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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-03-11 until 2006-27-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

27th July 1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)

Version / remarks:

July 2000

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 methacrylate

EC Number:

231-403-1

EC Name:

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

Cas Number:

7534-94-3

Molecular formula:

C14H22O2

IUPAC Name:

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

Test material form:

liquid

Specific details on test material used for the study:

TEST MATERIAL:
- Name of test material: Isobornyl Methacrylate (IBOMA)
- CAS Registry No: 7534-94-3
- Molecular weight: 222 g/mol
- Supplier: Rhodia Inc. (Cranbury, NJ USA)

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 Laboratories France, L’Arbresle, France
- Age at study initiation: males 8 weeks; females 10 weeks
- Weight at study initiation: mean body weight of 308 g; males (range: 277 to 338 g) and 229 g for the females (range: 214 to 246 g)
- Fasting period before study: no data
- Housing: individually (except during mating) in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm). A metal tray, containing autoclaved sawdust
SICSA, Alfortville, France) with wood shaving as nesting material, was placed under each cage. Shortly before parturition, the females were moved
to polycarbonate cages (43.0 x 21.5 x 20.0 cm) with wood shavings as nesting material.
- Diet: ad libitum, SSNIFF R/M-H pelleted maintenance diet, batch No. 6704545 (SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on mating procedure:

Females were paired with males from the same dose level. One female was placed with one male, in the latter's cage, durig the night, removed the following morning, and replaced with the same male the next night until confirmation of mating. Confirmation of mating was made in the morning by checking for the presence of a vaginal plug or of sperm in a vaginal lavage.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Validation of the analytical method
Specificity
The specificity of the analytical method was demonstrated as follows:
. analysis of a standard solution of IBOMA in mobile phase,
. analysis of a solution of corn oil diluted 10-fold in isopropyl alcohol and 5-fold in mobile phase.
No relevant interference between the test item peak and vehicle was observed on chromatograms.

Limit of quantification
The limit of quantification of the analytical method was established as 0.5 μg/mL for a standard solution of IBOMA.
This limit corresponds to a limit of quantification of 0.025 mg/mL for the test item in corn oil, the minimum dilution factor being 50-fold to avoid
interference.
L inearity
Linearity was checked by analysis of three different sets of seven standard solutions containing 0.5, 1, 5, 10, 20, and 50 μg/mL of IBOMA in mobile
phase. Satisfactory linearity was demonstrated in the range 0.5 - 50 μg/mL since the coefficients of determination obtained were higher than 0.999.

Duration of treatment / exposure:

males:
- 15 days before mating, during the mating and post-mating periods until sacrifice (at least 4 weeks in total)
females:
- 15 days before mating,
- during the mating period,
- during pregnancy and lactation, until day 5 post-partum inclusive (or until sacrifice, for unmated females).

Frequency of treatment:

7 days a week; once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

25 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 male and 10 female animals per dose group with appropriate randomization

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected following the results of a previously conducted study CIT/Study No. 28200 TSR, in which male and female rats were
dosed with 0, 100, 300 or 1000 mg IBOMA/kg/day for 10 consecutive days. In this study, weight loss and significantly increased liver and kidney
weights were observed at the 1000 mg/kg/day dose. Liver weight was also increased at 300 mg/kg/day. The high dose-level of 500 mg/kg/day was
selected because it was expected to elicit effects on body weight and food consumption during the in-life phase of the study and also to result in
increased liver weights in both males and females. As the dosing period was significantly longer than the 10 days used in the above mentioned study, higher dose-levels than 500 mg/kg/day were considered likely to produce marked effects on body weight as well as increased liver weights, and,
therefore, would endanger the survival of the animals.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

PARENT ANIMALS:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each animal was checked at least twice a day for mortality and signs of morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day; Animals were also observed in the afternoon on week days as part of the mortality check and any clinical signs were
recorded

BODY WEIGHT: Yes
- Time schedule for examinations: each male was recorded on the first day of treatment (day 1), then once a week until sacrifice; each female was
recorded on the first day of treatment (day 1), then once a week until mated (or until sacrifice) and on days 0, 7, 14 and 20 post-coitum and
days 1 and 5 post-partum

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

The quantity of food consumed by each animal was recorded once a week, over a 7-day period, from the first day of treatment through gestation
(days 0-7, 7-10, 10-14, 14-17 and 17-20 post-coitum intervals) and lactation (days 1-5 post-partum interval). During the mating period, the food
consumption was noted for neither males nor females.

Food intake per animal and per day was calculated by noting the difference between the food given and that remaining in the food hopper at the end
of the specified interval.

Oestrous cyclicity (parental animals):

Histopathological examination of ovary included any relevant changes in the follicular development, follicular atresia and corpora lutea formation.

Sperm parameters (parental animals):

Parameters examined in all male parental animals:
stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Examination of intact epididymis included head, corpus and tail, which could be accomplished by evaluation of longitudinal section. The epididymis was evaluated for luekocyte infiltration, change in prevalence of cell types, aberrant cell types and phagocytosis of sperm.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
The total litter size and number of pups of each sex was recorded as soon as possible after birth. Litters were observed daily in order to note the number of live, dead and cannibalised pups and any runts. A detailed external observation of pups was performed on days 1 and 5. The pups were observed daily for clinical signs or abnormal behaviour. The weight of each pup was recorded on day 1 and 5 post-partum.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: The males were sacrificed after the end of mating period (total treatment period was at least 4 weeks)
- Maternal animals: The females and their pups were sacrificed on days 6 post-partum (or shortly thereafter). The females showing no evidence of mating were sacrificed 24-26 days after the last day of mating period. The females which had not delivered by day 25 post-coitum were sacrificed when appropriate.

GROSS NECROPSY
- Complete macroscopic examination was performed on all adult animals and included the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvis cavities with their associated organs and tissues and the neck with its associated organs and tissues.
- The ovaries and uterus of parental females were examined to determine the number of corpora lutea and implantation sites
- in apparently non-pregnant or un-mated females, the presence of implantation scars was checked.
- for any pregnant female which had not delivered, the implantation sites were recorded according the following classification (uterine scar, early resorption, late resorption, dead fetus).

ORGAN WEIGHTS
The body weight of all adult animals was recorded and the following organs were weighed wet as soon as possible after dissection:
- epididymides
- kidneys
- liver
- testes

The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

MICROSCOPIC EXAMINATION
- Microscopic examination was performed on the ovaries, the testes and the epididymides of the control and high-dose groups in all males and females sacrificed at the end of treatment period.
- Livers (male and female) and kidneys (male only) were examined microscopically for all groups.

Postmortem examinations (offspring):

SACRIFICE AND EXAMINATION
- The F1 offspring was sacrificed on day 6 post-partum
- All animals were examined for gross external abnormalities and a macroscopic examination was performed.

Reproductive indices:

Pre- and post-implantation loss, mating index, fertility index, gestation index

Offspring viability indices:

Live birth index, viability index on day 4 post-partum

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Hypersalivation was observed in a dose-dependent manner in males and females given 100 or 500 mg/kg/day during the pre-mating period (from week 2) and then in females during gestation and lactation. This clincal sign was considered to be not adverse as t likely represents a reaction to dosing procedure and not direct to IBoMA toxicity. There were no clinical signs in the 25 mg/kg/day group.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no prematured deaths during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the first week of pre-mating period, males given 500 mg/kg/day gained less weight than controls. Neither food consumption nor body weight was affected during the gestation period. There were no other treatment-related effects on body weight, weight gain or food consumption during the study for males or females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During the first week of pre-mating period, males given 500 mg/kg/day gained less weight than controls. Neither food consumption nor body weight was affected during the gestation period. There were no other treatment-related effects on body weight, weight gain or food consumption during the study for males or females.

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic findings in the liver included biliary proliferation/ hypertrophy associated with fibrosis and macrophages infilatration (100 and 500 mg/kg/day males and females), disorganisation of the hepatic cords (500 mg/kg/d males and females), and necrosis in the parenchyma (500 mg/kg/d males) with a tendency towards higher severity in males were considered to be treatment-related. No treatment-related effects were observed at 25 mg/kg/day.
In the kidneys, acidophilic globules were observed in the cortical tubular epithelium (considered to be related to micro-2u-globulin) with higher severity in males given 100 and 500 mg/kg/day than in the controls. This latter finding was not considered relevant to human hazard assessment.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No relevant differences between females given 500 mg/kg/day and the control females were observed following a semi-quantitative evaluation of the follicular development, corpora lutea formation and follicular atresia.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

TESTES:
PAS/hematoxylin stained sections of the testes form all control and high-dose males were examined microscopically. No treatment-related abnormalities were found in the treated animals.
The tailed and round spermatids were unaffected and die different stages of spermatogenic cycle were undisturbed. Sloughing of germinal cells in lumen and vacuoles of seminiferous tubules were noted with similar incidence and/or severity in treated and control males. Retained spermatids were observed in the lumen of a few seminiferous tubules of stage X and/or onwards for a few control males but not in any treated male given 500 mg/kg/day.

EPIDIDYMIDES:
Minimal interstitial monocellular cell aggregation was noted in the epididymis of four males from the control group and one male given 500 mg/kg/day. As the above mentioned minor changes recorded in the testis and epididymis can be found spontaneously in the untreated laboratory rat of this strain and age, they were considered to be of no toxicological significance.

Reproductive performance:

no effects observed

Description (incidence and severity):

No effect on mating at any dose-level. Male and female fertility indices were unaffected, all pregnenant females had live pups.

Dose-level 0 25 100 500
Number of mated females 10 10 10 9
Number of pregnant females 10 10 10 8
Fertility index (%) 100 100 100 88.9
Duration of gestation 21.1 21.5 21.5 21.9**
Number of females with liveborn 10 10 10 8
Gestation index (%) 100 100 100 100
Mean number of pups delivered 14.1 13.9 14.8 13.3
Live birth index (%) 100 100 100 100
Mean number of pups alive on day 5 14.0 13.8 14.5 13.3

** p<0.01

The length of gestation in females given 500 mg/kg/day was not considered to be increased as individual values were equal to 21 or 22 days.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

2

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed

Remarks on result:

other: no adverse effect observed

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

All the clinical findings recorded in the pups were considered to be distributed among groups with no dose-related incidence, and as such, none of them were attributed to the test-item.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The number of pups which died during the 5 days of observation after birth was low and similar in all the groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No differences of toxicological importance were noted in the male and female pup body weight gain.

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

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no external abnormalities in the pups from the control or test item-treated groups. No relevant findings observed in the pups sacrificed on day 6 post-partum.

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Remarks on result:

other: no adverse effects observed

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the experimental conditions of the reproductive/developmental toxicity screening study according to OECD 421 the NOAEL for parental
toxcitity was 25 mg/kg bw/day and the reproductive NOAEL was 500 mg/kg bw/day.

Executive summary:

In a reproduction/developmental toxicity screening study according to OECD 421 Sprague-Dawley rats (10 male and 10 females per dose group) received Isobornyl Methacrylate (IBOMA), by daily oral (gavage) administration for 15 days before mating, through mating, gestation and the beginning of the lactation period (until day 5 post-partum, p.p.). The dose-levels were 25, 100 and 500 mg/kg/day. Another group of 10 males and 10 females received the vehicle, corn oil, alone, under the same experimental conditions and acted as a control group. The dosing volume was 5 mL/kg.

Based on the experimental conditions of the reproductive/developmental toxicity screening study the NOAEL for parental toxcitity was 25 mg/kg bw/day and the reproductive NOAEL was 500 mg/kg bw/day.