Isopropylcyclohexane

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-10-14 to 2015-11-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

Regulation (EC) No. 440/2008 (May 30, 2008)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Isopropylcyclohexane

EC Number:

211-792-4

EC Name:

Isopropylcyclohexane

Cas Number:

696-29-7

Molecular formula:

C9H18

IUPAC Name:

(propan-2-yl)cyclohexane

Test material form:

liquid

Details on test material:

Characteristics: Colourless liquid
Content: 99.62%
Batch no. B1507
Stability / Expiry date May 2018 (at least 3 years)

Specific details on test material used for the study:

The test item was suspended in the vehicle to the appropriate concentration.

Test animals

Species:

rat

Strain:

other: CD / Crl:CD (SD)

Details on test animals or test system and environmental conditions:

- Source: Charles River Laboratories, Research Models and Services, Germany GmbH Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age: 60 days
- Weight at study initiation: 188.1 - 238.1 g.
- Housing: Except during the mating period, the dams were kept singly in MAKROLON cages
- Diet: Pelleted maintenance diet, ad libitum, Commercial ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany,
- Water: Tap water, ad libitum
- Acclimation period: 11 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 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:

other: Sesame oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Vehicle: Sesame oil
- Concentration in vehicle: 100 mg / 300 mg /1000 mg/kg b. w. /day
- Total volume applied: 2 ml/kg bw/treatment
- Test item preparation: The test item formulations were freshly prepared every day.
The test item was suspended in the vehicle to the appropriate concentration 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

Details on analytical verification of doses or concentrations:

The results indicated correctly prepared and homogenised test item vehicle mixtures, which were stable for at least 24 hours.
In detail, the concentrations of test item Concentration, immediately after preparation and at study termination was 99.9% - 109.9%. Stability left at
room temperature for 8h or 24 h was 99.9% - 105.5%. Homogeneity at start of administration, during administration and before administration to the last animal was 93.9% - 104.2%.

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 presence 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:

- 1 mating day
- 14 administration days from gestation day 6 to 20

Frequency of treatment:

daily

Duration of test:

up to gestation day 21

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Treated animals: Groups 1 - 4: 25 animals per group
Evaluated litters: Groups 1 - 4: 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 study in pregnant rats (LPT study no. 32262).
- In the dose-range finding study, test item was administered to pregnant female rats at dose levels of 100, 300 or 1000 mg/kg b.w./day orally,
by gavage, once daily from gestation day 6 to 20.
- Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was above 1000 mg test item/kg b.w./day for the dams.
No test item-related influence was noted on behaviour or external appearance, body weight as well as intake of food and drinking water of the dams at any tested dose level. No premature deaths were noted. Necropsy revealed no changes for the dams of any test group.
- The no-observed-adverse-effect level (NOAEL) for the fetal organism was also above 1000 mg test item/kg b.w./day. No dead fetuses, no
malformations and no test item-related variations were noted at any of the tested dose levels. No embryotoxic properties of the test item were noted up to the highest tested dose of
1000 mg test item/kg b.w./day by oral administration.
- For the present rat embryotoxicity study (LPT Report No. 32263), dose levels of 100, 300 or 1000 mg test item/kg b.w./day, administered
orally, by gavage once daily (at a constant volume of 2 mL/kg b.w./day) from the 6th to the 20th day of pregnancy, were selected in agreement
with the Sponsor.

Examinations

Maternal examinations:

PARAMETERS ASSESSED DURING STUDY:
- Clinical signs: Individual animals were observed daily for behavioural changes, reaction to treatment, or illness
- Viability:Checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals
- Body weight: 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
- Body weight change: 0-3, 3 6, 6-9, 9-12, 12-15, 15-18 and 18-21, furthermore the carcass weight and the net weight change from day 6 is given
- Food consumption: Quantity of food consumed by each rat was recorded daily
- Water consumption: Daily monitoring by visual appraisal of the drinking water bottles was maintained throughout the study

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC):
- Macroscopic examination of the internal organs and placentae of the dams was carried out on the day of sacrifice (gestation day 21). The
following target organs of the dams were weighed: The gravid uteri (in toto), Heart, kidney (2), and liver.

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
- mean per litter
- mean per group
- mean per sex and group

Fetuses
- number per dam (alive)
- number per dam (dead)
- number of fetuses (alive + 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/femal ratio (alive + dead)

Runts
- number per dam
- mean per group

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

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

Fetuses with variations
- individual data per fetus
- type of variation
- 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 :

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



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

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) 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, varia-tions 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 an
internal computer program (e.g. findings during the fetal skeletal or soft tissue examination).

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.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At the intermediate dose level (300 mg test item/kg b.w./day) slight to extreme salivation and a slightly reduced motility were noted (see table below).
At the high dose level (1000 mg test item/kg b.w./day) slight to extreme salivation and pale faeces were noted for most or all dams of this group. Furthermore, for some dams a moderately to extremely reduced motility and an increased water consumption were noted additionally.
Start and ending of the symptoms:
Salivation started 0 to 5 min after administration of the test item and ended 20 to 60 min after administration.
Reduced motility was noted 0 - 5 min after administration of the test item and ended 5 to 60 min after administration.
See table below.

Mortality:

no mortality observed

Description (incidence):

No test item-related premature deaths were noted for the dams treated orally with 100, 300 or 1000 mg Hydrocumene/kg b.w./day from gestation day 6 to 20.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

However, the 3-day intervals of body weight gain revealed a delay in body weight gain after the start of treatment between gestation days 6 and 9 for the dams of the high dose group (1000 mg test item/kg b.w./day) (5.5 g in comparison to 13.8 g in the control group; statistically not significant). Further delays in body weight gain were noted for the dams of the high dose group for the whole study (gestation day 0 to 21(statistically not significant)) and for the period after the start of treatment (gestation days 6 to 21(p ≤0.05))
However, as the body weight gain in % from gestation day 6 - 21 for the high dose group was well within the limits of the LPTbackground data, the significant reduction of body weight gain is considered as not test item-related.
These delays in body weight gain were due to a slightly reduced gravid uterus weight and a marginally reduced carcass weight noted for the dams of the high dose group.

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 of the low and the intermediate dose group (100 or 300 mg test item/kg b.w./day).
At the high dose level (1000 mg test item/kg b.w./day) statistically significant (p ≤ 0.01) reductions in food consumption were noted on all 3 test days between gestation day 6 and 9 (23.4 to 36.4% below the values of the control group).
Thereafter, the amount of food consumption noted for the dams of the high dose group was again in the range of the control group.
This moderate reduction in food consumption, noted in the high dose group, is considered as test item-related.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

An increased drinking water consumption was noted for 8 of 20 high dosed dams by visual appraisal on 6 or 8 test days and considered as test item-related

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):

The heart, the liver and the kidneys were weighed and no test item-related differences for the absolute and the relative organ weights were noted between the control group and the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).
However, slight but statistically significant (p ≤ 0.05 / 0.01) increases were noted for the relative and the absolute organ weights of the liver and the kidneys in the high dose group and occasionally in the intermediate or the low dose group (see tables below). The small increases in the relative and the absolute organ weights are in the normal range of variability and not considered as test item-related. In case of the more significantly in-creased liver weight in the high dose group, the participation of an increased workload, caused by the high amount of administered test item, must also be considered as a trig-ger for the increased liver and kidney weights.

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

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 the carcass weight of the control dams and the dams of the low and the intermediate dose groups (100 or 300 mg test item/kg b.w./day).
A slight and statistically not significant reduction in the gravid uterus weight by 6.0% in comparison to the control group was noted for the dams of the high dose group (1000 mg test item/kg b.w./day).

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, pre- and post-implantation indices) were noted between the dams of the control group and the dams of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day)
See table below.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

see above

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

see above

Early or late resorptions:

no effects observed

Description (incidence and severity):

see above

Dead fetuses:

no effects observed

Description (incidence and severity):

see above

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

see above
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): see above

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

see above

Details on maternal toxic effects:

see table below

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No test item-related differences of the placental and fetal weights were noted between the control group and the test item-treated groups (100, 300 or 1000 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): No test item-related influence on the number of runts was noted for any of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).
In detail, 2 runts were noted in the high dose group, which is in the range of normal variability.

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 (100, 300 or 1000 mg test item/kg b.w./day).

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No test item-related differences between the ratio of male and female fetuses were noted between the control group and the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No test item-related differences of the placental and fetal weights were noted between the control group and the test item-treated groups (100, 300 or 1000 mg test item/kg b.w./day).

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related macroscopically visible external observations (malformations or variations) were noted for the fetuses of the test item-treated groups (100, 300 or 1000 mg test item/kg b.w./day) during the macroscopic inspection at laparotomy.
However, external observations (malformations - in 1 case combined with variations) were noted for altogether 4 fetuses (1 of the control group, 1 of the intermediate and 2 of the high dose group) (see table below).

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related skeletal malformations were noted for the fetuses of the test item-treated groups (100, 300 or 1000 mg test item/kg b.w./day) during the skeletal examination according to DAWSON.
The external malformations and variations, which were noted for altogether 3 fetuses (1 of the intermediate and 2 of the high dose group) during the macroscopic examination at laparotomy, were confirmed by skeletal malformations for those 2 fetuses that were examined according to DAWSON (see table below).

Skeletal variations were noted for the ribs (wavy) and the sternebrae (misaligned to a slight degree, misshapen).
No test item-related differences were noted in the incidences of the observed variations between the control group and the test item-treated groups. Hence, no test item-related variations were noted during the skeletal examination according to DAWSON in any of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).

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 lumbar vertebral bodies (bipartite or dumbbell-shaped), the lumbar vertebral arches (unossified), the caudal vertebral bodies (only one body ossified or all bodies unossified), the os ischii (incompletely ossified or unossified), the os pubis (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 differences in the incidences of the observed delays in skeletal ossification were noted between the control group and the test item-treated groups (100 or 300 mg test item/kg b.w./day).
A statistically significant (p ≤ 0.01) increase was noted in the incidence of incompletely ossified thoracic vertebral bodies (thoracic vertebral body/bodies dumbbell-shaped) for the fetuses of the high dose group (1000 mg test item/kg b.w./day).
All other observed retardations with statistically significantly different incidences in com-parison to the control group are also not considered as test item-related, as they showed no dose response relationship.

Visceral malformations:

no effects observed

Description (incidence and severity):

No test item-related malformations were noted for the fetuses of the control group and the fetuses of the test item-treated groups (100, 300 or 1000 mg test item/kg b.w./day) during the soft tissue examination according to WILSON.

Of the 3 fetuses with macroscopically visible external malformations, one fetus was examined by WILSON. The examination according to WILSON confirmed the external malformations that were noted at the head of the fetus during the macroscopic external examination.
Variations (noted during the examination according to WILSON)
During the examination of the internal organs and tissues according to WILSON variations were noted in the meninges (subdural haemorrhage), the cerebral ventricle (dilatation of the 4th ventricle), the kidneys (uni- or bilateral dilatation of the renal pelvis, malpo-sition) and the liver (haemorrhagic focus/foci).
No test item-related differences in the incidences of the observed variations were noted between the control group and the test item treated groups (100, 300 or 1000 mg test item/kg b.w./day).

Other effects:

no effects observed

Description (incidence and severity):

Gross inspection of the organs and tissues at laparotomy
The macroscopic inspection of the organs and tissues for gross alterations at laparotomy revealed no test item-related observations for the fetuses of the control group and the fetuses of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).

Details on embryotoxic / teratogenic effects:

ABSTRACT and ASSESSMENT of fetal alterations
No test item-related malformation was noted during the macroscopic inspection at laparotomy (including an external inspection and a gross inspection of the organs and tissues), the skeletal examination according to DAWSON and the soft tissue examination according to WILSON.
However, 4 fetuses with external malformations and/ or variations were noted (1 in the control group, 1 at the intermediate and 2 at the high dose level). The external malformations noted for the fetuses of the intermediate and the high dose group were confirmed by findings during the examinations of the fetuses according to DAWSON or WILSON. In all cases the incidence was in the background range of LPT and not con-sidered as test item-related.
No test item-related variation was noted during the macroscopic inspection at laparotomy, the skeletal examination according to DAWSON and the soft tissue examination according to WILSON.

A test item-related retardation was noted for the fetuses of the high dose group in the form of an increased incidence of incompletely ossified thoracic vertebrae (thoracic ver-tebrae body/bodies dumbbell-shaped). However, this finding is not regarded to be ad-verse.

In summary, none of the fetuses revealed a test item-related malformation or variation. A test item-related retardation was noted in the form of delays in the ossification of one or more thoracic vertebral bodies.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: prenatal developmental

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

 Examination of the dams

Summary of animals examined

Test item	Group 1 Control	Group 2 100 mg/kg	Group 3 300 mg/kg	Group 4 1000 mg/kg
Treated dams	25	25	25	25
Not pregnant dams	1	0	1	1
Dams without viable fetuses	0	0	0	0
Dams with early delivery	0	0	1	0
Not examined dams (spare animals)	4	5	3	4
Evaluated litters	20	20	20	20

Summary of animals evaluated

Test item dose in mg/kg b.w./day, p.o.		Animal nos. of mated rats	Animal nos. of rats with evaluable litters at laparotomy	Dams not pregnant (animal nos.)	Dams with total implantation loss (animal nos.)	Reserve animals, not examined (animal nos.)
Control		1 - 25	2 - 21	1	none	22 - 25
100		26 - 50	26 - 45	none	none	46 - 50
300		51 - 75	51 - 54 56 - 65 67 - 72	55	none	66 # 73 - 75
1000		76 - 100	77 - 96	76	none	97 - 100
#:	Dam no. 66 delivered early on gestation day 21.

Behaviour, external appearance, faeces

Test item-related observations in group 3 (300 mg test item/kg b.w./day)
Observation		Affected dams	Incidence (number of test days the observation was noted for the individual dams)	First to last seen (gestation days)
Salivation		6 of 20	Mostly one or 2 test days (only dam no. 54 showed salivation on 8 consecutive test days).	12 -19
Reduced Motility		1 of 20	Noted only on one test day.	19

Test item-related observations in group 4 (1000 mg /kg b.w./day)
Observation		Affected dams	Incidence (number of test days the observation was noted for the individual dams)	First to last seen (gestation days)
Salivation		16 of 20	On 2 up to 10 test days (mostly on 9 or 10 test days).	6 - 20
Reduced Motility		3 of 20	On one or 2 test days.	6 - 18
Pale faeces		20 of 20	On 7 up to 14 test days (mostly on 7, 8 or 9 test days).	8 - 21
Increased water consumption		8 of 20	On 6 or 8 test days.	13 - 21

Reproduction data of the dams

Parameter				Group 1 Control (n=20)	Group 2 100 mg/kg (n=20)	Group 3 300 mg/kg (n=20)	Group 4 1000 mg/kg (n=20)
Corpora lutea			total mean per dam	271 13.6	270 13.5	283 14.2	270 13.5
Implantation sites			total mean per dam	260 13.0	260 13.0	274 13.7	260 13.0
Resorptions			total mean per dam	8 0.4	3 0.2	14 0.7	5 0.3
Early resorptions			total mean per dam	8 0.4	2 0.1	12 0.6	5 0.3
Late resorptions			total mean per dam	0 0.0	1 0.1	2 0.1	0 0.0
Live fetuses			total mean per dam	252 12.6	257 12.9	260 13.0	255 12.8
Dead fetuses			total	0	0	0	0
Pre-implantation loss [%]			per group ##1 mean per dam	4.1 3.8	3.7 3.8	3.2 2.9	3.7 4.0
Post-implantation loss [%]			per group ##2 mean per dam	3.1 2.9	1.2 1.0	5.1 5.3	1.9 1.9
	*/**:	p ≤ 0.05/ 0.01 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.
	##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.

Macroscopic inspection of the fetuses at laparotomy

Fetuses with external observations (nottest item-related)
Group		Fetus	Observation	Classification
Control		3-3	Cleft palate	Malformation
Group 3: 300 mg/kg		67-8 #1	Head with multiple malformations: Encephalocele, Anophthalmia (left eye), Ablepharia (right eye), Protruding tongue	Malformation
Group 4: 1000 mg/kg		93-3 #2	Fetus with multiple malformations: Shortened trunk, Spina bifida (approx. 3/4 parts of the back), Encephalocele with partly prolapsing brain	Malformation
			Flexure of the fore- and hindlimbs	Variation
		94-10 #3	Proboscis	Malformation
#1:	The external multiple malformations at the head of fetus no. 67-8 were confirmed during the examination according to WILSON.
#2:	The shortened trunk and the spina bifida were confirmed by multiple malformations of the vertebral column noted during the examination according to DAWSON.
#3:	The observation of proboscis was confirmed by the examination according to DAWSON. Skeletal examination according to DAWSON

Fetuses with skeletal malformations
Group		Fetus	Observation
Group 4: 1000 mg/kg		93-4 #1	Multiple malformations: Mandible and maxilla reduced in size Cervical vertebral bodies absent or not discernible Thoracic and lumbar vertebral bodies reduced in size, misaligned or bipartite Cervical, thoracic and lumbar vertebral arches misaligned or partly fused (counting was not possible) Only 8 ribs on the left side and 12 ribs on the right side (the first and the eight left ribs were reduced in size and thickened)
		94-10 #2	Proboscis
#1:	The malformations of the vertebral column were confirmed by a shortened trunk and a spina bifida, which were noted during the external macroscopic examination at laparotomy.
#2:	Proboscis was also noted during the macroscopic external examination at laparotomy for the fetus 94-10.

Applicant's summary and conclusion

Conclusions:

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 300 mg test item/kg b.w./day for the dams.
The no-observed-adverse-effect level (NOAEL) for the fetal organism was 1000 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.

Executive summary:

Aim of the study is the examination of the influence of the 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 100, 300 or1000 mg/kg b.w./day from the 6th to 20th day of pregnancy.

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 300 mg test item/kg b.w./day for the dams.

None of the dams died prematurely.

Noteworthy signs of toxicity were noted at 1000 mg test item/kg b.w./day in the form of salivation, an increased motility, pale faeces and an increased water consumption. Food consumption was transiently reduced at 1000 mg Hydrocumene/kg b.w./day on the first 3 days after the start of dosing by up to 36%.

No test item-related influence on the body weight and the body weight gain was noted.

The macroscopic inspection of the organs and tissues of the dams and the weighing of the heart, the liver and the kidneys of the dams at necropsy revealed no test item-related differences.

 

The no-observed-adverse-effect level (NOAEL) for the fetal organism was 1000 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 were noted.

The examination of the fetuses revealed no test item-related malformations or variations.

At the slightly maternal toxic level of 1000 mg test item/kg b.w./day a slightly increased incidence of retardations was noted in the form of incompletely ossified thoracic vertebral bodies. However, this finding is not regarded to be adverse.