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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

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)

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

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on 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.

Details on mating procedure:

- M/F ratio per cage:1:1
- Length of cohabitation:overnight for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as [day 0] of pregnancy

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

Remarks:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

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.

Litter observations:

All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays.
The pups were weighed on the day after birth (PND 1) and on PND 4.
All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically.
All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding.

Statistics:

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

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.

REPRODUCTIVE PERFORMANCE
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all groups including the controls. Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter.
One control male (0 mg/kg bw/d - No. 7) generated neither F1 pups nor implants. Thus, the male fertility index ranged between 90% and 100% without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study.
The apparently infertile male rat of the control did not show relevant gross lesions.

The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm was detected (GD 0) varied between 1.8 and 3.7 days without any relation to dosing.
All sperm positive rats delivered pups or had implants in utero with the following exception:
• Control female No. 107 (mated with male No. 7) did not become pregnant.
The fertility index varied between 90% in the control and 100% in test groups 1 - 3. These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
The non-pregnant female had no relevant gross lesions. The mean duration of gestation was similar in all test groups (i.e. between 22.1 and 22.4 days). The gestation index was 100% in all test groups.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (12.0 / 12.4 / 11.1 and 12.2 implants/dam in test groups 0-3 (0, 100, 300 and 1000 mg/kg bw/d)). There were no statistically significant differences in post-implantation loss between the groups (10.0% / 5.3% / 7.2% / 1.5%), and the mean number of F1 pups delivered per dam remained unaffected (10.9 / 11.9 / 10.3 and 12.0 pups/dam at 0, 100, 300 and 1000 mg/kg bw/d).
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100% (test group 3 and test group 1) and 99% (test group 2 and control). Moreover, the number of stillborn pups was comparable between the groups.

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.
Fertility: No gross lesions were observed in the male No.7 as well as in its mating female no. 107, and are note 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.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Remarks on result:

other: Generation: F0 (migrated information)

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Remarks on result:

other: Generation: F0 (migrated information)

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Remarks on result:

other: Generation: F0 (migrated information)

The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were evenly distributed about the groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.
The viability index indicating pup mortality during lactation (PND 0 - 4) varied between 99.3% (test group 1), 99.1% (test group 3 and control) and 98.6% (test group 2) without showing any association to the treatment.
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.
No test compound-related influence on F1 pup body weights and pup body weight change were noted in all dose groups.
One male and two female runts were seen in test group 3 (1000 mg/kg bw/d).
A few pups showed spontaneous findings at gross necropsy, such as yellowish discolored liver lobe, abnormal lobation of liver, post mortem autolysis, situs inversus, hydroureter, hydronephrosis and dilated renal pelvis. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences.

Reproductive effects observed:

not specified

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.

• The NOAEL for reproductive performance and fertility was 1000 mg/kg bw/d for the F0 parental rats.

• The NOAEL for developmental toxicity in the F1 offspring was 1000 mg/kg bw/d.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Dihydrodicyclopentadienylacrylate was given daily as an oily solution to groups of 10 male and 10 female Wistar rats (F0 animals) by stomach tube at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d). Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (corn oil). 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.A detailed clinical observation (DCO) was performed in all animals before initial testsubstance administration and, as a rule, thereafter at weekly intervals.

Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for gestation days 0 - 7, 7 - 14, 14 - 20 and lactation days 1 - 4.

Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, on the day of parturition (postnatal day [PND] 0) and on PND 4.

Clinico-chemical and hematological examinations as well as urinalyses were performed in 5animals per sex and group towards the end of the administration period.At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental males and females per group. All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.The clinical examinations/Clinical Pathology/Pathology showed no test substance-related adverse findings.

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.

The NOAEL for reproductive performance and fertility was 1000 mg/kg bw/d for the F0 parental rats.

The NOAEL for developmental toxicity in the F1 offspring was 1000 mg/kg bw/d.

Short description of key information:
In an OECD Guideline 422 and GLP study with Dihydrodicyclopentadienylacrylate, the NOAEL for reproductive toxicity is considered to be 1000 mg/kg bw/day for all relevant endpoints, which was the highest dose tested.

Justification for selection of Effect on fertility via oral route:
The key study was selected

Effects on developmental toxicity

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

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

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on 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.

Details on mating procedure:

- M/F ratio per cage:1:1
- Length of cohabitation:overnight for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as [day 0] of pregnancy

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

Duration of test:

49 days

Remarks:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Maternal examinations:

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.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of implantations: Yes

Fetal examinations:

All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

PUP VIABILITY/MORTALITY
In general, a check was made for any dead or moribund pups twice daily.

CLINICAL OBSERVATION
The live pups were examined daily for clinical symptoms.

BODY WEIGHT
The pups were weighed on the day ater birth (PND1) an on PND 4.

All pups with scheduled sacrifice on PND 4. All pups were examined externally and eviscerated; their organs were assessed macroscopically.

Statistics:

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

Details on maternal toxic effects:

Details on maternal toxic effects:
CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY
1000, 300, and 100 mg/kg bw/d F0 PARENTAL ANIMALS

• No test substance-related adverse findings

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Remarks:

general, systemic toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
No toxicologically-relevant developmental differences in the F1 generation were detected during this study. At all doses, the pups developed normally.

Abnormalities:

not specified

Developmental effects observed:

not specified

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Justification for selection of Effect on developmental toxicity: via oral route:
The key study was selected

Justification for classification or non-classification

Based on the results of the rreproduction/developmental testing of Dihydrodicycloentadienylacrylate, the test item was not classified and labelled for toxicity of reproduction/developmental according to Directive 67/548/EEC (DSD) and Regulation (EC) No 1272/2008(CLP).