg test item/kg b.w./day). This was within the normal range of variation.

Details on embryotoxic / teratogenic effects:

No malformation and no test item-related variations were noted during the macroscopic inspection at laparotomy (including an external inspection and a gross inspection of the organs), the skeletal examination according to DAWSON and the soft tissue examination according to WILSON).
Also no test item-related retardations (delay in ossification) were noted in any of the treatment groups (30, 100 and 300 mg test item/kg b.w./day).

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

> 300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Reproduction data of the dams

Parameter				Group 1 Control (n=20)	Group 2 30 mg/kg (n=20)	Group 3 100 mg/kg (n=20)	Group 4 300 mg/kg (n=20)
Corpora lutea			total mean per dam	284 14.2	283 14.2	290 14.5	300 15.0
Implantation sites			total mean per dam	279 14.0	273 13.7	280 14.0	293 14.7
Resorptions			total mean per dam	7 0.4	5 0.3	10 0.5	6 0.3
Early resorptions			total mean per dam	5 0.3	3 0.2	7 0.4	6 0.3
Late resorptions			total mean per dam	2 0.1	2 0.1	3 0.2	0 0.0
Live fetuses			total mean per dam	273 #^3 13.7	268 13.4	270 13.5	287 14.4
Dead fetuses			total	0	0	0	0
Pre-implantation loss [%]			per group #^1 mean per dam	1.8 1.6	3.5 4.2	3.4 3.8	2.3 2.3
Post-implantation loss [%]			per group #^2 mean per dam	2.2 2.6	1.8 1.8	3.6 3.6	2.0 1.9
		Statistical analyses were performed for the mean values per dam using an ANOVA/DUNNETT test.
	#^1	The statistical comparison of the pre-implantation loss per group was done by comparing the values of implantation sites/corpora lutea of the test group with the ratio of implantation sites/corpora lutea of the control group using the Chi^2test (*/**: p ≤ 0.05/p ≤ 0.01).
	#^2	The statistical comparison of the post-implantation loss per group was done by comparing the values of live fetuses/implantation sites of the test group with the ratio of fetuses/implantation sites of the control group using the Chi^2test (*/**: p ≤ 0.05/p ≤ 0.01).
	#^3	Dam no. 13 was noted with live female twins (13-10 and 13-11).

Analysis of the test item-formulation

Parameter	Sampling / dealing	Range of % nominal concentration
Concentration	immediately after preparation	96.4% - 102.2%
	before administration to the last animal on test day 21	90.3% - 96.6%
Stability	immediately after preparation	96.4% - 102.2%
	8h after preparation	93.9% - 99.6%
	24h after preparation	95.1% - 100.9%
Homogeneity	before administration to the first animal	97.4% - 100.4%
	during administration to the animals	97.4% - 102.6%
	before administration to the last animal	96.2% - 102.0%

 

The measured actual concentrations of the test item in the test item vehicle-mixtures were between 90.3% and 102.6% of the nominal concentrations, indicating correctly prepared formulations.

 

Applicant's summary and conclusion

Conclusions:

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was above 100 mg test item/kg b.w./day for the dams.
At 300 mg test item/kg b.w./day, dam no. 90 was found dead in the morning of GD 13.
The no-observed-adverse-effect level (NOAEL) for the fetal organism was above 300 mg test item/kg b.w./day.
The reproductive parameters (number of implantation sites, number of resorptions and number of fetuses) were not influenced by the test item.
No dead fetuses, no malformations and no test item-related variations or retardations were noted.
Under the conditions of the study, test item did not show any teratogenic potential.

Executive summary:

The aim of this study was the examination of the influence of test item administered orally during the critical period of organogenesis and the fetal development (6th to 20th day of gestation) on the pregnant rat and the fetus.

 

In this prenatal developmental toxicity study, the test item was administered orally to female rats at dose levels of 30, 100 or 300 mg/kg b.w./day from the 6th to 20th day of pregnancy.

 Findings

Examination of the dams:
Mortality	No premature deaths were noted in the control group and in the low and intermediate dose group (30 or 100 mg test item/kg b.w./day). Dam no. 90 of the high dose group (300 mg test item/kg b.w./day), was found dead in the morning of GD 13. The death was considered to be test item-related.
Clinical signs	At 300 mg test item/kg b.w./day, test item-related signs of toxicity were noted in form of salivation in 6 of 21 animals.
Body weight and body weight gain	At 300 mg test item/kg b.w./day, a reduced body weight (statistically significant by p ≤ 0.05/0.01) was noted from GD 13 until the end of study (at maximum 7.4% below the value of the control group).
Food consumption	No toxicologically relevant differences were noted for the food consumption.
Drinking water consumption	No differences were noted.
Necropsy findings	No test item-related changes were noted during the macroscopic inspection of the dams at necropsy.
Uterus and carcass weights	At 300 mg test item/kg b.w./day, a reduced carcass weight (8.8% below the value of the control group, p ≤ 0.01) was noted. Accordingly, a reduction was noted for the net and absolute body weight gain from GD 6 (78% and 18.9% below the value of the control group, p ≤ 0.01).
Reproduction data	No test item-related influence was noted on the reproductive parameter (number of implantation sites, resorptions and fetuses).

Examination of the fetus:
Mortality	No dead fetuses were noted in any of the test groups.
Body weight of the fetuses and the placentae	No test item-related differences were noted between the control group and the treatment groups.
Fetal alterations
Malformations	No malformations were noted during the macroscopic examinations at laparotomy (external inspection and inspection of the organs and tissues for gross lesions), the skeletal examination according to DAWSON and the soft tissue examination according to WILSON.
Variations	The macroscopic examinations at laparotomy, the skeletal examination according to DAWSON and the soft tissue examination according to WILSON revealed no test item-related variations.
Unclassified observations	No test item-related unclassified observations were noted.
Retardations	No test item-related retardations (delays in ossification) were noted.
Analysis of test item formulations	The measured actual concentrations of the test item in the test item vehicle mixtures were between 90.3% and 102.6% of the nominal concentrations, indicating correctly prepared formulations.

Conclusion

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 100 mg test item/kg b.w./day for the dams.

At 300 mg test item/kg b.w./day, dam no. 90 was found dead in the morning of GD 13.

Treatment with 300 mg test item/kg b.w./day) led to salivation in 6 of 21 animals.

In the high dose group (300 mg test item/kg b.w./day) a reduced body weight and accordingly a decreased body weight gain was noted from GD 13 onwards until the end of the study.

No change in food consumption that was of toxicological relevance was noted in any treatment group.

No changes were noted in the macroscopic examination during laparotomy.

The no-observed-adverse-effect level (NOAEL) for the fetal organism was above 300 mg test item/kg b.w./day.

The reproductive parameters (number of implantation sites, number of resorptions and number of fetuses) were not influenced by the test item.

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

 

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