Hexamethylene diisocyanate, oligomers, reaction products with Bis-(Trimethoxysilylpropyl)amine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hexamethylene diisocyanate, oligomers, reaction products with N-(3-trimethoxysilyl)propylbutylamine and Bis-(Trimethoxysilylpropyl)amine

EC Number:

926-191-9

Molecular formula:

Unspecified

IUPAC Name:

Hexamethylene diisocyanate, oligomers, reaction products with N-(3-trimethoxysilyl)propylbutylamine and Bis-(Trimethoxysilylpropyl)amine

Test material form:

liquid

Details on test material:

-Storage conditions: room temperature, no direct sunlight, protect against humidity

Test animals

Species:

rat

Strain:

Wistar

Remarks:

75

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V.
- Age at study initiation: 12 weeks
- Weight at study initiation: Males: 293 to 332 g; Females: 187 to 217 g
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Seven days under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared daily using the test item as supplied by the Sponsor.

iGloss Crosslinker (ZQ54-2211) was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added and stirred for 60 minutes. Separate formulations were prepared for each concentration.

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): olive oil was a suitable vehicle
- Dose Volume: 4 mL/kg body weight

Details on mating procedure:

During the pairing period, females were housed with sexually mature males from the same dose group (1:1) until evidence of copulation was observed. The females were removed and housed individually when:
- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.
The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first treatment day samples from the control group (middle only) as well as three samples
(top, middle and bottom) of about 0.5 g of each concentration were taken prior to dosing for
analysis of concentration and homogeneity. Towards the end of the study, again three samples
(top, middle and bottom) were taken to confirm concentration and homogeneity. The
homogeneity was analysed also during the second sampling event, since it was difficult to
prepare a homogenous formulation with this test item. The aliquots for analysis of dose
formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Dr. B. Ludwig (Harlan
Laboratories Ltd., Zelgliweg 1, 4452 Itingen / Switzerland) and stored there at -20 ± 5 °C until
analysis.
The samples were analyzed by infrared spectroscopy (implementation of an analytical method
for dose formulation, D53904). The test item was used as the analytical standard. Analyzed
samples were discarded after approval of the data by the sponsor. Duplicates were taken of all
samples and were stored at Harlan Laboratories Ltd., Füllinsdorf / Switzerland. The samples
were discarded after approval of the data.

Duration of treatment / exposure:

Males: Minimum 4 weeks
Females: Approximately 6 weeks

Frequency of treatment:

Once daily

Details on study schedule:

N/A

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 75, 250 and 750 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

11

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on 14-day range finder.
- Rationale for animal assignment (if not random): Performed on the fifth day of acclimatization using a computer-generated random algorithm. Body
weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.

Positive control:

N/A

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS /DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Twice daily for mortality, Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy).
Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.
Detailed clinical observations:
Once prior to the first administration of the test item (day 6 of acclimatization) and weekly thereafter (in the gestation period on day 0, 6, 13 and 20 post coitum), detailed clinical observations were performed outside the home cage in a standard arena. Animals were observed
for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or
tonic movements, stereotypies or bizarre behavior were also reported.


BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily from treatment start to day of necropsy.

FOOD CONSUMPTION
Males: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14 and weekly after pairing period.
Females: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; gestation days 0 – 7, 7 - 14 and 14 – 21 and days 1 - 4 of the lactation.
No food consumption was recorded during the pairing period.

Oestrous cyclicity (parental animals):

N/A

Sperm parameters (parental animals):

Parameters examined in [P] male parental generations:
Organ weights:
Uterus (including cervix), Prostate, Ovaries (weighed as pairs), Seminal vesicles (inclusive coagulating gland)
Histopathology
Testes, epididymides, prostate, seminal vesicles, ovaries, oviduct, vagina and uterus from all animals of the control and high-dose group were examined.
Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Histological examination of ovaries was carried out on the females that did not give birth.

Litter observations:

The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.
Pups were sacrificed on day 4 post partum. Dead pups, except those excessively cannibalized, were examined macroscopically. All pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

Postmortem examinations (parental animals):

SACRIFICE
Males were sacrificed after treatment of at least 28 days, when no longer needed for the assessment of reproductive effects. Pups were sacrificed on day 4 post partum. Dams were sacrificed on day 5 post partum.

Since in several females a birth did not occur on the expected date (day 21 post coitum), these dams were sacrificed on day 25 post coitum.

GROSS NECROPSY
All animals sacrificed were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.
At the scheduled sacrifice, all animals were weighed and sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.
Dead pups, except those excessively cannibalized, were examined macroscopically.
All parent animals and pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.
For the parent animals, special attention was directed at the organs of the reproductive system.
The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of apparently non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

HISTOPATHOLOGY / ORGAN WEIGHTS
TISSUE PRESERVATION:
The following tissues from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution:
Prostate, Seminal vesicles with coagulating gland, Testes (in Bouin’s fixative), Epididymides (in Bouin’s fixative)
The following tissues from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution:
Ovaries (with oviduct), Uterus (with vagina)
In addition, from all males and females the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:
Gross lesions, Brain, Spinal chord (cervical, thoracic, lumbar), Small and large intestines[2] (incl. Peyer’s patches), Stomach (forestomach and glandular stomach), Liver, Kidneys, Adrenals, Lymph nodes (axillary and mesenteric), Urinary bladder, Aorta[1], Heart, Thymus, Thyroids and parathyroids, Trachea and lungs (preserved by inflation with fixative and then immersion), Pituitary gland[1], Spleen, Peripheral nerve (sciatic), Bone marrow (femur), Femur with knee joint[1], Mammary gland (male and female)[1], Pancreas[1], Eyes with optic nerve and harderian gland[1], Lacrimal gland[1], Larynx[1], Nasal cavity[1], Esophagus[1], Salivary glands – mandibular, sublingual[1], Skeletal muscle[1], Sternum with bone marrow[1], Pharynx[1]
[1] only examined by histopathology in case of macroscopic findings indicative of potential toxicity
[2] duodenum, jejunum, ileum, colon, caecum, rectum

HISTOPATHOLOGY:
All organ and tissue samples to be examined by the study pathologist were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin. Special stains were used at the discretion of the study pathologist.
Testes, epididymides, prostate, seminal vesicles, ovaries, oviduct, vagina and uterus from all animals of the control and high-dose group were examined. The same applied to all occurring gross lesions. The remaining organs/tissues of 5 randomly selected males and females of the control and high-dose group, respectively, were examined histopathologically. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure. Histological examination of ovaries was carried out on the females that did not give birth.

ORGAN WEIGHTS:
At the scheduled sacrifice, the testes and epididymides of all parental males were weighed separately.
In addition, from 5 males and 5 females killed at the end of the study which were selected for hematology and clinical chemistry examination from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken.
Adrenal glands (weighed as pairs), Brain, Heart, Kidneys (weighed as pairs), Uterus (including cervix), Prostate, Liver, Thymus, Spleen, Thyroid (after fixation), Ovaries (weighed as pairs), Seminal vesicles (inclusive coagulating gland)

Postmortem examinations (offspring):

SACRIFICE
Pups were sacrificed on day 4 post partum.

GROSS NECROPSY
Dead pups, except those excessively cannibalized, were examined macroscopically. All pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

Statistics:

The following statistical methods were used to analyze food consumption, body and organ weights, clinical laboratory and reproduction data and macroscopical findings:
• Means and standard deviations of various data were calculated.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Reproductive indices:

mating performance and fertility indices, median and mean precoital time, post-implantation losses (absolute and percent), duration of gestation, implantation rate, corpora lutea count

Offspring viability indices:

mean litter size, pup sex ratios and viability indices.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

A female treated with 750 mg/kg bw/day test item died spontaneously. For this female a hunched posture and ruffled fur to a slight degree were noted starting 14 days prior to the spontaneous death. This was accompanied by weakened conditions with increasing severity and visible body weight loss (13%, started from day 6 post coitum onwards) between days 6 and 25 of gestation.
A weakened condition, hunched posture, ruffled fur, labored breathing, salivation and pale skin were noted in one male that died consequently to the aspiration of test item during gavage. These findings are common observations of accidental aspiration of fluid in the lungs. One female showed no clinical signs. In all animals of both genders, clinical signs were limited to slight hair loss or wounds as well as slight scabs on the tail apex to maximally two animals per group. Additionally, one female showed slightly discolored eyes towards the end of the gestation period.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

At 750 mg/kg bw/day, one male and two females died spontaneously. In one male (found dead on day 14 of the post-mating period) and one female (found dead on day 16 of gestation), the cause of death was deemed to be caused by aspiration of test item during gavage. Therefore it was considered, that the animals died treatment-related but not test item-related. In the second female (found dead on day 25 post coitum), severe ulceration of the forestomach was recorded which was considered to be a test item-related effect.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight gain was dose-dependently reduced during the pre-pairing period (19% and 38% at 250 and 750 mg/kg bw/day compared to the control group). The reduction in mean body weight gain resulted in reduced absolute mean body weights in these groups until the end of the study. During the pairing period, mean body weight gain remained reduced in group 4, whereas in group 3 mean body weight gain was similar to the control group. These reductions of mean body weights and mean body weight gains were considered to be test item-related. The statistically significantly lower body weight gain observed at 75 mg/kg bw/day on day 3 and day 7 of the pre-pairing group was considered to be incidental, since the differences to the control values were minimal and transient.
The overall differences in mean body weight gain at the dose levels of 0, 75, 250 and 750 mg/kg bw/day were: +16%, +15%, +13% and +10% during the pre-pairing period and +4%, +2%, +3% and -1% during the pairing period (percentages refer to the body weight gain within the period).

No test item-related effects on mean body weight and mean body weight gain were noted.
The overall differences in mean body weight gain at the dose levels of 0, 75, 250 and 750 mg/kg bw/day were: +9%, +10%, +10% and +7% during the pre-pairing period, +51%, +59%, +57% and +50% during the gestation period and +0%, -1%, +1% and +0% during the lactation period (percentages refer to the body weight gain within the period).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related effect on mean food consumption was noted.
A slightly statistically significant increase in food consumption was noted in females treated at 75 and/or 750 mg/kg bw/day during the pre-pairing period, which was considered to be incidental due to the absence of a dose-dependency.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At 750 mg/kg bw/day, absolute and relative reticulocyte count was statistically significantly increased in males. Although differences in reticulocyte values in females did not reach statistical significance, a similar trend towards higher values was noted. In the absence of other related changes in red blood cell parameters, this finding was not considered adverse. The other statistically significant differences noted at 750 mg/kg bw/day (higher neutrophil counts, lower lymphocyte counts in males, higher basophil counts in females) were within the range of historical control values and are deemed incidental.
The assessment of the hematology data did not reveal any test item-related effects in males and females treated at 75 and 250 mg/kg bw/day.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At 750 mg/kg bw/day in males, statistically significantly increased cholesterol levels were observed. The values were slightly higher than the historical control values. Since cholesterol concentrations were not altered in females, and in the absence of any other findings, this difference was not considered to be toxicologically relevant.
In all other cases, where statistically significant differences occurred (males: higher urea values at 75 and 250 mg/kg bw/day, higher creatinine values at 75 mg/kg bw/day, lower bilirubin values at 250 and 750 mg/kg bw/day, higher triglyceride values at 250 mg/kg bw/day, higher sodium values at 250 and 750 mg/kg bw/day, higher potassium and chloride values at 750 mg/kg bw/day, lower protein values at 750 mg/kg bw/day; females: higher ALAT activities at 250 mg/kg bw/day, higher ALP activities at 75 and 750 mg/kg bw/day, lower albumin values at 750 mg/kg bw/day), the values were within the historical control range of the rat strain used, and/or were observed in one sex only. Therefore these differences were considered not to be compound-related.

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No test item-related findings were noted during the functional observational battery in males or females at any dose level.
The mean fore- and hind limb grip strength values and body temperature of the control rats were similar to those of the test item-treated groups. Findings were limited to one female that was slightly defensive during lifting and had a short spontaneous vocalization when held in hand.
Locomotor activity was assessed quantitatively in terms of low beam counts in activity monitor. Locomotor activity was not affected by the treatment with the test item in males or females at any dose level.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In animals that were sacrificed at scheduled necropsy, the incidence and severity of the ulceration/erosion of the glandular and forestomach, squamous hyperplasia and/or inflammatory cell infiltration in the submucosa of the stomach were increased in animals of group 3 and 4. Three animals (1 male and 2 females) of group 4 died spontaneously. Histopathologically, a respiratory disorder consisting of congestion, alveolar edema, alveolar hemorrhage, alveolitis and alveolar macrophages of the lung were recorded in two (1 male and 1 female) out of these three animals. It was deemed to be caused by aspiration of test item during gavage. In the second female, severe ulceration of the forestomach was recorded. No test item-related histological findings were recorded in the ovaries of females that did not give birth. At sperm staging, no differences on the completeness of stages or cell populations of the testes were recorded between controls and high dose animals.
The remainders of findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

All animals mated within the first pairing period. For one female in group 1, mating was not detected.
The median and mean precoital times were unaffected by treatment with the test item. Mean precoital times were 3.1, 2.8, 3.1 and 3.1 days in groups 1, 2, 3 and 4, respectively. The median precoital time was 3 days in groups 1 to 3 and 4 days in group 4.

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
At 750 mg/kg bw/day, one male and two females died spontaneously. In one male (no. 40, found dead on day 14 of the post-mating period) and one female (no. 85, found dead on day 16 of gestation), the cause of death was deemed to be caused by aspiration of test item during gavage. Therefore it was considered, that the animals died treatment-related but not test item-related.

In the second female (no. 79, found dead on day 25 post coitum), severe ulceration of the forestomach was recorded which was considered to be a test item-related effect.

Findings Related to Treatment with the Test Item:
For female no. 79 treated at 750 mg/kg bw/day, that died spontaneously, a hunched posture and ruffled fur to a slight degree were noted starting 14 days prior to the spontaneous death. This was accompanied by weakened conditions with increasing severity and visible body weight loss (13%, started from day 6 post coitum onwards) between days 6 and 25 of gestation.

Findings Unrelated to Treatment with the Test Item:
A weakened condition, hunched posture, ruffled fur, labored breathing, salivation and pale skin were noted in male no. 40 that died consequently to the aspiration of test item during gavage. These findings are common observations of accidental aspiration of fluid in the lungs. Female no 85 showed no clinical signs.

In all animals of both genders, clinical signs were limited to slight hair loss or wounds as well as slight scabs on the tail apex to maximally two animals per group. Additionally, one female showed slightly discolored eyes towards the end of the gestation period.

BODY WEIGHT (PARENTAL ANIMALS)
MALES:
Pre-pairing and Pairing Periods
Mean body weight gain was dose-dependently reduced during the pre-pairing period (19% and 38% at 250 and 750 mg/kg bw/day compared to the control group). The reduction in mean body weight gain resulted in reduced absolute mean body weights in these groups until the end of the study. During the pairing period, mean body weight gain remained reduced in group 4, whereas in group 3 mean body weight gain was similar to the control group.

These reductions of mean body weights and mean body weight gains were considered to be test item-related.

The statistically significantly lower body weight gain observed at 75 mg/kg bw/day on day 3 and day 7 of the pre-pairing group was considered to be incidental, since the differences to the control values were minimal and transient.

The overall differences in mean body weight gain at the dose levels of 0, 75, 250 and 750 mg/kg bw/day were: +16%, +15%, +13% and +10% during the pre-pairing period and +4%, +2%, +3% and -1% during the pairing period (percentages refer to the body weight gain within the period).

FEMALES:
Pre-pairing, Pairing, Gestation and Lactation Periods

No test item-related effects on mean body weight and mean body weight gain were noted.

The overall differences in mean body weight gain at the dose levels of 0, 75, 250 and 750 mg/kg bw/day were: +9%, +10%, +10% and +7% during the pre-pairing period, +51%, +59%, +57% and +50% during the gestation period and +0%, -1%, +1% and +0% during the lactation period (percentages refer to the body weight gain within the period).

FOOD CONSUMPTION (PARENTAL ANIMALS)
MALES:
Pre-Pairing Period

No test item-related effect on mean food consumption was noted.

A transient, slightly higher statistically significant increase in food consumption was noted in males treated at 75 and 250 mg/kg bw/day during the pre-pairing period, which was considered to be incidental due to the absence of a dose-dependency.

FEMALES:
Pre-pairing, Gestation and Lactation Periods

No test item-related effect on mean food consumption was noted.

A slightly statistically significant increase in food consumption was noted in females treated at 75 and/or 750 mg/kg bw/day during the pre-pairing period, which was considered to be incidental due to the absence of a dose-dependency.

MATING PERFORMANCE AND FERTILITY
All animals mated within the first pairing period. For one female in group 1, mating was not detected.

The median and mean precoital times were unaffected by treatment with the test item. Mean precoital times were 3.1, 2.8, 3.1 and 3.1 days in groups 1, 2, 3 and 4, respectively. The median precoital time was 3 days in groups 1 to 3 and 4 days in group 4.

One female each in groups 1 and 2 as well as 2 females in group 3 and 3 females in group 4 were not pregnant. As a result the fertility index in the control group and group 2 was 90.9%, in group 3 it was 81.8% and in group 4 it was 72.7%. All fertility indices were within the historical range of the rat strain used (60 to 100%).

Corpora Lutea Count
Mean number of corpora lutea per dam (determined at necropsy) was similar in all groups (14.3, 14.7, 14.8 and 13.1 in order of ascending dose level) and gave no indication of a test item-related effect.

Duration of Gestation
The mean duration of gestation was unaffected by exposure to the test item. Mean duration of gestation was 21.7, 21.4, 21.3 and 21.4 days, in order of ascending dose level.

Implantation Rate and Post-implantation Loss
No effects on implantation rate or post-implantation loss were observed at any dose level.

The mean number of implantations per dam was 11.1, 13.5, 13.4 and 11.7 in order of ascending dose levels. The mean incidence of post-implantation loss as a percentage of total implantations was 14.4%, 10.4%, 5.0% and 4.9%, in order of ascending dose level.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In males and females no test item-related effects on organ weights were noted.

In males, slight, but statistically significant differences in absolute testis and epididymides weights at 750 mg/kg bw/day were not confirmed by the relative values. Additionally, there were no compound-related histological findings in these organs. Therefore these differences were considered to be fortuitous. The same applies to higher adrenal weights at 75 mg/kg, and higher relative brain weights at 250 mg/kg bw/day, which occurred without dose-dependency.

In females, a statistically significant decrease in absolute and relative thymus weight was noted in animals treated at 75 and 750 mg/kg bw/day. Since one control animal (no. 54) had a higher thymus weight than average and due to the small number of animals in the group, this finding can be disregarded as being within the normal biological variance. The statistically significant differences in heart (lower weights at 250 and 750 mg/kg bw/day), kidney (higher relative weights at 75 mg/kg bw/day) and uterus weight (higher weights at 250 mg/kg bw/day) did not show a dose-relationship and were therefore considered to be incidental.


GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no test item-related findings noted at necropsy.

The macroscopical findings in males that were killed at planned necropsy consisted of a reddish discoloration of the pancreas (1 animal each in groups 1 to 4), reddish or dark red discoloration of the lymph nodes (1 animal each in groups 2 and 3, and 2 animals in group 4) and isolated dark red or brownish foci in the thymus. Additionally, one control male showed a watery cyst on the kidneys, two males of group 2 had foci on the left lung lobe and one animal of group 4 had dark red foci on the tongue.
In females killed at necropsy, the following macroscopical findings were noted: dark red discoloration of the ovaries (1 animal in each of groups 1 to 3) and reddish discoloration of the mandibular lymph nodes (one animal each in groups 1 and 2). Additionally, black firm nodules were noted in the liver of one control animal, pelvic dilation of the kidneys in one animal of group 2, a watery cyst on the ovaries of one group 3 animal and alopecia in the chest region with marked scars in one animal of group 4.

All these findings were considered to be incidental due to their low incidence and since there is no correlation to histopathological results.

In animals that died spontaneously similar findings as stated above were noted. Additionally, signs of autolysis were detected as well as signs of aspiration of fluid in the lungs.

HISTOPATHOLOGY (PARENTAL ANIMALS)
In animals that were sacrificed at scheduled necropsy, the incidence and severity of the ulceration/erosion of the glandular and forestomach, squamous hyperplasia and/or inflammatory cell infiltration in the submucosa of the stomach were increased in animals of group 3 and 4.

Three animals (1 male and 2 females) of group 4 died spontaneously. Histopathologically, a respiratory disorder consisting of congestion, alveolar edema, alveolar hemorrhage, alveolitis and alveolar macrophages of the lung were recorded in two (1 male and 1 female) out of these three animals. It was deemed to be caused by aspiration of test item during gavage. In the second female, severe ulceration of the forestomach was recorded.

No test item-related histological findings were recorded in the ovaries of females that did not give birth. At sperm staging, no differences on the completeness of stages or cell populations of the testes were recorded between controls and high dose animals.

The remainders of findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

750 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest tested dose

Remarks on result:

other: Generation: P and F1 (migrated information)

2

Dose descriptor:

NOAEL

Remarks:

reproduction/development

Effect level:

750 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest tested dose

Remarks on result:

other: Generation: P and F1 (migrated information)

3

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

75 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: This NOAEL is based on local irritation at the stomach, and the only additional effect was limited to a decreased body weight gain in males which is most likely due to these local effects.

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormal findings were noted at first litter check or during the first 4 days post partum.
Sex ratios at first litter check and on day 4 post partum were unaffected by exposure to the test item. The proportion of males at first litter check was 55, 48, 42 and 62%, in order of ascending dose level.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean pup weights on day 0 and day 1 post partum were unaffected by treatment with the test item.
On day 1 post partum mean pup weights were 6.5, 6.3, 5.9 and 6.2 g in order of ascending dose level. Also mean body weight gain was similar in all groups and on day 4 post partum and no effects on mean body weights were recorded.

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

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No findings were noted at macroscopic examination of F1 pups.

Histopathological findings:

not examined

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

LITTER SIZE AT FIRST LITTER CHECK
No effects on litter size were observed at any dose level.

Mean litter size at first litter check was 9.5, 12.2, 12.8 and 11.3 pups in order of ascending dose levels. No dead pups at first litter check were recorded in any group.

POSTNATAL LOSS DAY 0 - 4 POST PARTUM
No effects on postnatal loss were observed at any dose level.

Incidentally, 1 pup was lost in group 2 between days 0 - 4 post partum.

LITTER DATA - F1 PUPS
External Examination at First Litter Check and during Lactation:
No abnormal findings were noted at first litter check or during the first 4 days post partum.

Sex Ratios
Sex ratios at first litter check and on day 4 post partum were unaffected by exposure to the test item.

The proportion of males at first litter check was 55, 48, 42 and 62%, in order of ascending dose level.

Body Weights to Day 4 Post Partum
Mean pup weights on day 0 and day 1 post partum were unaffected by treatment with the test item.

On day 1 post partum mean pup weights were 6.5, 6.3, 5.9 and 6.2 g in order of ascending dose level. Also mean body weight gain was similar in all groups and on day 4 post partum and no effects on mean body weights were recorded.

Macroscopical Findings
No findings were noted at macroscopic examination of F1 pups.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

750 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest tested dose

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item to rats. The test item was administered in olive oil as vehicle at dosages of 75, 250, and 750 mg/kg body weight/day, and controls received the vehicle only. The test item was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

 

At 750 mg/kg bw/day, one female died spontaneously at the end of the gestation period after showing hunched posture and ruffled fur and visible body weight loss. During necropsy severe ulcerations of the forestomach were recorded. By histopathological examination it was also observed in the other animals at 250 and 750 mg/kg body weight/day that the incidence and severity of the ulceration/erosion of the glandular and forestomach, squamous hyperplasia and/or inflammatory cell infiltration in submucosa were increased.

 

In males, mean body weight gain was dose-dependently reduced during the pre-pairing period at 250 and 750 mg/kg bw/day, which resulted in reduced mean body weights until the end of the study. Body weight gain remained decreased at 750 mg/kg bw/day during the post-pairing period.

 

No compound-related adverse effects were observed by hematology and clinical chemistry. Likewise, no compound-related effects on reproduction or development were observed.

 

Based on these results a general NOAEL (No Observed Adverse Effect Level) was considered to be 75 mg/kg body weight/day. However, it should be noted that this NOAEL was primarily based on local irritation at the stomach, and the only additional effect was limited to a decreased body weight gain in males. No signs of systemic toxicity were evident by hematological and clinical chemistry examinations, organ weights, as well as macroscopic and microscopic examinations.

Therefore, the systemic NOAEL is set at 750 mg/kg body weight/day and 75 mg/kg body weight/day is considered as local NOAEL for irritation at the stomach.

 

The NOEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 750 mg/kg body weight/day.

Applicant's summary and conclusion

Conclusions:

Based on these results a general NOAEL (No Observed Adverse Effect Level) was considered to be 75 mg/kg body weight/day. However, it should be noted that this NOAEL was primarily based on local irritation at the stomach, and the only additional effect was limited to a decreased body weight gain in males. No signs of systemic toxicity were evident by hematological and clinical chemistry examinations, organ weights, as well as macroscopic and microscopic examinations.
The NOEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 750 mg/kg body weight/day.

Executive summary:

The purpose of this study was to generate preliminary information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provided information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

The test item was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

The following dose levels were applied:

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

Group 2: 75 mg/kg body weight/day

Group 3: 250 mg/kg body weight/day

Group 4: 750 mg/kg body weight/day

A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (olive oil).

The following results were obtained:

At 750 mg/kg bw/day, one female died spontaneously at the end of the gestation period. It showed a hunched posture and ruffled fur to a slight degree starting 14 days prior to the spontaneous death. This was accompanied by a weakened condition with increasing severity and visible body weight loss. Severe ulcerations/erosions of the forestomach were observed by histopathological examination. All these findings were considered to be test item-related. No other test item-related clinical signs were noted in males or females at any dose level. No effects on food consumption of males and females were observed at any dose level.

In males, mean body weight gain was dose-dependently reduced during the pre-pairing period at 250 and 750 mg/kg bw/day. The reduction in mean body weight gain resulted in reduced absolute mean body weights in these dose groups until the end of the study. During the following pairing period, mean body weight gain remained reduced at 750 mg/kg bw/day. In females, no test item-related effects on mean body weight and mean body weight gain were noted. No test item-related findings were noted. Mean precoital time, fertility index and conception rate were not affected by the treatment with the test item.

No effects on implantation loss or postnatal loss were observed. No effects on organ weights were noted in any group. There were no test item-related findings noted at necropsy. The incidence and severity of the ulceration/erosion of the glandular and forestomach, squamous hyperplasia and/or inflammatory cell infiltration in the submucosa of the stomach were dosedependently increased at 250 and 750 mg/kg bw/day.

The mean number of pups at first litter check was not affected by the treatment with the test item. The sex ratio was also not affected. No abnormal pup was noted at any dose level. No effects on pup weight and pup weight gain were observed. At necropsy of pups, there were no abnormal findings.

Based on these results a general NOAEL (No Observed Adverse Effect Level) was considered to be 75 mg/kg body weight/day. However, it should be noted that this NOAEL was primarily based on local irritation at the stomach, and the only additional effect was limited to a decreased body weight gain in males. No signs of systemic toxicity were evident by hematological and clinical chemistry examinations, organ weights, as well as macroscopic and microscopic examinations.

The NOEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 750 mg/kg body weight/day.