Hexahydro-4,7-methano-1H-indenyl acrylate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP 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 (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Hexahydro-4,7-methano-1H-indenyl acrylate

EC Number:

235-697-2

EC Name:

Hexahydro-4,7-methano-1H-indenyl acrylate

Cas Number:

12542-30-2

Molecular formula:

C13 H16 O2

IUPAC Name:

2-Propenoic acid hexahydro-4,7-methano-1H-indenyl ester

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Dihydrodicyclopentadienylacrylate
- Physical state: liquid/colorless, clear
- Analytical purity: 97.7 corr area% as a sum of four singel peaks. The main peak fo these four peaks has 91.3 corr area%
- Lot/batch No.: 110019P040

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany
- Age at study initiation: 11 - 13 weeks old
- Weight at study initiation: 314.3 - 345.7 g (male); 194.5 g - 220.7 g (female)
- Housing: housed individually, Makrolon type M II cages; During overnight matings, male and female mating partners were housed together in
Makrolon type M III cages. Pregnant animals and their litters were housed together until PND 4.
- Diet (e.g. ad libitum): ad libitum (Kliba maintenance diet mouse/rat "GLP" meal)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance solutions in corn oil were prepared at the beginning of the administration period and thereafter in intervals, which took into account the analytical results of the stability verification.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance in corn oil for a period of 7 days at room temperature were carried out prior to the start of the study.
Samples of the test substance solutions were sent to the analytical laboratory once at the beginning of the study for verification of the concentrations.

Duration of treatment / exposure:

The duration of treatment covered a 2-week pre-mating and a mating period in both sexes, 1 week post-mating in males, and the entire gestation period as well as approximately 2 weeks of the lactation period.

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw
Basis:

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CLINICAL OBSERVATION:
Morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each animal.
The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams.

BODY WEIGHT:
Body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice.
The following exceptions are notable for the female animals:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0) and on PND 4.

FOOD CONSUMPTION:
Food consumption was determined once a week for male and female parental animals:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0, 7, 14 and 20.
• Food consumption of F0 females, which gave birth to a litter was determined on PND 1 and 4.

CLINICAL PATHOLOGY: Yes
The blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba®, Essex GmbH Munich, Germany). For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. Urine samples were evaluated in a randomized sequence.
The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.
The following examinations were carried out in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group.

NEUROBEHAVIOURAL EXAMINATION: Yes
A functional observational battery was performed. The FOB started with passive observations without disturbing the animals, followed by removal
from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random..
Home cage observations: The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: Posture, Tremors, Convulsions, Abnormal movements, Impairment of gait, Other findings
Open field observations: The following parameters were examined: Behavior when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/stereotypy, impairment of gait, activity/arousal level)
Sensory motor tests/Reflexes: Approach response, touch response, vision (“visual placing response”), pupillary reflex, pinna reflex, audition (“startle response”), coordination of movements (“righting response”), behavior during “handling”, vocalization, pain perception (“tail pinch”), other findings, grip strength of forelimbs and hindlimbs, Landing foot-splay test
Motor activity measurement (MA): The examinations were performed using the Multi-Varimex system supplied by Columbus Instruments Int. Corp., Ohio, U.S.A. For this purpose, the animals were placed in clean polycarbonate cages for the time of measurement. The number
of beam interrupts was counted over 12 intervals for 5 minutes in each case.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

Statistics:

DUNNETT-test (twosided)
FISHER'S EXACT test
WILCOXON-test (onesided)
WILCOXON-test (twosided)
KRUSKAL-WALLIS test (two-sided)

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no test substance-related or spontaneous mortalities in any of the groups.
Several male and female animals of all dose groups showed salivation after treatment in all phases of the study. This transient salivation for a few minutes immediately after treatment was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It was not considered to be a sign of systemic toxicity. One sperm positive control female (0 mg/kg bw/d - No. 107) did not deliver F1 pups.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights and mean body weight change of the male and female F0 generation parental animals in all test substance-treated groups (test groups 1 - 3) were comparable to the concurrent control group during the entire study period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
With one exception, food consumption of the male and female F0 generation parental animals in all test substance-treated groups (test groups 1 - 3; 100, 300 and 1000 mg/kg bw/d) was comparable to the concurrent control group during the entire study period.
The observation period premating days 0 - 7 (females, test group 3) was the only time in which a statistically significantly decrease of food consumption was determined. Because of the singular observation of decreased food consumption and the small size of change (-7% deviation versus control) this observation was considered as spontaneous in nature and not treatment related.

CLINICAL PATHOLOGY
Concerning clinical pathology including the analyses of red and white blood cells, coagulations parameters, enzymes, substrates, electrolytes, and minerals no treatmentrelated, adverse effects were observed up to limit dose (1000 mg/kg bw/d). The higher incidences of transitional epithelial cells, granulated and epithelial cell casts in the urine sediment as well as a lower pH value of the urine found in test group 3 (1000 mg/kg bw/d)
and partially in test group 2 (300 mg/kg bw/d) in males only indicated a α2u-Globulinuria. It is a regular finding in treated males of this age, but is regarded as not relevant for humans (Hard et al., 1993).
Male and female animals of all dose groups (1000, 300 and 300 mg/kg bw/d) did not show any abnormalities.

NEUROBEHAVIOUR
The functional observational battery (FOB), and measurement of motor activity (MA) no significant differences to control were observed at any dose level.

PATHOLOGY
The significant absolute and relative weight increase in the kidneys of test groups 2 and 3 (300 and 1000 mg/kg bw/d) and in the liver of test group 3 (1000 mg/kg bw/d) is considered to be treatment-related. The relative liver weight increase of test group 2 (300 mg/kg bw/d) is not regarded as treatment-related due to the low increment and weak significance. The significant absolute and/or relative weight increase of the epididymides and testes is regarded as incidental since no correlating histopathological findings were observed. Furthermore, no dose-dependent relationship was found for the epididymides weight increase.

All gross findings noted at necropsy are regarded as incidental and spontaneous in nature and are not related to treatment.

Histopathology: Treatment-related findings were observed in the kidneys of males. Intracytoplasmic eosinophilic droplets in proximal convoluted tubules were positive for
Mallory-Heidenhain’s stain. A minimal to slight storage of eosinophilic cytoplasmic droplets was observed in the proximal tubules of control animals. Whereas the incidence and grading of eosinophilic cytoplasmic droplets in test group 1 (100 mg/kg bw/d) was comparable to the control group, this finding showed a tendency to increase from slight in test group 2 (300 mg/kg bw/d) to moderate in test group 3 (300 mg/kg bw/d). This change was characterized by an increase in the size and/or density of eosinophilic droplets, with increasing irregular forms, accompanied by an increase of affected tubules. A minimal increase in the incidence but not in the grading of basophilic tubules was also noted in the kidneys of males and females of test group 3 (1000 mg/kg bw/d) when compared with their respective control groups. However, this minimal change is considered to be within the
spectrum of normal background lesions and is not in relation with the storage of eosinophilic droplets. No histopathological correlate was found for the absolute and relative liver weight increase in males of test groups 3 (1000 mg/kg bw/d). All other findings noted were either single observations, or were biologically equally distributed between controls and treated rats. All of them were considered to be incidental and/or spontaneous in origin.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Under the conditions of this OECD 422 combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in Wistar rats no adverse effects were observed up to the limit dose tested (1000 mg/kg bw/d). Thereby, the following NOAEL (no

observed adverse effect level) of Dihydrodicyclopentadienylacrylate were determined:

• The NOAEL for general, systemic toxicity was 1000 mg/kg bw/d for the F0 females and males.