A mixture mainly based on: 2,3-dihydro-6-(2-hydroxy-2-methyl-1-oxopropyl)-1,1,3-trimethyl-3-[4-(2-hydroxy-2-methyl-1-oxopropyl)phenyl]-1H-indene; 2,3-dihydro-5-(2-hydroxy-2-methyl-1-oxopropyl)-1,1,3-trimethyl-3-[4-(2-hydroxy-2-methyl-1-oxopropyl)phenyl]-1H-indene

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Link to relevant study records

Reference

Reference 1

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 February 2002 - 28 June 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Version / remarks:

May 26, 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.34 (One-Generation Reproduction Toxicity Test)

Version / remarks:

30. May 1988

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Identity ESACURE KIP 150
Description Solid (at temperatures below 35°C)
Purity 99.999°/o (both batches, calculated as 100% - % of dichloromethane content)
Storage At room temperature, protected from light (uv)
Safety precautions Routine hygienic procedures were applied to ensure personnel security.

Species:

rat

Strain:

Wistar

Remarks:

WIST HanBrl: WIST (SPF Quality)

Details on species / strain selection:

Specified by the international guidelines as the recommended test system

Sex:

male/female

Details on test animals or test system and environmental conditions:

Number of animals 88 males, 22 per group
88 females, 22 per group

Age at delivery Males: 6 weeks
Females: 9 weeks

Body weights Males: 159 - 218 grams
(at commencement of treatment) Females: 161 - 208 grams

Acclimatization Ten days minimum under test conditions with an evaluation of the health status.
Identification Individual cage and corresponding ear number

Route of administration:

oral: feed

Vehicle:

acetone

Remarks:

See details on Study Design for description of diet preparation

Details on exposure:

Rationale for dose levels: Dose levels were based on the results of preceding toxicity studies.

Test item administration: The test item was administered orally in the diet. Control animals received diet without the test item.

Rationale Oral ingestion is the most likely route of human exposure to the test item.

Duration of administration P animals:
Males: The test item was administered during a 70-day prepairing period, during the pairing period and until necropsy.
Females: The test item was administered during a 14-day prepairing period and also during the pairing, gestation- and lactation periods until necropsy.

Details on mating procedure:

The P animals were paired one male I one female for a period of 21 days (maximum). The day on which spermatozoa were present or a copulation plug was found, was recorded as day 0 post coitum. If necessary, not-mated females were re-paired with alternative partners following completion of the original pairing period for a second period of 21 days (maximum).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of test item, its content, homogeneity and stability (3 weeks) in the food pellets were determined in a non-GLP trial.

Content, homogeneity and stability were determined at start of mating. Content and homogeneity were again determined at the end of gestation/start of lactation period. The analyses were performed by the FICC Ltd, Environmental Chemistry & Pharmanalytics Division using a method supplied by the Sponsor (HPLC) and the results included into the report (see Attachment IV).

Duration of treatment / exposure:

Males - 70 days prior to pairing and during the pairing period until necropsy.

Females - 14 days prior to pairing, during pairing, gestation and lactation periods until necropsy

(total 120 days)

Frequency of treatment:

Daily

Details on study schedule:

All dams were allowed to give birth and rear their litters (F1 pups) up to day 21 post partum.

The offspring were examined as soon as possible after completion of delivery and throughout the lactation period, as described in peri-postnatal observations. On day 4 post partum, the number of offspring was reduced to 8 per litter (equally divided as to sex), when possible, using a computer-generated random number table.

At or shortly after weaning, the P generation females and the remaining pups were sacrificed.

P generation males were sacrificed after necropsy of P generation females and pups.

Dose / conc.:

0 ppm

Remarks:

Control

Dose / conc.:

125 ppm

Dose / conc.:

750 ppm

Dose / conc.:

4 500 ppm

No. of animals per sex per dose:

22 male, 22 female

Control animals:

yes, concurrent vehicle

Details on study design:

DIET PREPARATION
The required amount of test item was weighed into a glass beaker and dissolved in a small amout of acetone. This solution was then mixed to granulated diet in a Buehler Mixer type DDMA-0.5 and pelleted in a Buehler pelleting machine type DFPL. Feed for Group 1 was prepared by adding a similar amount of acetone. Water was added to each feed preparation at a volume/weight ratio of approximately 1:10 to ensure pelleting, after which the pellets were dried with warm air for approximately 48 hours before storage. The feed preparations were stored in disposable paper bags at room temperature.

CONDITIONS
The study was performed under standard laboratory conditions: the animal room was air-conditioned with 10-15 air changes per hour; the environment was monitored continuously with hourly recordings of temperature (target range 22 13°C) and relative humidity (target range 30-70%), 12 hours artificial fluorescent light/ 12 hours dark with background music played at a centrally defined low volume for at least 8 hours during the light period. On two days the relative humidity was lower than 30%. These deviations were considered not to have affected the integrity or quality of the study.

ACCOMMODATION
Animals were housed in Makrolon cages (type-3) with wire mesh tops and standard granulated softwood bedding (Lignocel, Schill AG, CH-4132 Muttenz/ Switzerland).
During the prepairing period, males and females were housed individually one to a cage. Cages of males were interspersed amongst those holding females to promote the development of regular estrus cycles. During the pairing period, the rats were housed one male/ one female in Makrolon pairing cages.
After mating or at the end of the pairing period, males and females were housed individually again. Males until necropsy and the females for the birth and rearing of young until necropsy. On the day of weaning of each litter, the dam was separated from its litter.
Throughout the study, each cage was identified by a colored label according to the group and recording the study schedule number, animal number(s) and details of treatment.

DIET
Pelleted standard Kliba 3433 rat/mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/ Switzerland) was available ad lib/tum (Batch Nos. 119/02, 01/02 and 24/02). Flesults of analysis for contaminants are included in the report (see Attachment II, pp. 247-252).

WATER
Community tap water from Itingen was available ad libitum. Results of bacteriological. chemical and contaminant analyses scheduled to be conducted at least once yearly by RCC (contaminant analyses only) and by the Official Chemist of the Kanton Basel-Landschaft (bacteriological and chemical analyses) are included in the report (see Attachment I, pp. 243-245).

Positive control:

No positive control

Parental animals: Observations and examinations:

Mortality rate The animals were checked at least twice daily for any mortality. All rats found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death.
Signs and/or symptoms The animals were checked at least twice daily for signs of reaction to treatment and/or symptoms of ill health.

BODY WEIGHT
Animals were weighed at weekly intervals, with the exception of the pairing period. After mating, females were weighed on days 0, 7, 14 and 21 post coitum.
Dams which littered were weighed on days 0, 4, 7, 14 and 21 postpartum.

FOOD CONSUMPTION
Food consumption was measured weekly together with the recording of body weights, except during the pairing period when no food consumption was measured. During the lactation period, food consumption was recorded only until day 14 post partum. Relative food consumption ratios and intake of the test item expressed as mg/kg/day were calculated.

MATING
A record of mating of the females was made by daily examination of the vaginal smears for spermatozoa and/or appearance of a vaginal plug throughout the pairing period. The day on which evidence of mating was observed was considered to be day 0 post coitum.
Once evidence of mating had been noted, the females were housed individually. A period of 21 days was allowed for mating. Any male failing to copulate within 21 days was replaced by another male of the same group. However, no more than two males were used per female during a given pairing period.
The mating data were used to: detect whether or not pregnancy was interrupted after mating, detect marked anomalies of the estrus cycle, determine the mean precoital time for the group. Any female where no evidence of mating was detected after the two pairing periods, was housed individually.
If no pregnancy was detected, autopsy was performed three weeks after the last day of the second mating period.
Each uterus was placed in a solution of ammonium sulfide to visualize possible haemorrhagic alterations of implantation sites.

GESTATION AND PARTURITION
Towards the end of the gestation period, females were examined twice daily for signs of parturition. Duration of the gestation was calculated. Females without litters and females which lost their litter were killed and necropsied together with the dams after weaning of the pups.
The duration of gestation was taken as the time between the day of successful mating and parturition.

LACTATION AND LITTER DATA
Day 0 of lactation was the day on which a female had delivered all her pups. As soon as possible, the litters were examined for litter size, live birth, stillbirth and any gross anomalies. The sex ratio of the pups was recorded on day 0, day 4 and day 21 of lactation.
Litters were caged together with the dam until weaning on day 21 of lactation. Pups were weighed individually on days 0 (if possible) and / or 1, 4, 7, 14 and 21 of lactation. To prevent cannibalism immediately after birth the pups were weighed individually but without tattooing on day 0 post partum.

BLOOD SAMPLING
At sacrifice of all parental males and females, blood samples of 0.6 ml/animal were collected into heparinized tubes from all parental males and females. Plasma was prepared and retained at about ~80°C for possible future determination of exposure levels.
Additionally, from all animals as much blood as possible was collected into heparinized tubes. Plasma was prepared and divided into 300 1.1! aliquots (a maximum of 5 aliquots was prepared per animal). The samples were retained frozen between -16°C and -24°C for possible future hormone determination.
After finalization of the study report, the blood samples will be discarded without any analysis being performed.

Sperm parameters (parental animals):

Sperm analysis was performed for 10 males of both groups 1 and 4 utilizing sperm obtained from the left epididymldis

Motility
At necropsy of adult males the left epididymis was removed and weighed. An epididymal sperm sample was obtained from the left caudal epididymis. The sample was diluted with a prewarmed (about 35°C) physiological medium, and rapidly after being obtained, one hundred sperm were counted microscopically for determination of percentage progressively motile Sperm.

Morphology
A sample from the caudal epididymis was also used for morphological assessment after fixation and Eosin staining. 500 sperm per sample were evaluated microscopically and classified into the following categories:

Code Description
A Sperm with normal hook and tail
B Normal hook without tail
C Misshapen sperm hook with tail
D Sperm with abnormal curved hook with tail
E Sperm with reversed hook with tail
F Abnormal hook without tail

Sperm, spermatid count
The left caudal epididymis was taken for determination of caudal epididymal sperm reserve. The tissue was frozen at «20°C pending evaluation. For evaluation the weighed tissue was placed in Triton-X-100 solution and homogenized with a blender (Ultra Turax) and an ultrasonic waterbath. Sperm or spennatid heads were counted microscopically using a Neubauer chamber.

Postmortem examinations (parental animals):

From all P animals, samples of the following tissues and organs were collected at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution:

gross lesions
ovaries
pituitary
prostate
seminal vesicles with coagulating gland
testes with epididymides (in Bouin's fixative)
uterus and cervix
vagina
liver, kidneys

Histopathology was performed on the organs listed above for all high dose and control P animals and for all selected animals which died during the study, where practicable. Any organs demonstrating treatment-related histopathological changes were also examined for the animals in groups 2 and 3.

Additionally, reproductive organs of all infertile males and females of groups 2 and 3 were examined histopathologioally.

On day 1 post partum, pups were tattooed individually with Indian ink. At the onset of hair growth. the pups were identified by color spots on the fur. The dams and pups were observed daily for survival, behavioral abnormalities in nesting and nursing. Dead young. except those excessively cannibalized, were autopsied and/or preserved in fixative for possible further examination.
On day 4 post partum, the size of each litter was adjusted by eliminating extra pups by random selection to yield (as near as possible) 4 males and 4 females per litter. The surplus pups were sacrificed and examined macroscopically.
At weaning on day 21 post partum, all the remaining pups were sacrificed by CO; asphyxiation and examined externally and internally for abnormalities.
All P animals were necropsied when they were no longer necessary for the assessment of reproductive effects. P males were necropsied after the last litter had reached day 4 post partum, P females were necropsied after weaning.
All parent generation animals were examined macroscopically for any structural abnormalities or pathological changes either at terminal sacrifice or if death occurred during the study. Special attention was directed to the organs of the reproductive system.
Additionally, livers of parental animals were weighed at necropsy.
Implantation sites were counted for all dams. For this purpose, uteri were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites .

Postmortem examinations (offspring):

On day 4 p.p., after standardization of litter sizes, excess F1 pups were sacrificed, examined macroscopically and discarded. The remaining pups were sacrificed and examined macroscopically and discarded after weaning.

Statistics:

The following statistical methods were used to analyze body weights, food consumption, reproduction and breeding data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If the variables could be assumed to follow a normal distribution, the Dunnett many-one t-test, based on a pooled variance estimate. was used for intergroup comparisons (i.e. single treatment groups against the control group).
The Steel test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
Fisher‘s Exact test for 2X2 tables was applied if the variables could be dichotomized without loss of information.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

MALES

In groups 1, 2 and 4, no clinical observations or signs of discomfort were noted in all periods. In group 3, for male No. 63 wounds were noted caudal of the left pinna and on the right shoulder on days 22 to 30 of the prepairing period. From day 31 to 39 a crust on the right shoulder was noted for this male. For male No. 46 an distended abdomen was noted from day 7 to 31 of the after pairing period, macroscopic examination revealed that this finding was due to a general obesity. These findings were considered to be incidental.

FEMALES

Prepairing Period
During the prepairing period no clinical observations or signs of discomfort were noted in any group.

Gestation Period
During most days of the gestation period. ruffled fur was noted for all females in group 4 and considered to be test item related. The alopecia noted on the left flank in one female (No. 159) during the last third of the gestation period was considered to be incidental.
In group 2, alopecia was noted for one female (No. 117) during the second half of the gestation period. This finding was considered to be incidental.
No clinical signs were noted in groups 3 and 1.

Lactation Period
No clinical signs that were considered to be test item related were noted in groups 3. 2 and 1. The alopecia noted in one female (No. 117) was considered to be incidental. In group 4, ruffled fur was noted during the first 6 and 9 days, respectively, of the lactation period for the two dams that gave birth in this group.

(pp. 166-170 Figures and tables attachment)

Mortality:

no mortality observed

Description (incidence):

MALES

All animals survived until scheduled necropsy.

FEMALES

All animals survived until scheduled necropsy.

(pp. 166-170 Figures and tables attachment)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

MALES

Prepairing Period
Mean body weight gain in group 4 was clearly reduced (+92.1% compared with +114.1% in the vehicle control, resulting in a mean body weight of 367 g at the end of the prepairing period compared with 409 g in the vehicle control.
Body weight development in groups 3 and 2 gave no indication for a test item-related effect.

(pp. 48-50, 134-137 Figures and tables attachment)

After Pairing Period
Mean body weights were lower in group 4 in comparison to the vehicle control which was considered to be a consequence of the lower body weight gain during the prepairing period.
In terms of body weight gain during the after pairing period. there was no apparent test item- related effect.
Mean body weights as well as body weight gain in groups 3 and 2 were similar to the vehicle control.

(pp. 51-53, 138-141 Figures and tables attachment)

FEMALES

Prepairing Period
Mean body weight gain in group 4 was slightly reduced (+27% versus +69% in the vehicle control). Similar mean body weight gain was noted for groups 1-3.

(pp. 73-75, 154-157 Figures and tables attachment)

Gestation Period
Body weight gain in the two females that gave birth in group 4 was markedly reduced (+24.9% compared with +57.8% in the vehicle control). This was considered to be test item related as well as a consequence of the low number of fetuses per dam.
Body weight gain in groups 3 and 2 was similar to that of the vehicle control.

(pp. 7678, 158-161 Figures and tables attachment)

Lactation Period
Body weight gain in the two females that gave birth in group 4 was markedly reduced (+48% compared with +13.9% in the vehicle control).
Body weight gain in groups 3 and 2 was similar to that of the vehicle control.

(pp. 79-81, 162-165 Figures and tables attachment)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

MALES

Prepairing Period
Mean food consumption in group 4 was reduced during the prepairing period (-7.0% compared with the vehicle control).
Mean food consumption in groups 3 and 2 was similar to that of the vehicle control.

(pp. 38-40, 44, 45, 126-129 Figures and tables attachment)

After Pairing Period
Mean food consumption in group 4 was marginally reduced during the after pairing period (-2.3% compared with the vehicle control).
Mean food consumption in groups 3 and 2 was similar to that of the vehicle control.

(pp. 41-43, 46, 47. 130-133 Figures and tables attachment)

FEMALES

Prepairing Period
Mean food consumption in group 4 was markedly reduced during the first week of the prepairing period (28.8% compared with the vehicle control). During the second week of the prepairing period mean food consumption was marginally reduced (-2.0%).
Mean food consumption in groups 3 and 2 was similar to the vehicle control.

(pp. 53-60, 67, 68, 142-145 Figures and tables attachment)

Gestation Period
Food consumption in the two dams that gave birth in group 4 was clearly lower than that of the vehicle control (45.9%). This low food consumption was considered to be due to both treatment with the test item and a consequence of the low number of implantations.
Similar mean food consumption was noted for groups 1-3.

(pp. 61 -63. 69, 70, 146449 Figures and tables attachment)

Lactation Period
During the first 14 days of the lactation period markedly reduced food consumption was noted for the two dams which gave birth in group 4. This low food consumption was considered to be due to both: treatment with the test item and a consequence of the small litter size.
Mean food consumption in groups 3 and 2 was similar to that of the vehicle control.

(pp. 64-66, 71, 72l 150-153 Figures and tables attachment)

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological test item-related changes in rats administered Esacure KIP 150 were recorded at the ovaries, the vagina and the liver.
In the ovaries an atrophy was recorded in females of all test item-treated groups. Preceding or accompanying this finding, an interstitial cell hyperplasia was recorded in females of all test item-treated groups. Remarkably, in many of the ovaries of group 4 a total, bilateral absence of antral follicles was observed. So only follicles in early stages of development were noted. The primordial and primary follicles seemed not to ripe, and/or antral follicles get atretic. Additionally, in many ovaries, especially of group 4, small, persisting corpora lutea were recorded. The combination of both changes, an absence of antral follicles and a presence of persisting corpora lutea, both recorded in group 4. caused a blockade of the sexual cycle. At vaginal epithelium an increased incidence of diestrus was recorded in test item-treated groups. Together with the ovarian alterations this change was indicative for a persisting diestrus in group 4. The ovarian changes indicate effects of the test item on female gonadal function and on estrous cycle and influenced the reproductive function. So minimally to massively decreased conception rates were observed in group 3 and group 4 females.
The mechanism how Esacure Kip 150 induced these changes were unclear. As there were also test item-related findings in group 2, a NOEL could not be established. However. due to the lack of a recovery period, it can not be decided, if the ovarian changes would persist (and therefore would be of adverse character), or due to the regenerative capabilities of the gonadal stroma probably would disappear.
Additionally, a hepatocellular hypertrophy (zone 3 = centrilobular) was recorded in the liver of test item-treated animals. It was slightly more pronounced in male animals and was most likely due to an increased enzyme induction caused by the biotransformation of the test item in the liver. So the increased cytoplasmic basophilia of floccular/clumpy appearance was interpreted as rough endoplasmatic reticulum. Therefore it was regarded as an adaptive change. The hypertrophy was accompanied by a decrease in glycogen deposition in test item treated animals. This is a well known phenomenon, which very often accompanies a hepatocellular hypertrophy.

(pp. 275-447 Figures and tables attachment)

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

SPERM ANALYSIS

During sperm analysis no differences that were attributable to treatment with the test item were noted between group 4 and the vehicle control.
Mean epididymal weight, sperm count, motility and morphology assessment revealed similar results in group 4 and the vehicle control.

(pp. 105-108, 184-187 Figures and tables attachment)

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

FERTILITY AND MATING PERFORMANCE

There was no test item-related effect on mating performance. All animals mated during the first pairing period and the median precoital time was 3, 3. 3 and 4 days in groups 1-4, respectively.

In group 4, the conception rate (percentage of mated females that became pregnant) was markedly reduced (31.8% compared with 100% in the vehicle control). Furthermore, the gestation index (percentage of pregnant females that gave birth to living pups) was markedly reduced in this group (28.6% compared with 100% in the vehicle control).

In group 3, two out of 22 mated females (Nos. 134 and 139) did not become pregnant (corresponding to a conception rate of 90.9%). The two affected females mated with males (Nos. 46 and 51) for which macroscopic and/or histopathological changes of the reproductive organs were noted. However, since histopathological examination revealed pathologically altered ovaries in both non-pregnant females, it cannot finally be concluded whether their infertility was incidental (i.e. due to the incidentally infertile males) or test item related.

Fertility parameters in groups 3 and 2 gave no indication for a test item-related effect.

(pp. 88, 89, 171-174 Figures and tables attachment)

DURATION OF GESTATION

For the two females that gave birth in group 4, a slightly prolonged gestation period (23 days) was noted. Due to the low number of dams that gave birth and to the very small litter size, the duration of gestation cannot be evaluated.
A similar mean duration of gestation was noted in groups 1-3 (21.7. 21.4 and 21.7 days, respectively).

(pp. 91. 93-100 Figures and tables attachment)

6.3.3 IMPLANTATION RATE AND POST-IMPLANTATION LOSS

In group 4, a markedly decreased number of implantations per darn was noted (1.3 implantations per dam compared with 12.7 in the vehicle control).
In groups 3 and 2 the implantation rate was similar to that of the vehicle control (11.8 and 12.8 implantations per dam, respectively).
In group 4, total post-implantation loss was noted for 5 out of 7 pregnant females. All females for which total post—implantation loss was noted had only one implantation site. Since total resorption in pregnancies with only one or two implantations is a common finding, these total resorptions were considered not to be directly attributable to treatment with the test item but to be a consequence of the test item-related low number of implantations.
No total post-implantation loss was noted in groups 1-3 and the rate of partial post-implantation loss in these groups gave no indication for a test item-related effect (10.0, 5.7 and 8.9% of implantations sites in groups 1-3, respectively).

(pp. 90, 91, 93-100 Figures and tables attachment)

LITTER SIZE AT FIRST LITTER CHECK

In group 4, only two dams gave birth to live pups (one dam to two pups and one dam to one pup).
Mean litter sizes at first litter check in groups 3 and 2 did not give an indication for a test item-related effect (10.8 and 12.0 pups per darn, respectively, compared with 11.5 in the vehicle control).

(pp. 91, 93-100 Figures and tables attachment)

6.3.5 POSTNATAL LOSS - DAYS 0 - 4 POST PARTUM

In group 4, one dam lost the single pup it delivered. Due to the small number of litters and the small litter size it cannot be concluded whether this was attributable to treatment with the test item.
In group 3, no postnatal loss was noted. In both groups 2 and 1, four pups died within the first 4 days. This incidence of post implantation loss is considered to be within the normal biological range for rats of this strain.

(pp. 91, 93-100, 123 Figures and tables attachment)

6.3.6 BREEDING LOSS - DAYS 5 - 21 POST PARTUM

In group 4, one dam lost one of the two pups it delivered. Due to the small number of litters and the small litter size it cannot be concluded whether this was attributable to treatment with the test item.
In both groups 3 and 2 one pup died between day 5 and 21 post partum (compared to no breeding losses in the vehicle control). This incidence of post-implantation loss was considered to be within the normal biological range for rats of this strain.
The number of pups per dam on day 21 post partum was similar in groups 1-3 (8.0, 8.0 and 7.9, respectively).

(pp. 91, 93-100, 123 Figures and tables attachment)

Dose descriptor:

LOAEL

Effect level:

4 500 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Effect level:

750 ppm (analytical)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Dose descriptor:

LOAEL

Effect level:

125 ppm (analytical)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

125 ppm

System:

female reproductive system

Organ:

gonad

ovary

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

no effects observed

Description (incidence and severity):

LACTATION TO WEANING

During first litter check no abnormal findings were noted.
During the lactation period no abnormal findings or clinical signs were noted in groups 4 and 3.


(pp. 122, 202-236, 237 Figures and tables attachment)

SEX RATIOS
Sex ratios in group 4 could not be evaluated due to the low number of pups obtained in this group.
No indication for a test item-related effect of sex ratio was noted in groups 2 and 3.

(p. 91 Figures and tables attachment)

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

In group 2, one pup (female No. 12, dam No. 116) had several wounds on day 7 postpartum and was found dead and partially cannibalized on day 8 post partum.
In group 1, one pup that was found dead on day 1 post partum (female No. 11, dam No. 97) was partially cannibalized. An enlarged right eye was noted for one pup (female No. 11, dam No. 93) on days 19 and 21 postpartum.

(pp. 122, 202-236, 237 Figures and tables attachment)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

PUP WEIGHTS T0 WEANING (DAY 21 POST PARTUM)

In group 4, pup weights could hardly be evaluated due to the low number of pups. Pup weights appeared to be increased on day 1 post partum (6.6 9 versus 6.0 g in the vehicle control) which was attributable to the small litter size. From day 7 post partum onwards pup weights appeared to be markedly reduced.

Pup weights in groups 3 and 2 gave no indication for any test item-related effects.
The statistically significantly increased body weights noted for pups in group 3 from day 7 post partum onwards were considered to be incidental.

(pp. 110-121, 189-201 Figures and tables attachment)

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

During necropsy of F1 offspring no findings that were considered to be test item-related were noted.
For two pups, that were found dead, (one in group 1 and one in group 2) partial cannibalization was noted. And for one pup in group 2. a missing left testis was noted. This isolated finding was considered to be incidental.

(pp. 238, 202-236 Figures and tables attachment)

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

750 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

Critical effects observed:

no

Reproductive effects observed:

yes

Lowest effective dose / conc.:

750 ppm

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Summary of breeding performance

	Group 1 (0 ppm)	Group 1 (125 ppm)	Group 1 (750ppm)	Group 1 (4500 ppm)
Number of females paired	22	22	22	22
Number of females mated	22	22	22	22
Number of pregnant females	22	22	20	7
Number of females delivering pups	22	22	20	2
Number of females rearing pups to weaning	22	22	20	1

Conclusions:

The purpose of this study was to provide general information concerning the effects of the test item, Esacure KIP 150, on reproductive function of male and female rats.

Esacure KIP 150 was administered orally, by feed admix to males during a 70-day prepairing period, during the pairing period and until necropsy and to the females during a 14-day prepairing period, during pairing, gestation and lactation periods until necropsy.

Treatment at 4500 ppm was associated with effects on parental animals (reduced food consumption and body weight development as well as increased liver weights in males; clinical signs, reduced food consumption and body weight development as well as increased liver weights in females). Furthermore, treatment at this dosage had a negative effect on the reproductive performance (decreased conception rate, gestation index and implantation rate). Histopathologically, changes affecting the ovaries, the vagina and the liver were noted.

Treatment at 750 ppm was associated with increased liver weights in females. Histopathologically, changes affecting the ovaries, the vagina and the liver were noted. Furthermore, a negative effect on the conception rate could not be excluded.

Treatment at 125 ppm was associated with histopathological changes affecting the ovaries, the vagina and the liver. Although morphological changes of ovaries and vagina were noted at this dosage, they had no adverse effect on the reproductive parameters under investigation in this study.

Based on these results and considering both effects on parent animals and reproduction, an overall no-observed-effect-level (NOEL) could not be established.

Executive summary:

The purpose of this study was to provide general information concerning the effects of the test item on reproductive function as assessed by gonadal function, estrus cycles, mating behavior, conception, parturition, lactation, weaning, and the growth and development of the off-spring. The study also provided information about the effects of the test item on neonatal morbidity, mortality, behavior and preliminary data on teratogenesis and to serve as a guide for subsequent tests.

The test item was administered in the diet to males during a 70-day prepairing period, during the pairing period and until necropsy and to the females during a 14—day prepairing period, during pairing, gestation and lactation periods until necropsy.

The target dietary concentrations used were as follows:

Group 1: 0 ppm (control)

Group 2: 125 ppm

Group 3: 750 ppm

Group 4: 4500 ppm

The following results were obtained:

		Group 2 (mg/kg/day)	Group 3 (mg/kg/day)	Group 4 (mg/kg/day)
males	preparing period	8.1	48.8	292.5
	after pairing	6.2	38.0	238.1
females	preparing period	9.5	56.9	291.4
	gestation	9.4	56.5	315.8
	lactation	18.0	114.0	271.5

PARENT ANIMALS - GENERAL TOLERABILITY

MALES

All males survived until scheduled necropsy and no clinical signs that were considered to be test item related were noted during the study.

At 4500 ppm, mean food consumption was reduced during the prepairing period (-7.0% compared with the vehicle control) and marginally reduced during the after pairing period (-2.8% compared with the vehicle control).

At 4500 ppm, mean body weight gain was clearly reduced during the prepairing period (+92.1°/o compared with +114.1% in the vehicle control, resulting in a mean body weight of 367 g at the end of the prepairing period compared with 409 g in the vehicle control.

Consequently. mean body weights at this dosage were lower during the after pairing period in comparison to the vehicle control. In terms of body weight gain during the after pairing period, there was no apparent test item-related effect.

FEMALES

All females survived until scheduled necropsy.

During the prepairing period no clinical observations or signs of discomfort were noted in any group.

At 4500 ppm, ruffled fur was noted for all females during the gestation period and during the first 6 and 9 days, respectively, of the lactation period for the two dams that gave birth at this dosage.

At 4500 ppm, mean food consumption was markedly reduced during the first week of the prepairing period (-28.8% compared with the vehicle control). During the second week of the prepairing period mean food consumption was marginally reduced (-2.0%). During gestation, food consumption in the two dams that gave birth at this dosage was clearly lower than that of the vehicle control (-15.9%). This low food consumption was considered to be due to both: treatment with the test item and a consequence of the low number of implantations. During the first 14 days of the lactation period markedly reduced food consumption was noted for these two dams. This low food consumption was considered to be due to both: treatment with the test item and a consequence of the small litter size.

At 4500 ppm, mean body weight gain in group 4 was slightly reduced (+2.7% versus +6.9% in the vehicle control) during the prepairing period. During gestation body weight gain in the two females that gave birth was markedly reduced (+24.9% compared with +57.8% in the vehicle control). This was considered to be test item related as well as a consequence of the low number of fetuses per dam. During lactation body weight gain in the two females that gave birth was markedly reduced (+4.8% compared with +13.9% in the vehicle control).

PARENT ANIMALS - TERMINAL EXAMINATIONS

Macroscopical examination - No abnormal findings that were considered to be test item-related were noted during macroscopic examination of males and females At 4500 ppm, liver weights were clearly increased in males and females. At 750 ppm, female liver weights were increased, although to a lesser extent.

Sperm Analysis - During sperm analysis no differences that were attributable to treatment with the test item were noted between males dosed at 4500 ppm and the vehicle control.

Histopathology - Histopathological test item-related changes in rats administered Esacure KIP 150 were recorded at the ovaries, the vagina and the liver.

At the ovaries the changes consisted of atrophy, absence of antral follicles, interstitial cell hyperplasia, and persisting corpora lutea (especially at 4500 ppm). The vaginal epithelium showed changes indicative of a persisting diestrus. All these changes were regarded as test item-related effects. They indicate effects on female gonadal function and estrous cycles, influencing the reproductive function (decreased conception rates in test item-treated females at 4500 ppm).

Additionally, a hepatocellular hypertrophy (zone 3 = centrilobular) was recorded in the livers of animals from all groups treated with the test item. The hepatocellular hypertrophy was most likely due to an increased enzyme induction caused by the biotransformation of the test-item in the liver. Therefore it was regarded as an adaptive change.

PARENT ANIMALS - REPRODUCTION DATA

There was no test item-related effect on mating performance. All animals mated during the first pairing period and the median precoital time was similar in all groups.

At 4500 ppm, the conception rate (percentage of mated females that became pregnant) was markedly reduced (31.8% compared with 100% in the vehicle control). Furthermore, the gestation index (percentage of pregnant females that gave birth to living pups) was markedly reduced at this dosage (28.6% compared with 100% in the vehicle control).

At 750 ppm, two out of 22 mated females did not become pregnant (corresponding to a conception rate of 90.9%). The two affected females mated with males (Nos. 46 and 51) for which macroscopic and/or histopathological changes of the reproductive organs were noted.

However, since histopathological examination revealed pathologically altered ovaries in both non-pregnant females, it cannot finally be concluded whether their infertility was incidental (l.e. due to the incidentally infertile males) or test item related.

For the two females that gave birth at 4500 ppm, a 23-day gestation period was noted. Due to the low number of dams that gave birth and to the very small litter size, the duration of gestation cannot be evaluated.

At 4500 ppm, a markedly decreased number of implantations per darn was noted (1.3 implantations per dam compared with 12.7 in the vehicle control).

At 4500 ppm, total post-implantation loss was noted for 5 out of 7 pregnant females. All females for which total post-implantation loss was noted had only one implantation site.

Since total resorption in pregnancies with only one or two implantations is a common finding, these total resorptions were considered not to be directly attributable to treatment with the test item but to be a consequence of the test item-related low number of implantations.

At 4500 ppm, only two dams gave birth to live pups (one dam to two pups and one dam to one pup). One of these two dams dam lost the single pup it delivered on day 4 post partum.

The other dam lost one of the two pups it delivered on day 14 post partum. Due to the small number of litters and the small litter size it cannot be concluded whether this was attributable to treatment with the test item.

F1 OFFSPRING - OBSERVATIONS

During first litter check and lactation no abnormal findings that were considered to be test item related were noted.

At 4500 ppm, pup weights could hardly be evaluated due to the low number of pups. Pup weights appeared to be increased on day 1 post partum (6.6 9 versus 6.0 g in the vehicle control) which was attributable to the small litter size. From day 7 post partum onwards pup weight appeared to be markedly reduced.

F1 OFFSPRING - TERMINAL EXAMINATIONS

During necropsy of F1 offspring no findings that were considered to be test item related were noted.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because a pre-natal developmental toxicity study is available

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

9.5 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 January 2002 - 08 February 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Adopted: 22" January 2001.

Deviations:

yes

Remarks:

Due to the pilot status of this study a reduced number of animals is used.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Identity ESACURE KIP 150
Description Solid (at temperatures below 35°C)
Purity 99.999% (calculated as 100% - % of dichloromethane content)
Storage At room temperature, protected from light (uv)
Stability of the pure test item Stable under storage conditions
Stability in the vehicle Stable for at least 6 hours
Safety precautions Fioutine hygienic procedures will be applied to ensure personnel security.

Species:

rat

Strain:

Wistar

Remarks:

HanBrl:WlST (SPF)

Details on test animals or test system and environmental conditions:

TEST SYSTEM
Species Hat HanBrl:WlST (SPF)
Rationale Specified by the international guidelines as a recommended test system
Source RCC Ltd, Biotechnology & Animal Breeding Division, wolferstrasse 4, CH-4414 Fijllinsdori / Switzerland
Acclimatization Ten days minimum under test conditions with an evaluation of the health status.
Number of animals 20 mated females, 5 per group
Age at pairing 11 weeks minimum
Body weights 211 - 255 grams (day 0 post coitum)
identification Individual animal number tattooed on the pinnae (day 0 post coitum)

HUSBANDRY
Conditions
The animals were housed under standard laboratory conditions: air-conditioned with 10—15 air changes per hour; the environment monitored continuously with hourly recordings of temperature (target range 22 ± 3°C) and relative humidity (target range 30 - 70%), 12 hours artificial fluorescent light/ 12 hours dark with background music played at a centrally defined low volume for at least 8 hours during the light period.

Accommodation
The animals were housed individually in Makrolon cages (type-3) with wire mesh tops and standardized granulated softwood bedding (Lignocel, Schill AG, CH-4132 Muttenz/ Switzerland).

Diet
Pelleted standard Kliba 3433 rat/mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/ Switzerland) was available ad libitum (Batch Nos. 77/01 and 119/01).

Water
Tap water in bottles was available ad libitum.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
Identity Corn oil
Supplier Carl Roth GmbH
Batch No. 02938667
Corn oil was used as the vehicle for the test item in the dose groups and was administered as the control item to the females of the control group.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability of the test item/vehicle mixtures was determined during the first and last week of the administration period. Samples were taken after preparation and again 6 hours later. On each occasion samples (appr. 2 g per sample) were taken from the top, middle and bottom of each formulation. Each dose sample was individually identified. The samples were frozen (approx. -20°C) for analysis by (HPLC) by:
Dr. C. Knuppe, 900 Environmental Chemistry & Pharmanalytics Division, CH 4452 Itingen/ Switzerland.

The results of the analyses are attached (Attachment Vl, pp. 140 - 154)

The overall mean concentrations of the homogeneity samples were found to be 91.0%, 92.5%, and 101.2% of the nominal concentrations of dose group 2 (2 mg/ml), dose group 3 (20 mg/ml), and dose group 4 (50 mg/ml), respectively. The individual concentrations varied in the range from -5% to +2% of the mean concentrations. Therefore, the test item was found to be homogeneously distributed in the vehicle.

Details on mating procedure:

After acclimatization, the females were housed with the males (one male : one female) in special automatic mating cages, i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduces the variation in the copulation times of the different females. The females were removed and housed individually if:

a) the daily vaginal smear was sperm-positive, or
b) a copulation plug was observed.

This day was designated day 0 post coitum.
The male rats used for mating were in the possession of RCC. The fertility of these males was proved and continuously controlled.

Duration of treatment / exposure:

From day 6 through to day 20 post coitum.

Frequency of treatment:

Once daily.

Duration of test:

21 days.

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

Mated rats were assigned to the different groups using a computer-generated random algorithm.

All animals received a dose volume of 5mn body weight with a daily adjustment of the individual volume to the actual body weight. Control animals were similarly dosed with the vehicle alone.

Oral intake is one possible route of human exposure. International guidelines recognize the efficacy of oral administration.

Maternal examinations:

Mortality rate The animals were checked at least twice daily for any modalities.

Signs and/or symptoms The animals were observed at least twice daily for signs of reaction to treatment and/or symptoms of ill health.

Food consumption Food consumption was recorded for the following periods: days 0-3. 3-6. 6-9, 9-12, 12-15. 15-18 and 18- 21 post coitum (see Data Compilation and Data Processing).

Body weight Body weights was recorded daily from day 0 until day 21 post coitum (see Data Compilation and Data Processing).

Fetal examinations:

TERMINATION OF THE STUDY

On day 21 post coitum, the females were killed by CO2 asphyxiation and the fetuses removed by Caesarean section.

CAESAREAN SETION AND POST MORTEM EXAMINATION

Postmortem examination, including gross macrosco-pic examination of all internal organs, with emphasis on the uterus, uterine contents, position of fetuses in the uterus and number of corpora lutea, was performed and the data recorded. The uteri (and contents) of all females with live fetuses were weighed at necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain (see Data Processing).

Additionally, livers of females were weighed at necropsy. The fetuses were removed from the uterus, sexed, weighed individually, examined for gross external abnormalities, killed by subcutaneous injection of sodium pentobarbitone (Narcoren®) and allocated to one of the following procedures:

1) Microdissection technique (sectioning/dissection technique). Half of the fetuses from each litter were fixed in Bouin's fixative (one fetus per container). They were examined by a combination of serial sections of the head and microdissection of the
thorax and abdomen. This included detailed examination of the major blood vessels and sectioning of the heart and kidneys. After examination the sections were preserved in a solution of glycerine/ethanol (one fetus per container). Descriptions of any abnormalities and variations were recorded.

2) The remaining fetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. Carcasses were processed through solutions of ethanol. glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and agueous glycerin for preservation and storage. The skeletons were examined and all abnormal findings and variations recorded. They were preserved individually.

Statistics:

The following statistical methods were used to analyse body weights, food consumption and reproduction data:

- Means and standard deviations of various data were calculated and included in the report.
- If the variables could be assumed to follow a normal distribution, the Dunnett many-one t-test, based on a pooled variance estimate, was used for intergroup comparisons (i.e. single treatment groups against the Control group).
- The Steel test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.

All methods of analysis and the results are included in the report.

Historical control data:

Attachment V in 'attached background material'

Clinical signs:

no effects observed

Description (incidence and severity):

No mortalities or clinical symptoms were noted in any female throughout the study.

Mortality:

no mortality observed

Description (incidence):

No mortalities or clinical symptoms were noted in any female throughout the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weight gain was not affected by treatment with the test item.

Also, corrected body weight gain (corrected for uterus weight) gave no indication for any test item-related findings. The slightly decreased corrected body weight gain noted in group 4 was considered to be incidental.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Mean food consumption during the treatment period was clearly reduced in group 4 (-10.6% compared to vehicle controls). Food consumption in groups 2 and 3 was similar to that of vehicle controls.

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Mean liver weights were slightly but dose-dependently increased in groups 3 and 4 (17.6 and 17.9 9 compared to 15.8 g in vehicle controls). Liver weights in group 2 were similar to those in vehicle controls (15.4 g).

The increase in female liver weights in groups 3 and 4 was possibly test item-related since the liver is a known target organ of the test item.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

food consumption and compound intake

organ weights and organ / body weight ratios

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No test item-related effects on fetal body weights were noted.

Fetal body weights were slightly increased in group 4 (5.0 9 versus 4.8 g in vehicle controls, combined litter-based data for male and female fetuses), but this increase was considered to be incidental.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No test item-related effects on the sex ratio of fetuses were noted in any group.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No abnormalities were noted during external examination of fetuses

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were 8 fetuses with abnormal skeletal findings as follows:

None in group 1 (0/32), one in group 2 (1/35), one in group 3 (1/32) and six in group 4 (6/34). The sternebral and vertebral abnormalities noted in one fetus in group 2, one fetus in group 3 and two fetuses in 4 were common abnormal findings that are frequently seen in control rat fetuses of this strain. The incidence in this study was considered to be unrelated to treatment with the test item.

The abnormal finding of fusion of two components of the zygomatic arch noted in one fetus in group 3 and four fetuses (from 2 litters) in group 4, is also occasionally seen in control rat fetuses of this strain. The increased incidence in group 4 noted could well be due the low number of fetuses examined in this pilot study.

Examination of the stage of skeletal development revealed a slightly delayed ossification of limb structures in group 4 and to a lesser extent group 3. Also in group 4. a slight delay in ossification of bones of the skull was noted.

Furthermore the incidence of rudimentary supernumerary ribs was increased in group 4.

Visceral malformations:

no effects observed

Description (incidence and severity):

The frequency and type of common abnormal findings (mainly comprising elongated thymus, small additional liver lobes in the median cleft, median liver lobe with displaced cleft and left- sided umbilical artery) was similar in all groups.

There was no indication for any test item-related effects.

Other effects:

no effects observed

Description (incidence and severity):

During cartilage examination only one fetus with an abnormal cartilage finding (abnormally shaped cartilaginous parts of thoracic vertebral bodies 12 and 13) was noted in group 4.
This isolated finding was considered to be unrelated to treatment with the test item.

Examination of fetal cartilages for the stage of development gave no indication for any test item-related effects.
When calculated on an individual basis, two statistically significant differences at level 1% being due to a higher stage of development of cartilaginous sternebrae were noted in both groups 2 and 4. NO differences in the stage of development were noted in group 3.

When calculated on a litter basis, no significant differences in the stage of development of cartilages between groups treated with the test item and vehicle controls were noted.

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

skeletal malformations

Abnormalities:

effects observed, non-treatment-related

Localisation:

skeletal: skull

skeletal: supernumerary rib

Developmental effects observed:

no

Conclusions:

In order to detect effects on embryonic and fetal development in pregnant rats, Esacure KIP 150 was administered orally by gavage once daily from day 6 through to day 20 post coitum at dose levels of 10, 100 and 250 mg/kg body weight/day.
In females treatment with the test item caused increased liver weights at 100 and 250 mg/kg/day, and clearly reduced food consumption at 250 mg/kg/day.

At 250 mg/kg body weight/day an increased incidence of skeletal variations was noted. Also the stage of skeletal development was slightly decreased at 100 and 250 mg/kg body weight/day.

Due to the low number of litters examined in this pilot study it could not finally be concluded, whether these findings were incidental or caused by treatment with the test item.

Based on these data the following dose levels are considered appropriate for a consecutive main study: 10, 50 and a range between 100 and 250 mg/kg body weight/day, choosing 250 mg/kg body weight/day as a conservative approach, while 10 mg/kg body weight/day is expected to be the NOAEL.

Executive summary:

Fetal development when administered orally by gavage once daily to mated female rats from day 6 through to day 20 post coitum, inclusive.

Each group consisted of 5 mated female rats. Esacure KIP 150 was administered once daily at dose levels of:

Group 1: 0 mg/kg body weight/day (vehicle control)

Group 2: 10 mg/kg body weight/day

Group 3: 100 mg/kg body weight/day

Group 4: 250 mg/kg body weight/day

A standard dose volume of 5 ml/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil).

All females were sacrificed on day 21 post coitum and the fetuses were removed by Caesarean section. Examination of dams and fetuses was performed in accordance with international recommendations.

The following results were obtained:

MATERNAL DATA

General Tolerability

No mortalities or clinical symptoms were noted in any female throughout the study.

Mean liver weights were slightly but dose-dependently increased at 100 and 250 mg/kg/day (17.6 and 17.9 9 compared to 15.8 g in vehicle controls). This increase was possibly test item-related since the liver is a known target organ of the test item.

Food Consumption and Body Weights

Mean food consumption during the treatment period was clearly reduced at 250 mg/kg/day (-10.6% compared to vehicle controls). Food consumption at 10 and 100 mg/kg/day was not affected by treatment with the test item.

Mean body weight gain was not affected by treatment with the test item at any dosage employed.

Reproduction Data

No differences that were considered to be test item related were noted among the relevant mean reproduction data (post-implantation loss, number of fetuses per dam).

FETAL DATA

All results obtained are based on a low number of fetuses due to the pilot design of the study.

No test item-related effects on fetal body weights or fetal sex ratios were noted.

During external and visceral examination of fetuses no abnormalities that were considered to be attributable to treatment with the test item were noted.

At 250 and 100 mg/kg/day fusion of two of the components of the zygomatic arch were noted in four and one fetuses, respectively. Although this finding is occasionally noted in control rat fetuses of this strain. it cannot be finally concluded whether the increased incidence noted in this study is incidental or test item-related.

Examination of the stage of skeletal development revealed a slightly delayed ossification of limb structures at 250 mg/kg/day and to an even lesser extent at 100 mg/kg/day. Also at 250 mg/kg/day, a slight delay in ossification of bones of the skull was noted. Furthermore the incidence of rudimentary supernumerary ribs was increased at 250 mg/kg/day.

During cartilage examination no test item related findings were noted.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

1

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based upon the results seen the substance does meet the critria for classification (reproductive toxicant) in accordance with the classification, labelling and packaging regulation (EC 1272/2008)