Exo-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl 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

Study period:

2012-06-05 - 2012-07-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Exo-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl acrylate

EC Number:

227-561-6

EC Name:

Exo-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl acrylate

Cas Number:

5888-33-5

Molecular formula:

C13H20O2

IUPAC Name:

(1S,2S,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl prop-2-enoate

Test material form:

liquid

Specific details on test material used for the study:

Identity : Isobornyl acrylate
Alternative name : VISIOMER® IBOA
CAS number : 5888-33-5
EINECS number : 227-561-6
Appearance : Clear colourless fluid liquid
Storage conditions : Room temperature, protected from light

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italy S.p.A., Calco (Lecco), Italy

- Age at study initiation: (P) Males/females: approximately 8 - 9 wks
- Weight at study initiation: (P) Males: 196.5 - 204.7 g; Females: 166.1 - 189.3 g

- Fasting period before study:
- Housing: Pre mating period: no more than 5 per cage in clear polycarbonate cages measuring 59X39X20 cm with a stainless steel
mesh lid and floor (Techniplast - Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray will hold absorbent material which will be
inspected daily and changed at least three times a week.
During mating period: 1 male to one female per cage in clear polycarbonate cages measuring 36X19X24 cm with a stainless steel
mesh lid and floor (Techniplast - Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray will hold absorbent material which will be
inspected daily.
Pregnant females: will be transferred to individual cages after mating: solid bottomed, breeding cages (Techniplast - Gazzada S.a.r.l.,
Buguggiate, Varese), for the gestation period, birth and lactation.
Suitable nesting material will be provided and will be changed as necessary.
- Diet: ad libitum, commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4,
20019, Settimo Milanese (MI), Italy)
- Water: ad libitum, supplied via water bottles

- Acclimation period: aproximately 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±2 °C
- Humidity (%): 55 ±15 %
- Air changes (per hr): 15 - 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: A weighted amount of the test item was dissolved in the vehicle (distilled water) and brought to the final volume appropriate for each concentration. The test solutions were prepared daily at room temperature (concentrations of 5, 10, 20 and 40 mg/ml). All test item concentrations and dosages were based on the test item as supplied (dose volume of 10 ml/kg body weight).
Dose volumes for males were calculated according to individual body weight on the first day of treatment and adjusted according to individual body weight at weekly intervals thereafter. Dose volumes for females were calculated according to individual body weight on the first day of
treatment and adjusted according to individual body weight at weekly intervals up to positive identification of mating. Dose volumes were adjustced to body weight on Days 0, 7, 14 and 20 post-coitum and on Day 0 post-partum. Thereafter individual dose volumes remained constant.
Control animals received the vehicle alone at the same dose volume.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Once during week 1 and week 6 of treatment, samples of prepared formulations were analysed for verification of concentration.

Duration of treatment / exposure:

Males
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior
to pairing and thereafter through the day before necropsy.
Dose volumes were adjusted once per week for each animal according to the last recorded
body weight.

Females
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior
to pairing and thereafter during pairing, post coitum and post partum periods until Day 3
post partum or the day before sacrifice.
Dose volumes were adjusted once per week for each animal according to the last recorded
body weight. During the gestation period, dose volumes were calculated according to
individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum.
Thereafter individual dose volumes remained constant.

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 male and 10 female per dose group

Control animals:

yes

Details on study design:

Dose levels were selected in consultation with the Sponsor based on information from preliminary study.

Examinations

Observations and examinations performed and frequency:

Parental observations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
All clinical signs were recorded for individual animals. Examination of individual animals for signs of reaction to treatment was carried out daily
before dosing, immediately after, and approximately 30 minutes and 1 hour after dosing.
Animals were subjected to a detailed clinical examination at weekly intervals. For male animals the clinical signs were performed before treatment
started until necropsy. For female animals clinical signs were performed before treatment started, weekly during the treatment and the mating
periods and on days 0,7, 14 and 20 of gestation and on day 2 post-partum.
Animals were examined in an open arena for a period of three minutes.


BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed at allocation (Study Day -14), at treatment initiation
(Study Day I), and then weekly to pairing. Males were then weighed at weekly intervals up to the day sacrifice, females were weighed on Days 0, 7, 14
and 20 post-coitum and 0/1 and 4 post-partum.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption: The weight of food consumed by each cage of rats was recorded weekly following allocation to pairing. Food consumption
for each female was calculated on gestation Days 7, 14 and 20 and on post-partum day 4.

Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring: The total litter size (live and dead) was counted alter parturition (Day 0). Live
pups were identified individually within the litter by toe amputation, sexed, and examined for external abnormalities. All pups were examined daily for morbidity and abnormalities dead or abnormal young. All pups were individually weighed on post-partum days 0 and 4. The sexing at birth was
rechecked on Day 4 post-partum.

Sacrifice and pathology:

SACRIFICE
All adult animals were killed with carbon dioxide at the end of the scheduled treatment period (day 31 and day 32 of the study for males and on post partum day 4 for females). Nonpregnant females were killed after Day 25 post-coitum. The number of visible implantation sites were recorded for each dam. Uteri of nonpregnant females were immersed in a 10-20% solution of ammonium sulphide to reveal evidence of implantatlon.
All pups were killed by intracapular injection of "Tanax".

GROSS NECROPSY
- Gross necropsy: The clinical history of the animal was studied and a detailed post mortem examination was conducted (including examination of the external surfaces and orifices). Changes were noted and the requisite organs weighed.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs from all animals in all groups were dissected free of fat and weighed:
Testes, Epididymides, Ovaries with oviduct, Uterus with cervix, Prostate and seminal vescicles

In addition from at least 5 male and female animals per group the following organs were dissected free of fat and weighed:
Adrenals, Brain, Heart, Kidneys, Liver, Thymus, Spleen
This was a deviation from the protocol which indicated 5 animals per sex only.

Tissues fixed and preserved
From at least 5 male and female animals per group the following tissues listed below were fixed and preserved in 10% buffered formol-saline (except testes and epididymides which were fixed modified Davidson's fluid and preserved in 70% ethyl alcohol)
Abnormalities Lymph nodes - mesenteric
Adrenal glands Ovaries with oviduct
Brain (only for females) Prostate
Caecum Rectum
Colon Sciatic nerve
Duodenum Seminal vescivles
Heart Spleen
Ileum Stomach
Jejunum Testes
*Kidneys Thymus (where present)
*Liver Thyroid gland
Lungs Trachea
Lymph nodes - cervical Urinary bladder
Uterus with cervix
Vagina
This was a deviation from the protocol which indicated only 5 males and 5 females.

Histopathological examination

After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer
thickness and stained with haematoxylin and eosin.
In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.

Statistics:

Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means were assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopathological findings was carried out by means of the nonparametric Kolmogorov-Smirnov test if n was more than 5. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric version of the Williams test. The criteria for statistical significance were p<0.05 and p<0.01. The mean value, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

no mortality; clinical signs: salivation

Mortality:

mortality observed, treatment-related

Description (incidence):

no mortality; clinical signs: salivation

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

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

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

high proteinuria

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

moderate decrease the relative and absolute thymus weight

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

glandular stomach

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

glandular stomach; thymus

Histopathological findings: neoplastic:

not examined

Details on results:

Mortality and fate of females
No mortality occurred throughout the study.
All females proved to be pregnant.
Total litter loss was observed in 2 high dose females on Days 0 and 2 post partum.
The number of females with live pups on Day 4 post partum was 10 in each of the control,
low and mid-dose groups and 8 in the high dose group.

Clinical observations (Functional Observation Battery Tests)
Clinical observations for neurotoxicity assessment (removal of animals from the home cage
and open arena) did not reveal changes attributable to the test item.

Clinical signs
Salivation was the most relevant clinical sign detected in males and females of the mid- and
high dose group.

Body weight and body weight gain
No relevant difference in body weight and body weight gain was recorded in male animals
throughout the study.
Slight decrease in body weight and body weight gain was recorded in high dose females
during post coitum and post partum periods.

Food consumption
No effects on food consumption were observed in males.
Slight reduction was observed in high-dose females on Day 20 post coitum.

Motor activity and sensory reaction to stimuli
No effects of toxicological significance were noted in all parameters investigated between
control and treated groups.

Haematology
No changes of toxicological significance were recorded.

Clinical chemistry
The main relevant finding was the increase of urea in most of the animals dosed with 500
mg/kg/day (approximately 40%).
The other changes were considered of no toxicological importance.

Urinalyses
High proteinuria was recorded in males dosed with 100 and 500 mg/kg/day, with a doserelated
trend.

Oestrous cycle, reproductive parameters, pairing combination and mating
performance
Oestrous cycle, pre-coital intervals, copulatory index and fertility index did not show
intergroup differences.

Implantation, pre-birth loss data and gestation length of females
A slight decrease in the number of corpora lutea and implantation sites was detected in all
treated groups. In addition, a statistically significant reduction was recorded in the total litter
size at birth in the high dose group.
No significant differences were observed in pre-implantation and pre-birth loss.
All dams gave birth between Days 21 and 23 post coitum.

Litter data at birth, on Day 1 and on Day 4 post partum of females and sex ratio of pups
Statistically significant reduced litter size and litter weight were found in the high dose group
compared to controls, starting from birth up to Day 4 post partum.
In addition, the percentage of cumulative pup loss on Day 4 post partum, starting from the
total litter size of birth, was increased in the high dose group. No differences were found in sex ratio between the groups, both at birth and on Day 4 post partum.

Clinical signs of pups
Most of the clinical signs of pups were comparable between treated and control groups.
An increase number of pups were found dead in the high dose group.

Necropsy findings in decedent pups and in pups sacrificed on Day 4 post partum
No relevant differences were recorded in decedent pups between the groups.
No relevant findings were observed in pups sacrificed at term.

Terminal body weight and organ weights
At statistical analysis the following findings were observed with respect to the controls:
- slight decrease in terminal body weight in high dose females.
- slight increase in the relative kidney weight of mid- and high dose males.
- moderate decrease the relative and absolute thymus weight of high dose females.

Macroscopic observations
Treatment-related changes were noted in the glandular stomach of both sexes and in the
thymus of the females only.
In the glandular stomach of a single male, treated with the low dose, of a single male and a
single female treated with the mid- dose, and of two males and a single female treated with
the high dose, single or multiple depressed and/or thickened and/or dark areas were noted.
These lesions seen in the mid- and high dose animals correlated histologically with the
presence of single erosions.
In 4/10 and 7/10 females, respectively from the mid- and high dose, the thymus was reduced
in size. Only in 1/10 and 2/10 animals, respectively from the mid- and high dose, this
macroscopic change correlated with histological change.

Microscopic observations
Treatment-related findings were seen in the glandular stomach of both sexes and in the
thymus of the females only.
Stomach (glandular): in a single male and a single female, treated with the mid- dose and in
two males and a single female treated with the high dose, single erosion was seen in the
mucosa.
Thymus: in 1/10 and 2/10 animals, respectively from the mid- and high female dose groups,
different degrees of atrophy of the cortex were seen.
Spermatogenic cycle
Evaluation of the spermatogenic cycle did not show differences between the groups. Regular
layering in the germinal epithelium was noted.

Effect levels

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In this OECD TG 422 GLP study, the toxic effects on Sprague Dawley rats of both sexes after repeated dosing with Isobornylacrylate, as well as any effects of the test item on male and female reproductive function such as gonadal function, mating behaviour, conception, parturition and development of offspring up to Day 4 post partum were evaluated.
There was an increase of urea in most of the animals dosed with 500 mg/kg/day (approximately 40%). Statistically significant reduced litter size and litter weight were found in the high dose group compared to controls, starting from birth up to Day 4 post partum. In addition, the percentage of cumulative pup loss on Day 4 post partum, starting from the total litter size of birth, was increased in the high dose group. On the basis of these results the NOAEL (No Observed Adverse Effect Level) for both general toxicity and reproduction/developmental toxicity was determined to 100 mg/kg/day for males and females.

Executive summary:

In this OECD TG 422 GLP study, both potential repeated dose toxicity and potential reproduction / developmental toxicity of Isobornyl acrylate in Sprague Dawley rats was evaluated.

 

Isobornyl acrylate was administered orally, by gavage, at the dosages of 25, 100 and 500 mg/kg/day. The treatment schedule included 2 weeks before pairing, during pairing, postcoitum and post partum periods up to Day 3 post partum. Animals were administered for approximately 5 and 6 weeks for males and females, respectively.

The parental animals were monitored for daily clinical signs, weekly neurotoxicity assessment, body weight, food consumption and clinical pathology investigations. The dams were allowed to give birth and rear their offspring until Day 4 post partum.

The following results were obtained in the study:

Daily clinical signs: salivation was the relevant sign detected in both sexes of the mid- and high dose groups.

Clinical pathology: no findings of toxicological significance were recorded in the haematological investigation. At clinical chemistry evaluation, increased values of urea were noted in the high dose group of both sexes. At urinalyses high proteinuria was recorded in mid- and high dose males, with a dose related trend.

Reproductive / developmental parameters: no relevant differences were found in terms of mating performance including the pre-coital interval (number of days paired up to sperm positive day), the copulatory index (calculated as the percentage of animals mated respect to those paired) and the fertility index, these last two parameters with percentages of 100% in all groups.

All pregnant females in the groups, including controls, had comparable length of gestation period and gave births. Furthermore, sex ratio calculated as the percentage of males in the litter did not differ between the groups.

However, significant differences were found in terms of total litter size and litter weight in the high dose group compared to controls, from birth to Day 4 post partum.

In addition, the percentage of cumulative pup loss on Day 4 post partum, starting from the total litter size at birth, was increased in the high dose group.

No relevant findings were detected in pups sacrificed at term, nor in those found dead in all groups.

Organ weights:increased kidneys weight in mid- and high dose males and a decrease of thymus weight in high dose females were found.

Macroscopic and microscopic observations: treatment-related changes were found in

stomach of mid- and high dose animals of both sexes and in the thymus of mid- and high dose females only.

 

On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for both general toxicity and reproduction / developmental toxicity was determined to100 mg/kg/day for males and females.

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