Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
28 June 2012- 18 April 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was performed according to GLP and guideline. The study is not a developmental study in itself but laboratory investigations including litter responses to exposure to the test item were examined as part of the study.
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): (z)-hex-3-enyl acetate (leaf acetate)
- Physical state: liquid
- Analytical purity: 98.45 %
- Lot/batch No.: 1Z00151
- Expiration date of the lot/batch: 13 June 2015
- Stability under test conditions: stable at room temperature (15- 25 °C) for at least 8 d
- Storage condition of test material: at room temperature (15- 25 °C) away from direct sunlight, N2 into the container

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V. Kreuzelweg 53, 5961 NM Horst/ Netherlands
- Age at study initiation: (P) 10 wks
- Weight at study initiation: (P) Males: 310- 360 g; Females: 190- 234 g
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding. During the pairing period females were housed with sexually mature males (1:1) until evidence of copulation was observed.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 d

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dose formulations were not corrected for purity and were prepared as supplied. The dose formulations were prepared weekly as indicated by the results of stability analyses in the dose range-finding study (Harlan Laboratories Ltd, 2012). The test item was weighed into a glass beaker on a tared Mettler balance and the vehicle was added (w/v). The mixtures were stirred using a magnetic stirrer and stored at room temperature (15 - 25 °C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Lot/batch no. (if required): BCBG8285V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulations were analysed using HPLC. Concentration, homogeneity and stability of dose formulations were determined in samples taken after experimental start.
- Transport of dose formualtions to Analytical Laboratory: At ambient temperature (20 ± 5 °C)
- Storage of dose formulations in Analytical Laboratory: Frozen (ca. -20 ± 5 °C)
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 d
- Proof of pregnancy: daily vaginal smearwas sperm positive, a copulation plug was observed
- After 14 days of unsuccessful pairing, a second pairing of this female with a male in the same group, which had already mated successfully, was considered
- Further matings after two unsuccessful attempts: no. If mating was not recorded during this additional pairing period of a maximum of 14 days, the female was sacrificed and, if indicated, the reproductive organs examined histopathologically in order to ascertain the reason for the infertility
- After successful mating each pregnant female was removed and housed individually.
- 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.
Duration of treatment / exposure:
Males: minimum 4 weeks
Females: approximately 7 weeks
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
11/sex/ dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected bsed on the results of a non-GLP dose range-finding study (Harlan Laboratories Ltd, 2012)

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the first administration of the test item and weekly thereafter, detailed clinical observations were performed outside the home cage. In females, it was performed additionally, on days 0, 6, 13 and 20 of the gestation period.
- Females were observed for signs of difficult or prolonged parturition, and behavioural abnormalities in nesting and nursing
- 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:
- Time schedule for males: weekly during pre-pairing and after pairing periods
- Time schedule for females: pre-pairing period days 1-8 and 8- 14; gestation days 0- 7, 7- 14 and 14- 21 post coitum and days 1- 4 post partum
- No food consumption was recorded during the pairing period

HAEMATOLOGY: Yes
The following hematology parameters were determined:
- Complete Blood Cell Count: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Hemoglobin concentration distribution width, Leukocyte count, total, Differential leukocyte count, Platelet count
- Coagulation: Prothrombin time (= Thromboplastin time), Activated partial Thromboplastin time

CLINICAL BIOCHEMISTRY
- The following clinical biochemistry parameters were determined: Glucose, Urea, Creatinine, Bilirubin, total, Cholesterol, total, Triglycerides, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Gamma-glutamyl-transferase, Bile acids, Sodium, Potassium, Chloride, Calcium, Phosphorus, Protein, total, Albumin, Globulin, Albumin/Globulin ratio

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were obtained at the end of the pre-pairing period from 5 females from each group. Blood samples were drawn sublingually from all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms
- Animals fasted: Yes. The animals were fasted for approximately 18 h before blood sampling but allowed access to water ad libitum
- How many animals: Blood samples were drawn sublingually from all animals under light isoflurane

OTHER
- Termination of the Study: Dams were sacrificed on day 4 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

- Necropsy: All animals sacrificed or found dead were weighed and 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 sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated. All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

- Organ Weights: Five females killed at the end of the study which were selected 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), Liver, Thymus, Spleen

-Tissue Preservation: Five females per group selected for organ weights and from all animals found dead or killed in extremis, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution: Gross lesions, Brain, Spinal chord, Small and large intestines (incl. Peyer’s patches), Stomach, Liver, Kidneys, Adrenals, Spleen, Heart, Thymus, Thyroids, and parathyroids if possible, Trachea and lungs (preserved by inflation, with fixative and then immersion), Urinary bladder, Lymph nodes (mesenterial, mandibular), Peripheral nerve (sciatic), Bone marrow

-Histotechnique: All organ and tissue samples to be examined by the prinicipal investigator for histopathology were processed, embedded and cut at an approximate thickness of 2 - 4 µm and stained with hematoxylin and eosin. Additionally, the testes were stained by PAS-hematoxylin.

-Histopathology: Slides of all organs and tissues listed collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the prinicipal investigator for histopathology. The same applied to all occurring gross lesions and to all animals, which died spontaneously or had to be terminated in extremis. If test item-related morphologic changes were detected in organs of any high-dose animal, those same organs from the mid- and low-dose group were examined to establish a no-effect level, if possible.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- 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 non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites
-Tissue Preservation: The following tissues from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution: Ovaries. Five females per group selected for organ weights and from all animals found dead or killed in extremis, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution: Uterus (with vagina)
- Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, if necessary.
Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
- 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
Indices:
The following data were recorded on-line: reproduction and litter data (RCC-TOX LIMS or TOX CONTROL, as appropriate).

From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, post-implantation losses, mean litter size and pup sex ratios.

For reproduction data, group mean values were calculated both on a litter basis and on a percentage per group basis. Mean pup weights were calculated from the individual weights both on a per group and on a per litter basis.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
MORTALITY
One female (no. 80) of the dose group 1000 mg/kg bw/day was sacrificed in extremis on day 20 post coitum. One female (no. 67) was found dead on day 11 post coitum, treated with 300 mg/kg/day of the test item. Two females ( (nos. 57, 63) treated with 100 mg/kg/day of the test item were found dead on day 22 and on day 3 post coitum, respectively. These deaths were considered to be caused by aspiration during gavage procedures. The deaths were not related to systemic toxicity of the test item. All other animals survived until the scheduled necropsy.

CLINICAL SIGNS OR OBSERVATIONS
Almost females of all dose groups including controls showed slightly soft feces from treatment day 6 (pre-pairing period) onwards. This could be considered to be an effect of the vehicle.

No other clinical signs were noted in females treated with 100 mg/kg/day of the test item during the course of the study.

One female of the dose group 300 mg/kg bw/day had slight dyspnea on two days of the pre-pairing period. Another female of this group had slight breathing noises on four days of the pairing period and on two days of the gestation period as well as a third female had this on one day of the gestation period.

Slight breathing noises were noted in 4 females of the dose group 1000 mg/kg bw/day on up to six days at the end of the gestation period. Slight reddish nasal secretion and slight ptosis were noted in 1 female on one day of the gestation period.

All these findings could be observed by aspiration during gavage procedures and are therefore considered to be no adverse findings.

No clinical findings were noted in females during weekly observations in any period.

FUNCTIONAL OBSERVATIONAL BATTERY
No statistically significant differences in mean values of grip strength (fore- and hind paws) were noted in test item treated females when compared with the control animals. The mean body temperature of test item treated females showed no statistically significant differences when compared with the control animals.

LOCOMOTOR ACTIVITY
No changes in mean locomotor activity were noted in test item treated females when compared with the control animals.

FOOD CONSUMPTION
- Pre-Pairing, Gestation and Lactation Periods: No effects on mean food consumption of females treated with 100, 300 or 1000 mg/kg/day were noted when compared with the controls.

BODYWEIGHT
- Pre-Pairing, Pairing, Gestation and Lactation Periods: No statistically significant differences in mean body weight of test item treated females were noted when compared with the control females.

CLINICAL LABORATORY INVESTIGATIONS
Haematology
The mean value of total leucocytes was slightly decreased (p<0.05) in females treated with 100 mg/kg/day of the test item when compared with controls. The mean value of haemoglobin concentration distribution width and the mean value of large unstained cells were slightly decreased (p<0.05) in females treated with 300 mg/kg/day. No dose response relationship could be observed in these cases. Therefore the slightly decreased values were considered to be incidental.

Clinical biochemistry
No statistically significant differences in parameters of clinical biochemistry were noted in test item treated females when compared with control males.

ORGAN WEIGHTS
In females treated with 100 mg/kg/day the mean thymus weight, the mean thymus to body weight ratio and the mean thymus to brain weight ratio were decreased when compared with the controls (p<0.05, p<0.01, p<0.05; respectively). The mean thymus to body weight ratio was also decreased (p<0.05) in females treated with 1000 mg/kg/day of the test item. This was considered to be not test item related because no dose response relationship could be observed. The mean heart to body weight ratio was increased (p<0.05) in females treated with 100 mg/kg/day what was also considered to be not test item related.

MACROSCOPICAL FINDINGS
In animals found spontaneously dead or which were sacrificed in extremis in dose groups, reddish discoloration and incomplete collapse of the lung, and reddish discoloration and red focus/foci of the thymus were recorded. There were no gross lesions that could be attributed to treatment with the test item in sacrificed animals. All other gross lesions recorded were considered to be within the range of normal background alterations.

HISTOPATHOLOGY FINDINGS
Findings in Animals Decedents
The respiratory disorder consisted of necrosis and inflammatory cell infiltration of the trachea, congestion, alveolar edema, interstitial edema, alveolar hemorrhage and alveolar macrophages of the lung were recorded. Major microscopic findings with macroscopic findings in each animal were described as follow:
No.57
- Trachea: minimal inflammatory cell infiltration was recorded.
- Lung: reactive change consisting of slight congestion, slight alveolar edema, minimal alveolar hemorrhage and minimal alveolar macrophages was recorded. (Macroscopic findings: Incompletely collapse and Discoloration, dark red)
- Thymus: Multifocal slight hemorrhage as lesion during agonal period. (Macroscopic findings: Discoloration, dark red)

No.63
- Trachea: moderate mucosal necrosis and slight inflammatory cell infiltration were recorded and acute reactive and inflammatory changes were considered.
- Lung: reactive change consisting of slight congestion, slight alveolar edema, slight interstitial edema, minimal alveolar hemorrhage and slight alveolar macrophages was recorded. (Macroscopic findings: Discoloration, dark red)
- Thymus: Multi focal slight hemorrhage as lesion during agonal period. (Macroscopic findings: Focus/Foci, dark red)

No.67
- Trachea: marked mucosal necrosis and moderate inflammatory cell infiltration was recorded and acute reactive and inflammatory changes were considered.
- Lung: reactive change consisting of moderate congestion, slight alveolar edema, slight interstitial edema, slight alveolar hemorrhage and minimal alveolar macrophages was recorded. (Macroscopic findings: Discoloration, dark red)
- Thymus: Multi focal slight hemorrhage as lesion during agonal period. (Macroscopic findings: Focus/Foci, dark red)

No.80
- Trachea: moderate mucosal necrosis and moderate inflammatory cell infiltration was recorded and acute reactive and inflammatory changes were considered.
- Lung: minimal inflammatory exudate in Bronchi was recorded.

Findings in Schedule Sacrificed Animals
All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age

MATING PERFORMANCE AND FERTILITY
Within the first pairing period mated 10 control females and 10 females treated with 300 mg/kg/ day as well as 8 females treated with 100 mg/kg/day and 8 females treated with 1000 mg/kg/day. In the second pairing period, 1 female treated with 100 mg/kg/day mated, in 1 female of this group the mating was not detected as well as in one control female, in 1 female treated with 300 mg/kg/day and in 3 females treated with 1000 mg/kg/day.

The median and mean precoital times were unaffected by treatment with the test item. Mean precoital times were 3.3, 2.5, 4.0 and 2.9 days in the control and dose groups 100, 300 and 1000 mg/kg bw/day, respectively. The median precoital time was 4, 3, 3 and 3 days in order of ascending dose level. For the female in the dose group 100 mg/kg bw/day that mated during the second pairing period, precoital time was 4 days. The fertility index and conception rate were 90.9% in the control and dose group 300 mg/kg bw/day, 100.0% in dose group 100 mg/kg bw/day and 90.9% in dose group 1000 mg/kg bw/day. The gestation index was 90.0% in the control, 80.0% in dose groups 100 and 300 mg/kg bw/day and 87.5% in dose group 1000 mg/kg bw/day.

CORPORA LUTEA COUNT
No toxicologically relevant difference between the mean numbers of corpora lutea per dam(determined at necropsy) were noted between the groups (13.6, 13.5, 13.9 and 14.7 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 21.6, 21.3, 21.4 and 21.5 days, in order of ascending dose level.

IMPLANTATION RATE AND POST-IMPLANTATION LOSS
The mean number of implantations per dam was similar in all groups and test item treated groups showed no statistical significant changes when compared with the controls. The mean numbers of implantations per litter were 12.0, 13.1, 13.4 and 13.3 in order of ascending dose level. The mean incidence of post-implantation loss as a percentage of total implantations was 12.0, 0.0, 6.5 and 7.5%, in order of ascending dose level.

LITTER SIZE AT FIRST LITTER CHECK
In the control and dose groups 100, 300 and 1000 mg/kg bw/day the birth index was 88.0, 100.0, 94.4 and 92.5% and mean litter size at first litter check was 10.4, 13.1, 12.4 and 12.3, respectively.

POSTNATAL LOSS DAYS 0 - 4 POST PARTUM
Mean postnatal loss was 0.1, 0.0, 0.3 and 0.0% in the control and dose groups 100, 300 and 1000 mg/kg bw/day, respectively. No statistically significant changes in the viability index of test item treated groups compared with control animals were noted. In the control and dose groups 100, 300 and 1000 mg/kg bw/day the viability index was 98.9, 100.0, 98.0 and 100.0%.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:not examined

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Under the conditions of this study, four females of different dose groups were dead spontaneously or sacrificed in extremis. According to histological findings, aspiration during gavage procedures could be considered as the cause of these deaths, they are not related to systemic toxicity of the test item. Hemorrhage of the thymus was recorded and considered to be a lesion during agonal period.

All other animals survived the scheduled study period.

No gross lesions could be attributed to treatment in schedule sacrificed animals.

The test item produced no histological evidence of toxicological properties in the organs and tissues examined in schedule sacrificed animals.

The mean precoital time, fertility index, gestation index and conception rate were not affected by the treatment with the test item, also the implantation rate was unaffected. No impact on the post implantation loss was observed.

No test item related influence on pups (body weights, macroscopic findings, sex ratio, e.g.) was noted during the first litter check and on day four post partum.

Applicant's summary and conclusion

Conclusions:
The potential reproductive/ developmental toxicity of the test material was assessed according to OECD guideline 422. Based on the results of this study, the general NOAEL of the test material administered orally by gavage to Wistar rats, could be established at 1000 mg/kg bw/day as well as for reproduction/ developmental toxicity.