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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

IBOMA

OECD 421, GLP, rat, oral: NOAEL parental 25 mg/kg bw/day (based on liver and kidney findings); NOAEL developmental 500 mg/kg bw/day (no effects)

 

Metabolite data

MMA (metabolite donor for MAA)

OECD 414, GLP, rabbit, oral gavage: NOAEL maternal 50 mg/kg bw/day (food consumption); NOAEL developmental 450 mg/kg bw/day (no effects)

MAA

Similar OECD 414, rat, inhalation: NOAEC maternal 200 ppm (bw & food consumption); NOAEC developmental 300 ppm (no effects)

IBOAc (metabolite donor of IBO)

OECD 414 limit test, rat, oral: NOAEL maternal & developmental 1000 mg/kg bw/day (no effects)

CAM (metabolite of IBO)

NTP, rat, oral gavage: LOAEL maternal 100 mg/kg bw/day (water consumption); NOAEL developmental 800 mg/kg bw/day (no effects)

NTP, rabbit, oral gavage: LOAEL maternal & developmental 400 mg/kg bw/day (no effects)

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

27th July 1995

Deviations:

no

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

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)

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

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

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

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

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

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: 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.

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

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

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

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

Critical effects observed:

no

Reproductive effects observed:

no

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.

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-12-06 - 2009-04-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

22nd January 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

TEST MATERIAL:
- Name of test substance: Methyl Methacrylate
- CAS-No.: 80-62-6
- Physical state/appearance: Liquid /colorless, clear

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 GmbH
- Age at study initiation: (P) 37 (±1) days at the beginning of treatment; (F1) x wks
- Weight at study initiation: (P) Males: 127.5 - 151.0 g; Females: 110.5 - 145.1 g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study:
- Housing: During the study period, the rats were housed individually in Makrolon type M III cages (Becker & Co., Castrop-Rauxel, Germany), floor area of about 800 cm², with the following exceptions: 1) During overnight matings, male and female mating partners were housed together in Makrolon type M III cages. 2) Pregnant animals and their litters were housed together until PND 21 (end of lactation).
- Diet: ground Kliba maintenance diet mouse/rat “GLP” meal (Provimi Kliba SA, Kaiseraugst, Switzerland) ad libitum
- Water: ad libitum
- Acclimation period: (P) about 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 10 or 15 times
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The aqueous test substance suspensions were prepared at the beginning of the administration period and thereafter at intervals that took into account the analytical results of the stability verification. For the test substance preparation, the specified amount of test substance was weighed into an Erlenmeyer flask, topped up (shortly under the marking) with 1% Carboxymethylcellulose suspension in drinking water and four drops Cremophor EL and one drop of 32% hydrochloric acid. Afterwards the preparation was filled up with 1% Carboxymethylcellulose suspension in drinking water. The Erlenmeyer flask was sealed and the preparation was intensely mixed with a magnetic stirrer. A magnetic stirrer was used to keep the preparations homogeneous during treatment of the animals.

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 (= GD 0)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test substance preparations were sent to the analytical laboratory ten times during the study period (among other things at the beginning and towards the end) for verification of the concentrations. The samples, which were taken for the concentration control analyses at the beginning of the administration period, were also used to verify the homogeneity for the samples of the low and the high concentrations (50 and 400 mg/kg bw/d). Three samples (one from the top, middle and bottom in each case) were taken for each of these concentrations from the beaker with a magnetic stirrer running.

Duration of treatment / exposure:

until one day before sacrifice

Frequency of treatment:

once daily

Details on study schedule:

- F1 parental animals not mated until 75 days after selected from the F1 litters.
- Selection of parents from F1 generation after weaning (PND 21).
- Age at mating of the mated animals in the study: [...] weeks

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

In a Dose range finding study according to OECD 421 with male and female Wistar rats, the terminal body weight in F0 females dosed with 450 mg/kg bw/d was significantly decreased by 10% vs. Ctrl animals (p <= 0.01) and also the food consumption of females in the F0 and the F1 generation (last, during gestation) was significantly reduced by 86-91%. Dosing with 50 and 150 mg/kg bw/d was not related to observed effects. Based on these findings, the highest dose for the main study was set to 400 mg/kg bw/d.

No. of animals per sex per dose:

F0 generation parental animals: 25
F1 generation parental animals: 25

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
In a dose range finding study according to OECD 421 with male and female Wistar rats, the terminal body weight in F0 females dosed with 450 mg/kg bw/d was significantly decreased by 10% vs. Ctrl animals (p <= 0.01) and also the food consumption of females in the F0 and the F1 generation (last, during gestation) was significantly reduced by 86-91%. Dosing with 50 and 150 mg/kg bw/d was not related to observed effects. Based on these findings, the highest dose for the main study was set to 400 mg/kg bw/d.

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working days or once daily (Saturday, Sunday or on public holidays)
- Parturition and lactation behaviour of dams: once daily in combination with the daily clinical inspection

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: first day of the premating period and then once a week at the same time of the day (in the morning) until sacrifice. The following exceptions are notable for the female parental animals: 1) During each gestation period the F0 and the F1 generation parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. 2) Females showing no positive evidence of sperm in vaginal smears were weighed once a week during the mating interval (solely for calculation of dose volume). 3) Females with litter were weighed on the day after parturition (PND 1) and on PND 4, 7, 14 and 21. 4) Females without litter were weighed once a week during the lactation phase (solely for calculation of dose volume).

OTHER:
- Food consumption: In general, food consumption was determined once a week for the male and female F0 and F1 parental animals. After the 10th test week, food consumption of the females during pregnancy (animals with evidence of sperm) was determined weekly for GD 0-7, 7-14 and 14-20. During the lactation period (animals with litter) food consumption was determined for PND 1-4, 4-7, 7-14 and 14-21.

Oestrous cyclicity (parental animals):

Estrous cycle length and normality were evaluated daily for all F0 and F1 female parental rats for a minimum of 3 weeks prior to mating. The evaluations were continued throughout the mating period until the female exhibited evidence of mating. Moreover, at the scheduled necropsy a vaginal smear was microscopically examined to determine the stage of the estrous cycle for each F0 and F1 female.

Sperm parameters (parental animals):

Immediately after necropsy and organ weight determination, the right testis and cauda epididymis were taken from F0 and F1 males of all dose groups.
Parameters examined in all male parental generations:
testis weight, epididymis weight, cauda epididymis weight, prostate weight, seminal vesicles including coagulation glands weight, sperm head count in testis, sperm head count in cauda epididymis, sperm motility, sperm morphology

Sperm motility and sperm head count (cauda epididymis and testis) were evalutated for the control and highest dose group.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups/ sex ratio, stillbirths, live births, postnatal mortality, presence of gross anomalies, body weight, body weight gain, clinical symptoms, sexual maturation (vaginal opening and preputial separation), organ weight (brain, spleen and thymus) of 1 pup/sex and litter from F1 and F2 pups

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE AND GROSS NECROPSY
All F0 and F1 parental animals were sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
Weight assessment was carried out on all animals sacrificed at scheduled dates. The following weights were determined: Anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, cauda epididymis, prostate, seminal vesicles including coagulation glands, ovaries, uterus, spleen, brain, pituitary gland, thyroid glands (with parathyroid glands). The following organs or tissues of the F0 and F1 generation parental animals were fixed in 4% neutral buffered formaldehyde solution or in BOUIN’s solution, respectively: Vagina, cervix uteri, uterus, ovaries (BOUIN), oviducts, left testis (BOUIN), left epididymis (BOUIN), seminal vesicles, coagulation glands, prostate, pituitary gland, adrenal glands, liver, kidneys, spleen, brain, thyroid glands (with parathyroid glands), all gross lesions. After fixation, the organs fixed in BOUIN´s solution were embedded in Paraplast. Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings.

OTHER:
- Differential Ovarian Follicle Count (DOFC) in F1 generation: From both ovaries (”ovary 1” and “ovary 2”) of F1 female animals (control and top dose), five sections were taken from the proximal and the distal part of the ovaries, respectively, at least 100 µm apart from the inner third of the ovary. All ovarian sections were prepared and evaluated. Primordial follicles and growing follicles were counted by light microscope (magnification: 100x) on each of these slides, – according to the definitions given by Plowchalk et al. (PLOWCHALK, D. R., B. J. SMITH, and D. R. MATTISON: Assessment of Toxicity to the Ovary Using Follicle Quantitation and Morphometrics. In: Methods in Toxicology, Vol. 3, Part B: Female Reproductive Toxicology (J. J. HEINDEL and R. E. CHAPIN, Editors), p. 57-68, 1993, Academic Press). To prevent multiple counting on serial slides – especially of the growing follicles – only follicles with an oocyte with visible chromatin on the slide were counted. The number of each type of follicle was recorded individually for ovary 1 and ovary 2 of every animal on any of the slide levels (level 1-10), giving in summary the incidence of each type of the follicles by using EXCEL sheets for the reporting of the results. Finally, the results of all types of follicles were summarized for all animals per group in dose groups 10 and 13. As primordial follicles continuously develop into growing follicles, the assessment of the follicles was extended to the combined incidence of primordial plus growing follicles. In general, the fifth slide of the left and right ovary was evaluated for histological findings. Whenever the diagnosis: ”no abnormalities detected” was used for the ovaries, this implicates all functional statuses of follicles, especially corpora lutea, were present. An attempt was made to correlate gross lesions with histopathological findings.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 4 days of age. All F2 offspring were sacrificed after weaning (~ PND 21).
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- All pups were examined externally and eviscerated; their organs were assessed macroscopically.

HISTOPATHOLOGY / ORGAN WEIGHTS
Animals with notable findings or abnormalities were further evaluated on a case-by-case basis, depending on the type of finding noted. After the scheduled sacrifice the brain, spleen and thymus of 1 pup/sex and litter from the F1 and F2 pups were weighed. Normally, the first male and the first female pups/litter were taken for these determinations. For the calculation of the relative organ weights, the pup body weights, determined routinely during the in-life phase on PND 21, were used.

Statistics:

Simultaneous comparison of all dose groups with the control group using Dunnett-test (2-sided) for the hypothesis of equal means:
- food consumption (parental animals)
- body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used)
- oestrus cycle duration
- number of mating days
- duration of gestation
- number of implantation sites
- postimplantation loss and % postimplantation loss
- number of pups delivered per litter
- duration of sexual maturation (days to vaginal opening or preputial separation)
Pairwise comparison of each dose group with the control group using Fisher's exact test for the hypothesis of equal proportions:
- male and female mating indices
- male and female fertility indices
- gestation index
- females with stillborn pups
- live birth index
- pups stillborn, died, cannibalised, sacrificed moribund
- viability index
- lactation index
- number of litter with affected pups at necropsy
- sexual maturation (vaginal opening and preputial separation)
- males with certain amount of abnormal sperm (cutoff value: 0.9-quantile of control groups)
Pairwise comparison of each dose group with the control group using the Wilcoxon-test (1-sided) for the hypothesis of equal medians:
- Proportion of affected pups per litter with necropsy observations
Pairwise comparison of each dose group with the control group using the Wilcoxon-test (1-sided) for the hypothesis of equal medians:
-Total spermatids/ g testis, total sperm/g cauda epididymis
Pairwise comparison of each dose group with the control group using the Wilcoxon-test (1-sided) with Bonferoni-Holm-Adjustement for the hypothesis of equal medians:
- Sperm motility(%)
Non parametric one-way analysis using Kruskal-Wallis-test (2-sided). If resulting p-value was <= than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (2-sided) for the equal medians:
-pup organ weights (absolute and relative)

Reproductive indices:

- Male reproduction data: The mating partners, the number of mating days until vaginal sperm could be detected in the female, and the gestational status of the female were noted for F0 and F1 breeding pairs. For the males, mating and fertility indices were calculated for F1 and F2 litters according to the following formulas: Male mating index (%) = (number of males with confirmed mating)/(number of males placed with females) x 100. Male fertility index (%) = (number of males proving their fertility)/(number of males placed with females) x 100.
- Female reproduction and delivery data: The mating partners, the number of mating days until vaginal sperm were detected, and gestational status were recorded for F0 and F1 females. For the females, mating, fertility and gestation indices were calculated for F1 and F2 litters according to the following formulas:
Female mating index (%) = (number of females mated)/(number of females placed with males) x 100.
Female fertility index (%) = (number of females pregnant)/(number of females mated) x 100.
Gestation index (%) = (number of females with live pups on the day of birth)/(number of females pregnant) x 100.
The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 and F2 litters according to the following formula:
Live birth index (%) = (number of liveborn pups at birth)/(total number of pups born) x 100.
The implantations were counted and the postimplantation loss (in %) was calculated according the following formula:
Postimplantation loss (%) = (number of implantations – number of pups delivered)/(number of implantations) x 100.

Offspring viability indices:

The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7, 8-14 and 15-21 (lactation period) were determined; however, pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation days 4, 7, 14, and 21. Furthermore, viability and lactation indices were calculated according to the following formulas:
Viability index (%) = (number of live pups on day 4 (before culling) after birth)/(number of live pups on the day of birth) x 100.
Lactation index (%) = (number of live pups on day 21 after birth)/(number of live pups on day 4 (after culling) after birth) x 100.

Clinical signs:

no effects observed

Description (incidence and severity):

Clincial signs of males and females except gestation and lactation period:
A male and 7 females of the high dose group showed transient salivation during major parts of the treatment period. Salivation persisted in the respective animals only for some minutes after daily gavage (i.e. upto 15 minutes). This symptom is initially observed during study week 5.
The temporary salivation is considered to be test item-related. Form the temporary, short appearance immediately after dosing, it is likely that this finding was induced by the bad taste of the test item or local affection of the upper digestivve tract. It was clearly considered to be not an adverse toxicological finding. There were no clinical signs in low and mid-dose animals.

Clinical signs of females in females during gestation of F1 litters:
NIne out of 25 high dose females showed transient salivation during major parts of the gestation period (GD 0-16 and GD 20). There were no clinical signs in low and mid-dose females.

Clinical signs of females and offspring during lactation of F1 litters:
Seven out of 24 high dose females showed transient salivation during major parts of the lactation period (OND 0-8; PND 10; PND 14-16 and PND 18-21). The F0 animals and the low and mid-dose females showed no clinical signs.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no test item-related mortalities in any of the male and female parental animals in any of the groups. During study week 11, one high dose female was found dead, after showing hypothermia, salivation and labored respiration. Pathological examination revealed pleuritis and pericarditis as the cause of death, most likely secondary to a gavage error.
During lactation period, two high dose females were found dead. There were no macroscopic or histopathologic findings that could explain the death of these animals.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights and body weight change of F0 parental males and females in the treated groups were comparable to the control group throughout the entire study. The statistically significantly increased or decreased body weight change in the low-dose females during week 0-1 and weeks 4-5, in the mid-dose females during study week 1-2 and weeks 4-6 and in high dose females during weeks 4-6 was considered as spontaneous in nature.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test group 03 (400 mg/kg bw/d)
• Statistically significantly decreased food consumption in parental males during premating weeks 5 - 10 (up to 7%)
• Statistically significantly decreased food consumption in parental females during premating weeks 1 - 3 (up to 6%), weeks 5 - 8 (up to 7%) and weeks 9 - 10 (about 6%)
• Statistically significantly decreased food consumption in parental females during gestation days 0 - 7 (about 10%)
• Statistically significantly decreased food consumption in parental females during lactation days 4 - 7 (about 7%)

Test group 02 (150 mg/kg bw/d)
• Statistically significantly decreased food consumption in parental females during premating weeks 1 - 2 (about 5%)
• Statistically significantly decreased food consumption in parental females during gestation days 0 - 7 (about 7%)

Test group 01 (50 mg/kg bw/d)
• no test substance-related adverse effects/findings

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:

no effects observed

Description (incidence and severity):

All findings were either single observations, or were similar in distribution pattern and severity in control rats compared to treatment groups. All of them were considered to be incidental and/or spontaneous in origin and without any relation to treatment. Male animals and their female mating partners did not show any histopathologic findings that could explain their infertility. One male animal revealed oligospermia and debris within the tubules of the testes and the epididymides. This could have caused the infertility in this mating pair. The female mating partner did not show any histopathologic finding that could explain the infertility.

Information on descendents: see in section mortality

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrus cycle data, generated during the last three weeks prior to mating for the F1 litter, revealed regular cycles in the females of all test groups. The mean oestrus cycle duration in the different groups was comparable: 4.0 (control), 4.5 (low-dose), 5.7 (mid-dose) and 5.3 (high-dose).

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No treatment-related effects were noted for the sperm measures. The number of homogenisation resistant testicular spermatids of cauda epididymal sperm, the percentages of abnormal and normal sperm and sperm motility data were comparable between the groups.

Reproductive performance:

no effects observed

Description (incidence and severity):

For all F0 males, copulation was confirmed. Fertility was proven for most of the F0 males. 3 control , 3 low-dose, 3 mid-dose and 1 high-dose male did not generate F1 pups. The male fertility index ranged from 88-96% without showing any relation to the dosing. The infertile male rats did not show histopathological findings that could explain infertility. One male of the low-dose had an oligosperma and debris within the tubules of the testes.
The female mating index calculated after mating period of F1 litter was 100% in all groups. The mean duration until sperm was detected varied from 2.3 and 2.6 days without any relation to dosing. All sperm positive rats delivered pups or had implants in utero (except 3 control 3 low-dose, 3 mid-dose and 1 high-dose female which did not become pregnant). The fertility index varied between 88 and 96%. There were no corroborative histopathological findings in the sexual organs of the non-pregnant females. The mean duration of gestation varied between 22.0 and 22.2 days without any relation to dosing.
Implanation was not affected by treatment since the number of implanation sites was comparable to control. There were no indications for test item-related intrauterine embryo-/fetolethality since postimplantation loss did not show any statistically significant differences between the groups. The mean number of F1 pups delivered per dam remained unaffected. The rate of liveborn pups was also not affected by treatment with the test item, as indicated by live birth index of 99 (control, mid- and high-dose) and 100% (low-dose). Moreover, the number of stillborn pups was comparable between the groups.

Under the conditions of the present 2-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicity is 400 mg/kg bw/d for the F0 parental rats, the highest dose tested.
The NOEL (no observed effect level) is 50 mg/kg bw/d for the F0 parental rats based on effects on food consumption being a consequence of reduced appetite observed at the LOEL (Lowest Observed Effect Level) of 150 mg/kg bw/d in the F0 parental females.
The NOAEL for fertility and reproductive performance for the F0 parental rats is 400 mg/kg bw/d, the highest dose tested.

Dose descriptor:

NOAEL

Remarks:

General systemic toxicity

Effect level:

400 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

Dose descriptor:

NOAEL

Remarks:

fertility and reproductive performance

Effect level:

400 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

Dose descriptor:

NOEL

Remarks:

food consumption

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

food consumption and compound intake

Dose descriptor:

LOEL

Remarks:

food consumption

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

food consumption and compound intake

Remarks on result:

other: consequence of reduced appetite observed in the F0 parental females

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

24 out of 25 males and 12 out of 25 females of the high dose F1 parental animals showed transient salivation during major parts of the treatment period. Salivation persisted in the respective animals only for some minutes after daily gavage (i.e. upto 15 minutes). The salivation persisted in the respective animals only for some minutes after daily gavage dosing (i.e. up to 15 minutes). This symptom was initially observed during study week 6 in males and towards the end of the study in females.

The temporary salivation is considered to be test item-related. Form the temporary, short appearance immediately after dosing, it is likely that this finding was induced by the bad taste of the test item or local affection of the upper digestivve tract. It was clearly considered to be not an adverse toxicological finding. Other clinical findings like labored respiration, microphthalmia, poor general state, palpable mass in the neck and throat region in some low-and mid-dose individuals were considered as spontaneous in nature and not as test item-related.

Clinical signs of females in females during gestation of F2 litters:
Five out of 25 high dose females showed transient salivation during major parts of the gestation period (GD 4, 6-7, 9 and 11-12). There were no clinical signs in low and mid-dose females.

Clinical signs of females and offspring during lactation of F2 litters:
There were no clinical findings in F1 females during the lacation period for the F2 litters at all dose levels.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were not deaths observed in the F1 parental dose groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

HIgh dose F1 parental males had statistically significantly lower body weights during week 0-5 (upt o 17%) and 10-11 (up to 6%). Mean body weight gain of these parental males was comparable to control group throughout the entire study.
Mean body weights and body weight gain of F1 parental males in the low and mid dose were comparable to control throughout the entire study.

HIgh dose F1 parental females had statistically significantly lower body weights during premating week 0-1 (up to 16%). High dose females' body weights were comparable to control group during remaining premating, entire gestation and lacation period. Mean body weight gain of these parental females was comparable to control animals.
Mean body weights and body weight gain of F1 parental males in the low and mid dose were comparable to control throughout the entire study. The statistically significantly increased body weight gain of high dose females during premating weeks 1-2 and premating weeks 0-10 was considered as spontaneous in nature.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test group 03 (400 mg/kg bw/d)
Statistically significantly decreased food consumption in parental males during premating weeks 0 - 1 (about 14%) and weeks 8 - 10 (up to 7%)
• Statistically significantly decreased food consumption in parental females during premating weeks 0 - 1 (about 10%) and weeks 9 - 10 (about 8%)
• Statistically significantly decreased food consumption in parental females during gestation days 0 - 14 (up to 8%)
• Statistically significantly decreased body weights in parental males during weeks 0 - 5 (up to 17%) and weeks 10 - 11 (up to 6%)
• Statistically significantly decreased body weights in parental females during premating weeks 0 - 1 (up to 16%)

Test group 02 (150 mg/kg bw/d)
• no test substance-related adverse effects/findings

Test group 01 (50 mg/kg bw/d)
• no test substance-related adverse effects/findings

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, non-treatment-related

Description (incidence and severity):

Absolute organ weights

Group male animals female animals
50 150 400 50 150 400
_________________________________________________________________________________________
Body weight -1% -5% -7%**
Liver +3% +9%** +10%**
Kidney +7%** +12%** +12%**
Brain 0% -2% -3%*
Cauda epididymis +7%* +7%* +6%*

*:p<=0.05 **:p<=0.01

All other mean absolute weight parameters did not show any significant differences compared to control.
The increase of absolute liver and kidney weights of females were considered as treatment-related. The increase of cauda epididymis weight in all treated males is considered incidental and not treatment-related as there was no histopathologic correlate observed. The decrease of brain weights in high-dose males is regarded to be a consequence of the reduced terminal body weight.

Relative organ weights

Group male animals female animals
50 150 400 50 150 400
_________________________________________________________________________________________
Liver -1% +3% +5%* +3%* +9%** +13%**
Kidney 0% +5%* +10%** +6%** +12%** +15%**
Epididymides +5%* +7%* +7%**
Cauda epididymides +8%* +13%** +14%**

*:p<=0.05 **:p<=0.01

All other parameters did not show significant differences in comparison to control.
The increase relative liver and kidney weights of females of all treated groups was regarded as treatment-related. The increase in liver weight in high-dose males and of kindey in mid- and high-dose males were considered to be treatment-related. The increase of epididymides in all treated males were thought to be reflective to the lower body weight. In addition, there was no histopathologic correlate corresponding to the weight decrease in the high-dose group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All gross lesions observed in test animals occured singularly. They are considered to be spontaneous lesions in origin and are not related to treatment.
The females that were not pregnant and the mating partners did not show relevant gross lesions.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All findings were single observations either or were similarly in distribution pattern and severity in control rats compared to treatment groups. All of them are considered to be incidental and/ or spontaneous in origin and without any relation to treatment.

The non-pregant females and the male mating partners did not show any histopathologic lesion that could explan the infertility.

Descendents: In the F1 generation, all animals survived until the end of the study.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrus cylce data, generated during the last 3 weeks prior to mating for the F2 litter, revealed regular cycles in the females of all groups. The mean oestrus cycle duration was similar: 4.1 (control), 4.2 (low-dose), 4.3 (mid-dose) and 4.1 days (high-dose).

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No test item-related effects were noted for the different sperm measures examined at or after the sacrifice of the parental F1 males. The number of homogenisation resistend testicular spermatids or caudal epididymal sperm, the percentages of abnormal and normal sperm and sperm motility data were comparable to control animals.

Reproductive performance:

no effects observed

Description (incidence and severity):

For all F1 males, copulation was confirmed. The male mating index is 100%. Fertility was proven for most of the F1 males within the scheduled mating interval for F2 litter. One control, one low-dose and one high-dose male did not generate F2 pups. The male fertility index ranges from 96-100 % without any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for the study. All respective values are within the range of historical control data of the test laboratory. The apparently infertile males did not show histopathological findings that could explain infertility.

The female mating index calculated after the mating period for F2 litter was 100% in all dose groups. The mean duration until sperm was detected (GD 0) varied between 2.2 and 2.8 days without any relation to dosing. All sperm positive rats delivered pups or had implants in utero, except one control, one low-dose and one high-dose female rat.

The female fertility index ranges from 96 (control, low and high-dose) to 100 (mid-dose) % without any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for the study. All respective values are within the range of historical control data of the test laboratory. The apparently non pregnant females did not show histopathological findings.
The mean duration of gestation values varied from 22.0 and 22.2 days without any relation to dosing. The gestation index was 100% in all dose groups.

Implanation was not affected by treatment since the number of implanation sites was comparable to control. There were no indications for test item-related intrauterine embryo-/fetolethality since postimplantation loss did not show any statistically significant differences between the groups. The mean number of F2 pups delivered per dam remained unaffected. The rate of liveborn pups was also not affected by treatment with the test item, as indicated by live birth index of 99% in all dose groups. Moreover, the number of stillborn pups was comparable between the groups.

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

400 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

Dose descriptor:

NOAEL

Remarks:

fertility and reproductive performance

Effect level:

400 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

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

The F1 pups did not display any treatment-related clinical signs until weaning. One pup of the control group displayed a domed head on PND 14-16 and was cannibalised on PND 16.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The viability index indication pup mortality during early lactation (OND 0-4) varied between 100 (low-dose) and 99% (other dose groups). The lacation index indicating pup mortality on PND 4-21 varied between 100 (low-dose), 99 (control), 97 (mid-dose) and 92%**(high-dose). The statistically significant decreased lactation index in the high-dose group was caused by the non test item-related death of 2 dams on PND 13 and 18, the litters of which had to be sacrified. These litters losses were considered as spontaneous in nature.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item-related influence on F1 pup body weight was noted in all treated groups. Mean body weight gain of low and mid-dose pups was comparable to control group. A temporary decrease of mean body weight gain was noted for the high dose pups on PND 4-7. Considering the absence of significant differences in weights themselves, these decreases were regarded as incidential and non treatment-related.

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

The food consumption of the parental F1 animals is reported above.

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:

no effects observed

Description (incidence and severity):

The first day when vaginal opening was observed was PND 28, the last was PND 42. The mean age at vaginal patency was 31.6, 33.0, 33.2* and 33.9* days in the control, low-, mid- and high-dose, respectively. The mean body weight on the day, when vaginal patency was noted, amounted to 95.5, 101.4, 101.5 and 98.1 g in the control, low-, mid- and high-dose, respectively. This minimal apparent increase in the high-dose group is considered secondary to the lower female body weight during major phase of sexual maturation.
The first day of preputial separation was observed was PND 39, the last was PND 49. The mean number of days to reach the criterion in the control and 50, 150 and 400 mg/kg bw/d test group was 41.6, 41.4, 41.6 and 42.5 days, indication that male sexual maturation was not influenced by the test item. The mean body weight, when preputial separation was recorded, amounted to 171.1, 168.9, 166.9 and 162.2*g. All ages and corresponding body weight values were either below or at the lower end of the historical control range. The significantly low high-dose body weight corresponds to the slightly, but not-significantly, later high-dose male sexual maturation.

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The absolute organ weight of the brain were statistically significant changed in comparison to the control.
absolute brain weight (male +female): 99%( low-dose); 97% (mid-dose); 97% * (high-dose)
absolute brain weight (male): 99% (low-dose); 95%*(mid-dose); 96%*(high-dose)

These marginal differences in absolute brain weights reflect normal biological variation in this strain of rats as they are comparable to historical control data. Relative brain weights remained unchanged. These findings are neither adverse nor toxicologially relevant.

Gross pathological findings:

no effects observed

Description (incidence and severity):

A few F1 pups showed spontaneous findings in gross necropsy (e.g. situs inversus, supernumerary lung lobe, small kidney, enlarged kidney). There findings occured without any relation to dosing and/ or can be found in the historical control data at comparable or even higher incidences. Therefore, these findings were not considered to be associated to the test item.

Histopathological findings:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

The NOEL (no observed effect level) is 50 mg/kg bw/d for the F1 parental rats based on effects on food consumption being a consequence of reduced appetite observed at the LOEL (Lowest Observed Effect Level) of 150 mg/kg bw/d in the F0 parental females.
The NOAEL for fertility and reproductive performance for the F1 parental rats is 400 mg/kg bw/d, the highest dose tested.
The NOAEL for developmental toxicity, in the F1 of the test substance is 400 mg/kg bw/d, the highest dose tested.

Dose descriptor:

NOAEL

Remarks:

prenatal developmental toxicity

Generation:

F1

Effect level:

400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Remarks on result:

other: no adverse effects observed

Dose descriptor:

NOEL

Remarks:

food consumption

Generation:

F1

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

food consumption and compound intake

Remarks on result:

other:

Remarks:

effects on food consumption beeing a consequence of reduced appetite observed at the LOEL of 150 mg/kg bw/d in the F0 parental females

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

The F2 pups did not show any treatment-related clinical signs until weaning. Female pup of one control dam had multiple malformed left hindlimb and for one female pup of a high-dose dam a lateral position on one occasion during lactation (OND 19) was recorded.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The viability index indicating pup mortality during early lactation (OND 0-4) varied between 98 (low-dose) and 99% (other dose groups). The lactation index indicating pup mortality on OND 4-21 varied between 98 (low-and mid-dose), 99 (high-dose) and 100% (control). The statistically significantly increased number of cannibalised pups in the low-dose was considered as spontaneous in nature.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item-related influence of F2 pup body weight was noted in all dose groups. Temporary decreases of mean body weight change were noted in F2 male and/or female pups of high-dose group on PND 1-4 and PND 4-7 as well as in low-dose group on PND 14-21. Considering the absence of significant differences in the weights themselves, these decreases were regardrd as incidental and not treatment-related.

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:

no effects observed

Description (incidence and severity):

There were no changes in absolute or relative organ weights noted in any of the dose groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Single F2 pups showed some spontaneous findings at gross necropsy such as incisors sloped, hemorrhagic thymus, diaphragmatic hernia, empty stomach, dilated renal pelvis, dilated ureter, small testis, haematoma and multiple malformed hindlimb. Every pup necropsy finding occured without any relation to dosing and/ or can be found in the historical control data at comparable or even higher incidences.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The sex distribution and sex ratios of live F2 pups on day of birth and PND 21 were comparable to control group.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Remarks:

prenatal developmental toxicity

Generation:

F2

Effect level:

400 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

Critical effects observed:

no

Reproductive effects observed:

no

- Analytics:

The various analyses:

• demonstrated the stability of the test substance preparations over a period of 7 days at room temperature
• confirmed the homogeneous distribution of the test substance in the vehicle (1% Carboxymethylcellulose suspension in drinking water and a few drops Cremophor EL and one drop hydrochloric acid)
• showed that the prepared concentrations were close to the expected values ranging between 86.8 and 113.2% of the nominal concentrations

Analytical values (range):

Test group	Nominal Dose (mg/kg bw/d)	Analytical Dose (mg/kg bw/d) [minimum]	Analytical Dose (mg/kg bw/d) [maximum]	% Nominal Dose [minimum]	% Nominal Dose [maximum]
00 / 10	0	0	0
01 / 11	50	43.40	46.61	86.8	93.2
02 / 12	150	132.90	169.80	88.6	113.2
03 / 13	400	359.20	379.90	89.8	95.0

 

Conclusions:

The NOAEL for general, systemic toxicity is 400 mg/kg/d for the parental rats in the highest dose tested.
The NOEL 50 mg/kg bw/day for the P parental rats, based on effects on food consumption observed at the LOAEL of 150 mg/kg bw/day in the P parental females.
The NOAEL for fertility and reproductive performance for the P and F1 parental rats is 400 mg/kg bw/day, the highest dose tested.
The NOAEL for developmental toxicity, in the F1 and F2 progeny, of the test substance is 400 mg/kg bw/day, the highest dose tested.

Executive summary:

The study was performed according to OECD TG 416 in compliance with GLP. Methyl Methacrylate was administered to groups of 25 male and 25 female healthy young Wistar rats (P parental generation) as an aqueous preparation by stomach tube at dosages of 0; 50; 150 and 400 mg/kg body weight/day. At least 73 days after the beginning of treatment, P animals were mated to produce a litter (F1). Mating pairs were from the same dose group and F1 animals selected for breeding were continued in the same dose group as their parents. Groups of 25 males and 25 females, selected from F1 pups to become F1 parental generation, were treated with the test substance at dosages of 0; 50; 150 and 400 mg/kg body weight/day post weaning, and the breeding program was repeated to produce F2 litter. The study was terminated with the terminal sacrifice of the F2 weanlings and F1 adult animals.

Control parental animals were dosed daily with the vehicle (1% Carboxymethylcellulose suspension in drinking water and four drops Cremophor EL and one drop hydrochloric acid).

The mid- and high-dose parental animals (400 mg/kg bw/d) showed clinical signs of systemic toxicity. The only relevant clinical observation was temporary salivation during a short period after dosing, which is considered to be test substance-induced. From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse toxicologically relevant finding.

In the mid- and high-dose (150 and 400 mg/kg bw/d) P generation animals, dose-related intermittent reductions of food consumption were noted, either during premating, gestation and lactation phases of this study. Less significant changes were noted for the F1 generation animals where the effects were limited to the high-dose group.

High dose F1 parental males had statistically significant lower body weights during several study segments, which led to a statistically significant reduction of the mean terminal body weight resulting in secondary weight changes of brain.

High dose parental females had statistically significant lower body weights during the first weeks after weaning. This weight decrease during major phases of sexual maturation led to an apparent marginal delay of vaginal patency. This minor delay did, however, not result in any corroborative pathological findings nor did it adversly effect F1 female cyclicity, fertility and reproduction. Thus, an influence of the test substance on female sexual maturation is not assumed.

Pathological examinations revealed no test-substance-related changes in organ weights, gross lesions, changes in differential ovarian follicle counts or microscopic findings, apart from an increase in kidney and liver weights in male and female animals in both generations which is presumably related to the treatment. There was no histopathologic lesion observed, that could explain the weight increase. It is regarded to be an adaptive change, most likely caused by an increase in metabolic activity in the two organs, which does not lead to histopathologic findings. It is not regarded to be an adverse effect.

There were no indications from clinical examinations as well as gross and histopathology, that the administration of methyl methacrylate via the diet adversely affected the fertility or reproductive performance of the P or F1 parental animals up to and including a dose of 400 mg/kg bw/day. Estrous cycle data, mating behavior, conception, gestation, parturition, lactation and weaning as well as sperm parameters, sexual organ weights and gross and histopathological findings of these organs (including differential ovarian follicle counts in the F1 females) were comparable between the rats of all test groups and ranged within the historical control data of the test facility.

All data recorded during gestation and lactation in terms of embryo-/fetal and pup development gave no indications for any developmental toxicity in the F1 and F2 offspring up to a dose level of 400 mg/kg bw/day. Up to this dose level, the test substance did not adversely influence pup viability and pup body weights. Sex ratio and sexual maturation was not directly affected at any dose level, inclusive the high-dose group (400 mg/kg bw/day).

Conclusion:

Under the conditions of the present 2-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicityis 400 mg/kg bw/d for the parental rats, the highest dose tested.

The NOEL (no observed effect level) is 50 mg/kg bw/d for the F1 parental rats based on effects on food consumption being a consequence of reduced appetite observed at the LOEL (Lowest Observed Effect Level) of 150 mg/kg bw/d in the F0 parental females.

The NOAEL for fertility and reproductive performance for the F1 parental rats is 400 mg/kg bw/d, the highest dose tested.

The NOAEL for developmental toxicity, in the F1 of the test substance is 400 mg/kg bw/d, the highest dose tested.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Reference 3

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

TEST MATERIAL:
- Name of test material: Isobornyl Acetate, CAS # 125-12-2
- Clear and colorless liquid
- Supplier: Takasago International Corp, Rockleigh, New Jersey, USA
- Batch No.: J145

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc, Portage, Michigan
- Age at study initiation: (P) x wks; (F1) x wks
- Weight at study initiation: (P) Males: 133 to 159 g; Females: 212 to 253 g
- Housing: individually in stainless steel, wire-bottomed cages, except during cohabitation when each pair of male and female rats were housed in the male rat’s cage.
- Diet (e.g. ad libitum): Certified Rodent Diet #5002 meal (PMI Nutrition International, Inc, St Louis, Missouri)
- Water (e.g. ad libitum): automatic watering access system and/or individual water bottles attached to the cages
- Acclimation period: "short"

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Formulations (suspensions) were prepared at least once weekly at the testing facility and a 10-day stability of IBOAc was established.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Male rats were treated beginning 84 days before cohabitation, throughout cohabitation period (max. 14 days) and continued to the day before euthanasia.

Female rates were treated beginning 14 days prior to cohabitation, through the cohabitation period (max. 14 days) and continuing through the day of euthanasia
(through Gestation Day 25 for rats that did not deliver or Lactation Day 22 for rats that delivered a litter.

Frequency of treatment:

once daily

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25 of each sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

F1 generation rats selected for continued evaluation were sacrificed on day 60 (+/- 3) postpartum

Positive control:

no

Parental animals: Observations and examinations:

The following parameters were evaluated in P generation males and females:
- viability
- body weight
- clinical observations
- food consumption
- mating and fertility
- organ weights
- gross pathology
- histopathological examination

The following parameters were evaluated in P generation males:
- sperm assessments (motility and concentration)

The following parameters were evaluated in P generation females:
- oestrus cycle
- natural delivery
- litter observations
- ovarian follicle counts

Oestrous cyclicity (parental animals):

see above (chapter Parental animals: Observations and examinations)

Sperm parameters (parental animals):

sperm motility and concentration

Litter observations:

The following parameters were evaluated on F1 pups:
- viabilty
- food consumption
- body weight
- body weight gain
- feed consumption
- organ weight
- nipple eruption (day 12 postpartum)
- sexual maturation
- preputial separation and vaginal opening
- anogenital distance (days 1 and 22 postpartum)
- gross observations

Postmortem examinations (parental animals):

see above (chapter Parental animals: Observations and examinations)

Postmortem examinations (offspring):

see above (chapter Litter observations)

Statistics:

Clinical observations and other proportional data: variance test for homegeneity of the binomial distribution
Continuous data: Bartlett test for homegeneity of the variances and ANOVA for analysis of variance
Individual groups: Dunnett test to identify statistical significance of individual groups
Individual groups: Kuskal-Wallis Test or Dunn test to identify statistical significance of individual groups (if ANOVA is not appropriate)
if there were >75% ties, Fisher exact test was performed
group means and standard deviations of primordial follicles: Kruskal-Wallis non parametric ANOVA Test to calculate and compare across groups
if significant result occured (P<0.05): Wilcoxon Test (Mann-Whitney U Test) for pairwise group comparison to controls.

Reproductive indices:

male mating index, male fertility index
female mating index, female fertility index, gestation index,

Offspring viability indices:

live birth index, viability index, lactation index

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The number of rats (male and female) with excess salivation (slight and/ or moderate) was significantly increased at 300 mg/kg bw/d in comparison to control. Slight excess salivation was observed as early as SD 2 and persisted into the lactation period, although it was more pronounced during pre-cohabitation and gestation and was also accompanied by a low incidence of transient urine-stained abdominal fur (P<=0.01; N=4 ot of 25) during the gestation period.
At 100 mg/kg bw/d, excess salivation was significantly increased only during gestation, in comparison to the control group.
All other clinical observations during the pre-cohabitation, gestation, and lactation period were considered unrelated to the treatment.

The only clinical attributed to treatment with the test item included slight to moderate salivation at 300 mg/kg bw/d-group. Among males, slight increase in salivation was observed as early as SD 27 and occured in 22 of 25 males of the highest dose group. Moderate increase in salivation occured in 6 of 25 males in the same dose group. Among females, slight increase in salivation was observed as early as SD 2 and persisted into the lactation period. The clinical sign was more pronounced during the pre-cohabitation and gestation period.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female rat at 100 mg/kg bw/d-group and one female of the high dose-group were euthanised during the gestation period due to adverse clinical observations (vocalisation and an apparent tail injury in the mid-dose rat and sings of dystocia, including delivery of 9 live pups, and the presence of 5 dead fetuses in utero in the high-dose rat. These findings were not considered to be related to the test substance because they occured only in single animals and were dissimilar in nature.
One male rat in the vehicle control group was found dead on SD 42 following apparent gavage accident and one male rat (100 mg/kg bw/d) was euthanised on SD 107 with adverse clinical signs (scab, ulceration, localised alopecia, abrasion on the neck and red discharge from the neck. In addition, localised alopecia on the limbs and underside were also observed and the rat was apprehensive when or at handling). The deaths were considered to be unrelated to the treatment .

Body weight and weight changes:

no effects observed

Description (incidence and severity):

In female rats, absolute and relative food consumption, body weights, and body weight gains during pre-cohabitation, gestation, and lactation period were unaffected by treatment. All values were comparable among the 4 dosage groups. Statistically significant increase in mean body weight gains at 100 and 300 mg/kg bw/d on SD 5-8 and a statistically significant increase in body weight gains at 300 mg/kg bw/d for the gestation period (GD 1-22) also were considered unrelated to the test substance because the increases were not dosage dependent and/or reflected an overall reduction in body weight gain or net loss in body weight in the corresponding control group.
In male rats, the absolute and relative food consumption, body weights and body weight gains were unaffected by the treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

In female rats, absolute and relative food consumption, body weights, and body weight gains during pre-cohabitation, gestation, and lactation period were unaffected by treatment. All values were comparable among the 4 dosage groups. The statistical significant increase in absolute and relative food consumption that occured in the 100 and 300 mg/kg bw/d -group on Lactation Day 8-11 were considered unrelated to the test substance because the increases were transient and not dosage dependent.
In male rats, the absolute and relative food consumption, body weights and body weight gains were unaffected by the treatment.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test substance-related histopathological findings were observed in any of the dose groups.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The oestrus cycling (e.g. the length of the oestrus cycle) was unaffected by treatment. Furthermore, the pregnancy rate is close to 100% in all groups.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

All evaluated sperm parameters were unaffected by the test item. The number and percentage of motile/ non motile sperm, total sperm count from vas deferens, sperm count and densitiy from cauda epiddidymal were comparable to control group. Although the morphology of the sperm was not examined, this endpoint was considered to be of no interest as the pregnancy rate in all dose group was close to 100%.

Reproductive performance:

no effects observed

Description (incidence and severity):

Male rats: All mating and fertility parameters (number of days in cohabitation, number of rats mated, male fertiltiy index, rats with confirmed mating dates during the first and second weeks of cohabitation and number of pregnancies per number of rats in cohabitation) are comparable to the control animals.

Female rats: All mating and fertility parameters (number of days in cohabitation, number of rats mated, female fertiltiy index, rats with confirmed mating dates during the first and second weeks of cohabitation and number of pregnancies per number of rats in cohabitation) are comparable to the control animals. Pregnancy occured in 23/22/24 and 22 of 25 mated females, respectively. Gestation index: 92/ 88/ 96 and 88 %. All pregnant dams delivered litters. Natural delivery and lactation parameters were comparable to control group.
Information on the number of implanations, corpora lutea, litter size, pre- and post-implantation loss in rats and rabbits after treatment with Isobornyl Acetate (OECD 414 in rats; Hoechst AG, 1992) and Camphor (OECD 414 in rats and rabbits, NTP, 1992) were given in the IUCLID section 7.8.2 Developmental toxicity/ teratogenicity.

No deaths related to isobornyl acetate occurred in either the P or F1 generation rats. One male rat in the control group was found dead at day 42 due to an apparent gavage accident. One male rat in the 100 mg/kg bw per day group and one female rat from each of the 100 and 300 mg/kg bw per day groups were euthanized during the study for adverse clinical signs unrelated to the administration of the test substance. P generation male and female rats in the 300 mg/kg bw per day group showed slight to moderate excess salivation that was attributed to dosing. In addition, female rats in the 300 mg/kg bw per day group showed low incidences of urine-stained fur during the gestation period.
There were 23, 22, 24, and 22 pregnant rats in the four dosage groups; all pregnant rats delivered litters.
Isobornyl acetate did not affect body weight, body weight gain or feed consumption values in any of the P or F1 generation rats. In male P generation rats, isobornyl acetate dosing produced no apparent effects on mating and fertility, reproductive or non-reproductive organ weights or sperm motility and concentration. In female P generation rats, isobornyl acetate dosing produced no apparent effects on reproductive and non-reproductive organs, estrous cycles, mating and fertility parameters or natural delivery, and there were no dosingrelated macroscopic lesions or microscopic changes at any dose tested.

Dose descriptor:

NOAEL

Effect level:

> 300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

At birth and during lactation period, there were no clinical signs observed that were related to the treatment.

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

No deaths occured during the post weaning period. One male rat was euthanised due to a broken palate on PPD 63. All other F1 pups survived until scheduled euthanasia.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The absolute and relative food consumption, body weights and body weight gains were unaffected by the treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The absolute and relative food consumption were unaffected by the treatment.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

no effects observed

Description (incidence and severity):

Sexual maturation was unaffected by the test item. The day of the preputial separation and vaginal opening were comparable to control animals.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The AGD in the treated F1 pups were comparable to the control pups.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipple eruption occured in any of the treated male pups. All female pups had nipples.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The weights of the examined reproductive (for males: prostate, seminal vesicles, testes; for females: uterus with cervix, ovaries) and non-reproductive organs (brain, paired kidneys and paired adrenals) were comparable to the control group. Except the relative weight of the adrenals of the 100 mg/kg bw group of F1 males was significantly reduced. The reduction was not considered to be treatment-related as the absolute adrenal weights were unaffected, the reduction was not observed in female rats and it was not dose-dependent.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related lesions observed. One pup of the highest dose group had a complete situs inversus. All other pups appeared normal.

Histopathological findings:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Evaluation of F1 generation male and female rats showed no dosing-related lesions or changes in body weight, body weight gain, feed consumption or organ weights at any of the doses tested. Nipple eruption (day 12 postpartum) and sexual maturation in F1 male and female rats were unaffected by isobornyl acetate dosing of the P generation at all doses tested.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reproductive effects observed:

no

Dosage (mg/kg bw/d)	0	30	100	300
Delivered one or more liveborn pups, N	23	22	24	22
Pups delivered (total) N Mean (SD)	302 13.1 (2.7)	299 13.6 (2.7)	317 13.2 (2.6)	302 13.7 (1.7)
Liveborn (total) Mean (SD) N (%)	13.0 (2.8) 299 (99.0)	13.6 (2.7) 299 (100.0)	12.9 (2.7) 309 (97.5)	13.6 (1.8) 299 (99.0)
Live birth index	99.0	100.0	97.5	99.0
Stillborn, mean (SD)	0.1 (0.3)	0.0 (0.0)	0.3 (0.6)	0.1 (0.5)
Viability index N (%) % N/N	3 (1.0) 99.7 298/299	0 (0.0) 99.7 298/299	8 (2.5) 99.7 308/309	3 (1.0) 98.7 295/299
Lactation index % N/N	98.3 293/298	96.3 287/298	98.7 304/308	99.0 292/295

Conclusions:

Based on the findings of this study, the NOAEL of isobornyl acetate is 300 mg/kg bw per day, the highest dose tested, for general and reproductive toxicity in the parental generation as well as viability and growth of the F1 generation.

Executive summary:

In an one-generation reproduction toxicity study according OECD 415 Sprague-Dawley rats (25 of each sex per dose group) were treated by oral gavage with Isobornyl acetate dosing 0, 30, 100 and 300 mg/kg/day. No dosing related effects were observed up to the highest ose group of 300 mg/kg/d. Based on the findings of this study, the NOAEL of isobornyl acetate is 300 mg/kg bw per day, the highest dose tested, for general and reproductive toxicity in the parental generation as well as viability and growth of the F1 generation.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 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

Reproductive toxicity of IBOMA has been investigated in OECD 421 combined reproductive/ developmental toxicity study on a screening level.

IBOMA is rapidly hydrolized by non-specific carboxylesterases to MMA and IBO (see chapter “Toxicokinetics”) so that the systemic toxicity of the parent ester can be assessed with information from the metabolites and their donor substances (i.e. MMA for MAA and IBOAc for IBO).

Here, the reproductive toxicity of the primary metabolite MAA is assessed bya reliable, oral 2-generation study with MMA as metabolite donor substance for MAA.From MAA as such, the investigations on reproductive organs of a 90 day inhalation study were selected as suppurtive information. The reproductive toxicity of the primary metabolite IBO is assessed by a reliable, oral 1-generation study with IBOAc.

 

IBOMA

In a reproduction/developmental toxicity screening study according to OECD 421 and GLP requirements, Sprague-Dawley rats (10 male and 10 females per dose group) received 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 (based on liver and kidney findings) and the reproductive/ developmental NOAEL was 500 mg/kg bw/day as there were no findings on reproductive/ developmental toxicity.

 

MAA

In a OECD 413 90-day inhalation study on the metabolite MAA, 10 male and 10 female Sprague Dawley rats were whole body exposed to target concentrations of 20, 40, 100 and 350 ppm (corresponding to 72, 143, 358 and 1253 mg/m³) during 6 hrs/d, 5 days/wk for 90 days (65 exposures). In addition to the examination of the standard organs and tissues, the sexual organs and parameters of fertility were examined. These examinations included weighing of sexual organs in both males and females and sperm mobility, morphology and sperm head count in one testis and one epididymis of each male animal of the high dose and control group. Substance-related changes of the sexual organs were not noted in any of the exposed animals (males or females), nor were there any changes of sperm mobility and sperm head counts. Under the current test conditions, the NOAEC for fertility related parameters was 350 ppm (1253 mg/m³) for male and female rats.

 

MMA (metabolite donor substance for MAA)

Methyl methacrylate has been tested in an OECD TG 416 oral two-generation reproduction toxicity study in rats, in which both, parental and F1 animals were dosed with 0, 50, 150 and 400 mg/kg body weight/day.

In mid- and high-dose parental animals (150 and 400 mg/kg bw/d) temporary salivation, presumably due to a bad taste of the test substance and associated dose-related intermittent reductions of food consumption were noted. Less significant changes were noted for the F1 generation animals where the effects were limited to the high-dose group and not associated with effects on histopathology or reproductive performance.

The NOAEL for fertility and reproductive performance for the P and F1 parental rats was determined to be 400 mg/kg bw/day, the highest dose tested. The NOAEL for developmental toxicity, in the F1 and F2 progeny, of the test substance was determined to be 400 mg/kg bw/day, the highest dose tested. There were no signs of systemic toxicity other than reduced body weight gain associated with reduced food consumption, presumably due to bad palatability (NOEL of 50 mg/kg bw/day for the P and F1 parental rats and LOEL of 150 mg/kg bw/day in the P parental females).

 

 IBOAc (metabolite donor substance for IBO)

In a one-generation reproduction toxicity study according to OECD 415, Sprague-Dawley rats (25 of each sex per dose group) were treated by oral gavage with IBOAc dosing 0, 30, 100 and 300 mg/kg/day. Suspensions of the test substance or the vehicle corn oil were administered via gavage to the P generation male rats once daily beginning 84 days before the cohabitation period, through the cohabitation period (maximum of 14 days), and continuing until the day before euthanasia; and to the P generation female rats once daily beginning 14 days before the cohabitation period, through the cohabitation period (maximum of 14 days), and continuing through the day of euthanasia. After weaning, 25 F1 generation pups/sex/dosage group were randomly selected for evaluation until sexual maturity. In the P generation (males and females), viability, clinical signs, body weights, feed consumption, mating and fertility, organ weights, gross and microscopic observations, sperm assessments (motility and concentration), natural delivery and litter observations, and ovarian follicle counts were evaluated. In the F1 generation (pups), viability, body weights, sexual maturation, anogenital distance (days 1 and 22 postpartum), nipple eruption (day 12 postpartum), and gross necropsy observations were recorded.

IBOAc did not adversely affect any of the investigated parameters. Increased incidences of excess salivation occurred in P generation at male and female rats at 100 mg/kg/d and/or higher doses throughout the dosage period, and low incidences of urine-stained abdominal fur were seen in high dosed females during the gestation period. These clinical signs were not considered as adverse effects of IA administration. Based on the results of this investigation, the no observable adverse effect level (NOAEL) for general toxicity of IBOAc is considered to be 300 mg/kg/d. The NOAEL for reproductive toxicity in the P generation as well as the NOAEL for viability and growth of the F1 generation is considered to be 300 mg/kg/d.

 

Read across assessment according to ECHA’s Read Across Assessment Framework (RAAF)

Reproductive toxicity of IBOMA has been investigated in an OECD 421 combined reproductive/ developmental toxicity study.Existing data gaps are covered using the read across approach mentioned in chapter1considering the ECHA guidance on Read Across (RAAF, ECHA 2017a). For this endpoint, the common metabolic pathway of the read across substances (rapid hydrolysis of IBOMA by ester cleavage, leading to the primary metabolites MAA and IBO) is considered as the most relevant aspect of the read across approach. The relatively short half-life shown for IBOMA as parent ester leads to the conclusion that systemic effects after repeated exposure are mainly driven by the products of ester hydrolysis, namely MAA and IBO. Local effects after inhalation are determined by local release of MAA. MMAand IBOAc are known to hydrolyse also rapidly so that these substances serve as donor substances for the primary metabolites MAA and IBO.

Qualitatively, this aspect can be categorized as “(Bio) transformation to common compound(s)” (RAAF scenario #1). The read across approach is done with a high level of confidence.

 

 

Effects on developmental toxicity

Description of key information

IBOMA

OECD 421, GLP, rat, oral: NOAEL parental 25 mg/kg bw/day (based on liver and kidney findings); NOAEL developmental 500 mg/kg bw/day (no effects)

 

Metabolite data

MMA (metabolite donor for MAA)

OECD 414, GLP, rabbit, oral gavage: NOAEL maternal 50 mg/kg bw/day (food consumption); NOAEL developmental 450 mg/kg bw/day (no effects)

MAA

Similar OECD 414, rat, inhalation: NOAEC maternal 200 ppm (bw & food consumption); NOAEC developmental 300 ppm (no effects)

CAM (metabolite of IBO)

NTP, rat, oral gavage: LOAEL maternal 100 mg/kg bw/day (water consumption); NOAEL developmental 800 mg/kg bw/day (no effects)

NTP, rabbit, oral gavage: LOAEL maternal & developmental 400 mg/kg bw/day (no effects)

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 421

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

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

Species:

rat

Strain:

Sprague-Dawley

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

Route of administration:

oral: gavage

Vehicle:

corn oil

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

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.

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

Abnormalities:

no effects observed

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effects observed

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

IBOMA did not show developmental toxicity via oral gavage to rats at doses as high as 500 mg/kg bw/day in the decribed reproductive/developmental toxicity screening study according to OECD 421.

Executive summary:

IBOMA did not show developmental toxicity via oral gavage to rats at doses as high as 500 mg/kg bw/day in the decribed reproductive/developmental toxicity screening study according to OECD 421 (for more details please also see chapter 7.5.1 and 7.8.1). There were no signs of neonatal toxicity or external malformations at doses of 500 mg/kg bw/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

before 27th June 2018

Deviations:

no

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

TEST MATERIAL:
- Name of test material: Methacrylic acid
- CAS: 79-41-4
Obtained from Fluka Chemie AG

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Nulliparous female Sprague-Dawley rats, weighing 180-200 grams obtained from IFFA Credo Breeding Labs.
Age at Start of Test: sexually mature females; age not specified. Mated females were housed inclear polycarbonate cages with stainless steel wire lids and hardwood shavings for bedding. 
Food and water available adlibitum except during exposures.
12 hours light-dark photocycle
Acclimatization of test animals: 2 weeks

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

Exposures were whole body and conducted in a 200 L chamber. Chamber temperature was 23 °C +/- 2 °C, and the relative humidity was 50% +/- 5%. Food and water were withheld during exposure. Dynamic and adjustable laminar air flow (6 - 20 m³/h). Air was passed through a heated bubbler containing test material. The vaporized material was then introduced into the exposure chambers.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations were determined 3 times at regular intervals during each 6 hr exposure by collecting the material and analyzing against a standard using GC.

Target concentrations [ppm] Analytical concentrations [ppm]
mean +/- SD
--------------------------------------------------------------------------------
50 55.3 +/- 8.1
100 101.5 +/-16.9
200 207.3 +/- 24.7
300 316.0 +/- 36.7
--------------------------------------------------------------------------------

Details on mating procedure:

2-3 females were caged with one male rat for mating. The onset of gestation was based upon the presence of sperm in the vaginal smear and this was designated gestation day 0. After confirmation of mating, females were turned to an individual cage. 

Duration of treatment / exposure:

6 hours per day

Frequency of treatment:

day 6 to 20 of gestation

Duration of test:

Mated females were exposed 6 hr/day on days 6 through 20 of gestation.

Dose / conc.:

50 ppm

Remarks:

corresponds to 179 mg/m3

Dose / conc.:

100 ppm

Remarks:

corresponds to 358 mg/m3

Dose / conc.:

200 ppm

Remarks:

corresponds to 716 mg/m3

Dose / conc.:

300 ppm

Remarks:

corresponds to 1076 mg/m3

No. of animals per sex per dose:

23 to 27 female rats were bred resulting in 22 to 23 pregnant rats used for experiment.

Control animals:

yes, concurrent no treatment

Maternal examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: GDs 0, 6, 13 and 21

FOOD CONSUMPTION: Yes
- Time schedule for examinations: GDs 6-13 and 13-21

Ovaries and uterine content:

Uterine content was examined after termination: Yes
Examinations included:
- uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: No data
- Number of viable and dead fetuses: Yes

At necropsy, the uterine horns and ovaries were exposed to count the C.L., implantation sites, resorption sites, and viable and dead fetuses.   FERTILITY AND REPRODUCTIVE PERFORMANCE: The following data were recorded for each group of numbers of CL, and implantation sites o number of resorptions and viable and dead fetuses. O mean fetal body weights o  fetuses examined for gross malformations and skeletal abnormalities of sex and of fetuses.

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: No data

Live fetuses were weighed, sexed, and examined for external  anomalies. 50% of the live fetuses from each litter were preserved in Bouin's solution and examined for internal soft-tissue changes. The remaining fetuses (50%) were fixed in ethanol (70%), eviscerated and then processed for skeletal staining with alizarin red S.

Statistics:

The number of corpora lutea, implantation sites, and live fetuses, maternal food consumption and various body weights were analyzed by ANOVA, followed by Dunnett's
test. The percentage of non-live  implant, resorptions, and males and the proportion of fetuses with alterations in each litter were evaluated by Kruskal-Walles test followed by Dixon-Massey test. Rates of pregnancy and percentage of litters with any malformations or external, visceral, or skeletal variations were analyzed using Fisher's test. Where appropriate, least squares analysis was performed. The level of significance was p < 0.05. The litter was used as the basis of analysis of fetal variables.

Indices:

Pre- and postimplantation loss; live birth index

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

All animals survied the exposure.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Absolute weight gain was significantly reduced at 300 ppm.

Change in weight during gestation in Sprague-Dawley rats inhaling Methacrylic acid on days 6 to 20 of gestation and euthanized on day 21:
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Concentrations No of dams Mean Body weight [g] +/- SD Mean body weight gain on GD [g] Mean absolute weight gain [g]
[ppm], 6 h/day on GD 6 6 - 13 13 - 21 6 - 21
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------
0 23 276 +/- 19 32 +/- 9 109 +/- 32 141 +/- 36 36 +/- 13
50 22 278 +/- 18 32 +/- 8 115 +/- 13 147 +/- 15 35 +/- 11
100 22 283 +/- 19 34 +/- 7 110 +/- 19 144 +/- 23 36 +/- 14
200 22 284 +/- 22 29 +/- 8 107 +/- 20 136 +/- 24 32 +/- 13
300 23 276 +/- 16 20 +/- 7** 91 +/- 26* 111 +/- 29** 12 +/- 14**
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------
*, ** Denote significant differences from the control (0 ppm), p < 0.05 and p < 0.01, respectively.

Mean absolute weight gain: Mean body weight gain corrected by gravid uterine weight

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was reduced at 300 ppm.

Food consumption of Sprague-Dawley rats inhaling Methacrylic acid on days 6 to 20 of gestation and euthanized on day 21:
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Concentrations No of dams Mean food consumption [g/dam/day] on GD
[ppm], 6 h/day 6 - 13 13 - 21 6 - 21
------------------------------------------------------------------------------------------------------------------------------------------------------------------------
0 23 23 +/- 2 27 +/- 3 25 +/- 2
50 22 24 +/- 1 28 +/- 2 26 +/- 2
100 22 24 +/- 3 28 +/- 3 26 +/- 3
200 22 22 +/- 2 27 +/- 2 25 +/- 2
300 23 20 +/- 2** 24 +/- 2** 22 +/- 26*
------------------------------------------------------------------------------------------------------------------------------------------------------------------------
*, ** Denote significant differences from the control (0 ppm), p < 0.05 and p < 0.01, respectively.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Number of abortions:

no effects observed

Description (incidence and severity):

There were no abortions reported.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no significant changes in number of implantations across control and exposed groups.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Total litter loss by resoprtion was not reported.

Early or late resorptions:

no effects observed

Description (incidence and severity):

The number of early and late resorptions was unaffected by treatment.

Dead fetuses:

no effects observed

Description (incidence and severity):

The number of dead fetuses was not reported.

Changes in pregnancy duration:

not specified

Description (incidence and severity):

In the OECD 414, a Caesarian section on GD 21 is performed.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The number of pregnant females is comparable to the control.

Details on maternal toxic effects:

Maternal toxic effects: yes

Details on maternal toxic effects:
All animals survived the exposure. Exposure to 300 ppm led to significant decreases in maternal weight gain and food consumption throughout exposure. Absolute weight gain was significantly reduced at 300 ppm.

Dose descriptor:

NOAEL

Effect level:

200 ppm

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Remarks on result:

other: corresponds to 716 mg/m³

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The fetal body weight was unaffected by treatment.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Reproductive parameters in Sprague-Dawley rats inhaling Methacrylic acid on days 6 to 20 of gestation and euthanized on day 21:
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Concentrations No of litters No. of live fetuses per litter
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
0 22 14.73 +/- 3.92
50 22 14.86 +/- 1.93
100 22 14.05 +/- 3.17
200 22 13.77 +/- 3.99
300 22 14.05 +/- 3.76
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Reproductive parameters in Sprague-Dawley rats inhaling Methacrylic acid on days 6 to 20 of gestation and euthanized on day 21:
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Concentrations No of litters Average fetal body weight [g/per litter]
[ppm], 6 h/day All Males Females
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
0 22 5.71 +/- 0.56 5.86 +/- 0.57 5.42 +/- 0.37
50 22 5.66 +/- 0.27 5.82 +/- 0.32 5.49 +/- 0.27
100 22 5.79 +/- 0.30 5.92 +/- 0.32 5.62 +/- 0.32
200 22 5.76 +/- 0.47 5.92 +/- 0.47 5.54 +/- 0.45
300 22 5.67 +/- 0.49 5.71 +/- 0.34 5.54 +/- 0.52
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Changes in litter size and weights:

not specified

Description (incidence and severity):

Litter size and weights were not reported.

Changes in postnatal survival:

not specified

Description (incidence and severity):

The pups were euthanised before birth.

External malformations:

no effects observed

Description (incidence and severity):

No significant increase of external fetal malformations was observed after exposure to methacrylic acid. There was no difference between the control and exposed groups.
Description please see attached document (Incidence of malformations and variations in fetuses of Spraque-Dawley rats inhaling Methacrylic acid on days 6 to 20 of gestation).

Skeletal malformations:

no effects observed

Description (incidence and severity):

No significant increase of skeletal fetal malformations was observed after exposure to methacrylic acid. There was no difference between the control and exposed groups.
Description please see attached document (Incidence of malformations and variations in fetuses of Spraque-Dawley rats inhaling Methacrylic acid on days 6 to 20 of gestation).

Visceral malformations:

no effects observed

Description (incidence and severity):

No significant increase of visceral fetal malformations was observed after exposure to methacrylic acid. There was no difference between the control and exposed groups.
Description please see attached document (Incidence of malformations and variations in fetuses of Spraque-Dawley rats inhaling Methacrylic acid on days 6 to 20 of gestation).

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: no effects

Details on embryotoxic / teratogenic effects:
There were no significant changes in number of implantations and live fetuses, in the incidence of non-live implants and sorptions, or in fetal weights across groups. One fetus of 200 ppm and two of the 300 ppm group showed different types of malfomations. There was no consistent pattern of changes to suggest any treatment-related effects. The difference of fetuses with external, visceral, and skeletal variations did not differ between the control and the treated groups. No significant increase in embryo/fetal lethality or fetal malformations were observed after exposure to methacrylic acid. While maternal toxicity was observed, methacrylic acid caused no evidence of developmental toxicity up to 300 ppm.

Dose descriptor:

NOAEL

Remarks:

prenatal developmental toxicity

Effect level:

> 300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Abnormalities:

no effects observed

Developmental effects observed:

no

		Methacylic acid (Saillenfait 1999, OECD 414 rat inhalativ 6 h/day)
	control	50 ppm (corresponding 179 mg/m³)	100 ppm (corresponding 358 mg/m³)	200 ppm (corresponding 716 mg/m³)	300 ppm (corresponding 1076 mg/m³)
number of pregnant/ non pregnant dams	25 (total) / 23 (= 92%) pregnant / 2 non pregnant	25 (total) / 22 (= 88%) pregnant / 3 non pregnant	26 (total) / 23 (= 84.6%) pregnant / 3 non pregnant	25 (total) / 22 (= 88%) pregnant / 3 non pregnant	26 (total) / 23 (= 88.5%) pregnant / 3 non pregnant
number of dams with abortions/ early deliveries/ stillbirths/ resorptions and/or dead fetuses	number of abortions not explicitly specified early deliveries were not reported (assumed to be 0) number of stillbirth were not reported (assumed to be 0) number of resorptions: 8.96 +/- 20.73	number of abortions not explicitly specified early deliveries were not reported (assumed to be 0) number of stillbirth were not reported (assumed to be 0) number of resorptions: 3.22 +/- 3.34	number of abortions not explicitly specified early deliveries were not reported (assumed to be 0)  number of stillbirth were not reported (assumed to be 0) number of resorptions: 6.76 +/- 8.14	number of abortions not explicitly specified early deliveries were not reported (assumed to be 0) number of stillbirth were not reported (assumed to be 0)  number of resorptions: 5.67+/- 12.77	number of abortions not explicitly specified early deliveries were not reported (assumed to be 0) number of stillbirth were not reported (assumed to be 0) number of resorptions: 10.61 +/- 21.45
pre-implantation loss (as no individual data is given in the publication, the pre-implantation loss is calculated based on the means of number of corpora lutea and number of implantation sites)	12.55 mean	4.6 mean	7.58 mean	12.01 mean	9.81 mean
postimplantation loss (as no individual data is given in the publication, the postimplantation loss is calculated based on the means of number of implantation sites and number of live offspring)	1.21 mean	3.26 mean	6.33 mean	4.11 mean	2.63 mean
Mean Body weight [g] on GD6	276 +/- 19 SD	278 +/- 18 SD	283 +/- 19 SD	284 +/- 22 SD	276 +/- 16 SD
Mean Body weight change [g]	141 +/- 36 on GD 6-21	147 +/- 15 on GD 6-21	144 +/- 23 on GD 6-21	136 +/- 24 on GD 6-21	111 +/- 29** on GD 6-21 significant different from the control (0 ppm)
mean gravid uterine weight including optionally body weight change corrected for gravid uterine weight (difference between body weight gain from GD 6-21 and absolute body weight gain)	105 g mean	112 g mean	108 g mean	104 g mean	99 g mean
mean number and percent of live offspring	14.73 live pups/litter percentage not specified	14.86 live pups/litter percentage not specified	14.05 live pups/litter percentage not specified	13.77 live pups/litter percentage not specified	14.05 live pups/litter percentage not specified
mean foetal/ pup weight by sex and sexes combined	5.71 mean (g/litter) 5.86 males (g/litter) 5.42 females (g/litter)	5.66 mean (g/litter) 5.82 males (g/litter) 5.49 females (g/litter)	5.79 mean (g/litter) 5.92 males (g/litter) 5.62 females (g/litter)	5.76 mean (g/litter) 5.92 males (g/litter) 5.54 females (g/litter)	5.67 mean (g/litter) 5.71 males (g/litter) 5.54 females (g/litter)
number and percent of foetuses and litters with malformations (including runts) and/ or variations	number of malformations: -                 litter affected: - number of variations: 37/324 or 11.4% litter affected: 21/22 (95.4%)	number of malformations: -                        litter affected: - number of variations: 35/327 or 10.7%                        litter affected: 20/22 (90.9%)	number of malformations: -                  litter affected: - number of variations: 41/309 or 13.3% litter affected: 15/22 (68.2%)	number of malformations: 1/303 or 0.3%                                        litter affected: 1/22 or 4.5%              number of variations: 29/303 or 9.6% litter affected: 16/22 (72.7%)	number of malformations: 2/309 or 0.6%                                                               litter affected: 2/22 or 9.0%                                       number of variations: 39/309 or 12.6%                         litter affected: 16/22 (72.7%)
description and incidences of malformations and main variations (and/or retardation)	(detailed information see attached document to the IUCLID entry of this study (Incidence of malformations and variations in fetuses of Spraque-Dawley rats  inhaling Methacrylic acid on days 6 to 20 of gestation))	(detailed information see attached document to the IUCLID entry of this study (Incidence of malformations and variations in fetuses of Spraque-Dawley rats  inhaling Methacrylic acid on days 6 to 20 of gestation))	(detailed information see attached document to the IUCLID entry of this study (Incidence of malformations and variations in fetuses of Spraque-Dawley rats  inhaling Methacrylic acid on days 6 to 20 of gestation))	(detailed information see attached document to the IUCLID entry of this study (Incidence of malformations and variations in fetuses of Spraque-Dawley rats  inhaling Methacrylic acid on days 6 to 20 of gestation))	(detailed information see attached document to the IUCLID entry of this study (Incidence of malformations and variations in fetuses of Spraque-Dawley rats  inhaling Methacrylic acid on days 6 to 20 of gestation))

Conclusions:

Using a valid scientific method, no significant increase in embryo/fetal lethality or fetal malformations were observed after exposure to methacrylic acid. While maternal toxicity was observed, methacrylic acid caused no evidence of developmental toxicity up to 300 ppm.

Executive summary:

In an OECD 414 prenatal developmental toxicity study using whole body inhalation methacrylic acid at test concentrations of 50, 100, 200 and 300 ppm, corresponding to 179, 358, 716 and 1076 mg/m³ methacylic acid did not produce any embryo - or foetal lethality, nor fetal malformations, despite overt maternal toxicity (decreased body weight and feed consumption). The NOEC (teratogenicity) was considerd to be 300 ppm (1076 mg/m³).

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

April 22th to May 28th 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 12 May 1981

Deviations:

no

GLP compliance:

yes

Limit test:

yes

Specific details on test material used for the study:

- Physical state: liquid

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoe: WISKf (SPF71)
- Age at study initiation:65-70 days
- Weight at study initiation: 191 + / - 6 g
- Housing: individually in plastic cages
- Diet (e.g. ad libitum): ad libitum (Altromin 1310)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 7 days prior the test
- Sex: female

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24 ° C
- Humidity (%): 49-53%
- Air changes (per hr): 16-20/h
- Photoperiod (hrs dark / hrs light): 12 light hours a day

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: the substance was dissolved fresh daily in a concentration of 200 g/l in sesame oil. The applied fluid volume was in all animals 5 ml/kg body weight.
VEHICLE
- Amount of vehicle (if gavage): 5 ml/kg bw dose

Details on mating procedure:

Proof of pregnancy: sperm in vaginal smear referred to as day 1 of pregnancy

Duration of treatment / exposure:

days 7 to 16 of gravity

Frequency of treatment:

once daily, administered within three hours

Duration of test:

Sacrifice at day 21 of gravity

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

limit test

No. of animals per sex per dose:

20 mated females: test group
21 mated females: control group

Control animals:

yes

Details on study design:

- Dose selection rationale: Limit dose of the respective guideline

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
BODY WEIGHT: Yes
- Time schedule for examinations: once a week and a day after the last application
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: uterus, ovaries
OTHER:
Macroscopic examination and weighing of the organs: heart, liver, kidneys and spleen

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes

Fetal examinations:

- External examinations: Yes
- Skeletal examinations: Yes: half per litter
- Body cross-sections for Wilson, photography: half per litter

Statistics:

Comparisons between body weights and organ weights in the test and control group were performed with a classical analysis of variance (MANOVA); to evaluate the relative feed intake, a analysis of variance according to Puri & Sen (1985).
Corpora lutea and implantations were performed with the Mantel-Haenszel x2 test (Mantel & Haenszel, 1959), as well as live and dead fetuses and resorptions. Other parameters according to an analysis of variance.
The body cross-sectional study of fetuses and skeletal findings were studied with the Fisher Exact test.

Historical control data:

To compare the data with historical controls, normal ranges were calculated for groups.

Clinical signs:

no effects observed

Description (incidence and severity):

One dam had local hair loss on the forelimbs at day 11 of gravity and at day 19 also on stomach, flanc and hindlimbs. The authors of the study indicated the hair loss as a randome event.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weight changes of the treated group is comparable to the control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Dams had a similar food consumption compared to the control animals during the treatment time. After the treatment time the animals exposed to the substance had a slightly increased uptake of food compared to the control group. The body weight change of the treated and untreated animals were thereof unaffected.

Behaviour (functional findings):

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

In two dams of the high dose group the right renal pelvis was slightly increased. But there were no macroskopic alterations observed. Therefore the findings were neither considered as relevant nor adverse.

Histopathological findings: neoplastic:

not examined

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

One treated dam had no live fetuses but only 5 implation sites indecation a preimplantation loss. In the report it was reported as an abort.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

pre-/ postimplantation loss (number and percent): 8,69 / 5,37 (1000 mg/kg group)
: 11,52 / 7,74 (control group)

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There was no litter loss observed in the study.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There was no early or late resorptions observed in the study.

Description (incidence and severity):

There was one dead fetus found in the uterus of a dam of the control group.

Description (incidence and severity):

In a OECD 414 guideline study a Caesarian section is performed.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

All females were pregnant.

Details on maternal toxic effects:

Maternal toxic effects: no effects

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

other: no adverse effects observed

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean pup weight (sexes combined: 3,3g (1000 mg/kg dose group) and 3,4g (control group))

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Mean number of live offspring:
237 total in the 1000 mg/kg group
256 total in the control group

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex ratio in the treatment group and the control group was relative even. In both groups the male fetues predominated.
Treated group: 58,2 % male pups
Control group: 53,1 % male pups

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Mean litter size:
12,5 pups (1000 mg/kg group)
12,8 pups (control group)
Mean pup weight please see above

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

There was no change in post natal survival observed in the study.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Description please see attached document.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Description please see attached document.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Description please see attached document.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: no effects

Dose descriptor:

NOAEL

Remarks:

embryotoxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effects observed

Dose descriptor:

NOAEL

Remarks:

teratogenicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effects observed

Abnormalities:

effects observed, non-treatment-related

Description (incidence and severity):

Description please see attached document.

Developmental effects observed:

no

All findings did not differ from those of control group out of the spontaneous rate.

Conclusions:

No maternal toxic effects, no embryonic/fetal toxic effects and no teratogenic effect at oral doses of 1000 mg/kg bw/day in rats.

Executive summary:

Isobornylacetate was tested in a oral limit test according to OECD guideline 414 of a group of 20 female Wistar rats from 7 to 16 day of gravity, administered once daily in a dose of 1000 mg / kg bw. As a control, was an equally large group of control animals that received the vehicle without substance addition. On 21 days of gestation the mother animals were killed and examined. Fetuses were morphologically observed for developmental disorders. The morphological examination of fetuses revealed no evidence for embryotoxic and teratogenic effects of the substance. The findings are assigned to the spontaneous rate. Neither maternal nor embryonic/fetal toxicity was found at oral doses of 1000 mg/kg bw/day IBA in rats. An indication of teratogenic effect could not be noticed.

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-04-02 until 2009-06-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 22nd January 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ECC Directive 2004/73/EC, Part B: Method for the determination of Toxicity: Prenatal Developmental Toxicity Study; Official Journa of the European Communities; L 216, pp.227-235.

Version / remarks:

29th April 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

TEST MATERIAL:
- Name of test substance: Methyl Methacrylate
- CAS No.: 80-62-6

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 16-21 weeks
- Weight at study initiation: 2187-2917 g
- Fasting period before study: no
- Housing: the rabbits were housed singly in type 12.2395.C stainless steel wire mesh cages supplied by Draht-Bremer GmbH, Marktheidenfeld, Germany (floor area about 3,000 cm²)
- Diet: pelleted “Kliba maintenance diet for rabbits & guinea pigs, GLP”, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12 (6.00 p.m. to 6.00 a.m. dark, 6.00 a.m. to 6.00 p.m. light)

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The aqueous test substance preparations were prepared at the beginning of the administration period and thereafter at maximum intervals of 7 days, which took into account the analytical results of the stability verification. For the test substance preparation, an specific amount of test substance was weighed depending on the dose group, into a graduated flask (conical Erlenmeyer flasks with groundin stopper), topped up (shortly under the marking) with 1% Carboxymethylcellulose solution in drinking water and a few drops Cremophor EL and one drop of 32% hydrochloric acid. Afterwards the preparation was filled up with 1% Carboxymethylcellulose suspension in drinking water. The flask was sealed and the preparation was intensely mixed with a magnetic stirrer. During administration, the preparations were kept homogeneous with a magnetic stirrer and the vessels were kept closed between the withdrawals of the preparations.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test substance preparations were sent to the analytical laboratory twice during the study period (at the beginning and towards the end) for verification of the concentrations. Samples were analyzed by GC with external calibration.

Details on mating procedure:

After an acclimatization period of at least 5 days, the female rabbits were fertilized by means of artificial insemination. This implied that 0.2 mL of a synthetic hormone which releases LH and FSH from the anterior pituitary lobe (Receptal) were injected intramuscularly to the female rabbits about 1 hour before insemination. The ejaculate samples used for the artificial insemination were derived from male Himalayan rabbits of the same breed as the females. Each female was inseminated with the sperm of a defined male donor. The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study. The day of insemination was designated as gestation day (GD) 0 and the following day as GD 1.

Duration of treatment / exposure:

from implantation to one day prior to the expected day of parturition (GD 6-28)

Frequency of treatment:

once daily

Duration of test:

until GD 29

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

actual dose: 41 mg/kg bw/d

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

actual dose: 132 mg/kg bw/d

Dose / conc.:

450 mg/kg bw/day (nominal)

Remarks:

actual dose: 406 mg/kg bw/d

No. of animals per sex per dose:

25 artificially inseminated females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose volume was 10 mL/kg body weight. The calculation of the volume administered was based on the most recent individual body weight.

Maternal examinations:

CLINICAL EXAMINATIONS
- Mortality:
Mortality was checked in the females twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-29).

- Clinical symptoms:
A clinical examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occurred, the animals were examined several times daily (GD 0-29).

- Food consumption:
The food consumption was determined daily on GD 1–29.

- Body weight data:
All animals were weighed on GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29. The body weight change of the animals was calculated.

- Corrected (net) body weight gain:
The corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6).

TERMINAL EXAMINATIONS OF THE DOES:
After the does had been sacrificed on GD 29, they were necropsied and assessed by gross pathology in randomized order. Details see chapter ovaries and uterine content.

Ovaries and uterine content:

On GD 29, the surviving does were sacrificed in randomized order by an intravenous injection of pentobarbital (Narcoren; dose: 2 mL/animal). After the does had been sacrificed, they were necropsied and assessed by gross pathology in randomized order.
The uterus and the ovaries were removed and the following data were recorded:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
1) live fetuses
2) dead implantations:
a) early resorptions (only decidual or placental tissues visible or according to SALEWSKI (Salewski, 1964) from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy);
b) late resorptions (embryonic or fetal tissue in addition to placental tissue visible);
c) dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened).

After the weight of the uterus had been determined, all subsequent evaluations of the does and the gestational parameters were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose the animal numbers were encoded.
Furthermore, calculations of conception rate and pre- and postimplantation losses were carried out:
The conception rate (in %) was calculated according to the following formula: (number of pregnant animals)/(number of fertilized animals) x 100.
The preimplantation loss (in %) was calculated based on each individual pregnant animal with scheduled sacrifice according to the following formula: (number of corpora lutea – number of implantations)/(number of corpora lutea) x 100.
The postimplantation loss (in %) was calculated based on each individual pregnant animal with scheduled sacrifice from the following formula: (number of implantations – number of live fetuses)/(number of implantations) x 100.

Fetal examinations:

All fetal analyses were conducted by technicians unaware of the treatment group, in order to minimize bias.
- Examination of the fetuses after dissection from the uterus: Each fetus was weighed and examined macroscopically for any external findings.
Furthermore, the viability of the fetuses and the condition of the placentae, the umbilical cords, the fetal membranes, and fluids were examined. Individual placental weights were recorded. Thereafter, the fetuses were sacrificed by a subcutaneous injection of phenobarbital (Narcoren; 0.2 mL/fetus).

- Soft tissue examination of the fetuses: After the fetuses had been sacrificed, the abdomen and the thorax were opened in order to examine the organs in situ before they were removed. The heart and the kidneys were sectioned in order to evaluate the internal structure. The sex of the fetuses was determined by examination of the gonads in situ. After these examinations, the heads of approximately one half of the fetuses per litter and the heads of those fetuses, which revealed severe findings during the external examination (e.g. anophthalmia, microphthalmia or hydrocephalus) were severed from the trunk. These heads were fixed in BOUIN's solution and were, after fixation, processed and evaluated according to WILSON's method (Wilson and Warkany, 1965). About 10 transverse sections were prepared per head. After the examination these heads were discarded. All fetuses (partly without heads) were skinned and fixed in ethyl alcohol. After fixation for approx. 1-5 days, the fetuses were removed from the fixative for awhile. With a scalpel, a transversal incision was made into the frontal / parietal bone in the heads of the intact fetuses. The two halves of the calvarium were then cauteously bent outward and the brain was thoroughly examined. Subsequently, the fetuses were placed back into the fixative for further fixation.

- Skeletal examination of the fetuses: After fixation in ethyl alcohol the skeletons were stained according to a modified method of KIMMEL and TRAMMELL (Kimmel, C.A., and Trammell, C., 1981). Thereafter, the stained skeleton of each fetus was examined. After the examination the stained skeletons were retained individually.

Statistics:

- DUNNETT-test (two-sided) for food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight.
- FISHER'S EXACT test (one-sided) for female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings.
- WILCOXON-test (one-sided) for proportions of fetuses with malformations, variations and/or unclassified observations in each litter.

Indices:

Conception rate; Pre-implantation loss; Postimplantation loss

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related clinical signs or any disturbances of general behaviour observed in any rabbits.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related or spontaneous mortalities in any group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights of the low-, mid- and high-dose rabbits (50; 150 and 450 mg/kg bw/d) were not significantly different from the concurrent control throughout the course of the study. The average body weight gain of the mid- and high-dose rabbits was statistically significantly reduced by about 27% and 31% during the treatment period. A significant reduction of mean body weight gain was also noted for the the high-dose rabbits on GD 19-21.

Corrected (net) body weight gain: Mean carcass weights and the corrected body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) were comparable among all groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption in the high-dose females (450 mg/kg bw/d) was distinctly and statistically significantly reduced during a significant part of the treatment period (GD 15-23). During the entire treatment period (GD 6-28) the total average food consumption of the high dose rabbits was about 18% below controls. The food consumption of the mid dose females (150 mg/kg bw/d) was similarly affected in terms of magnitude and course of reduction, however the reduction of food consumption reached statistical significance only on GD 22-24. During the treatment period (GD 6-28) the total average food consumption of the mid-dose rabbits was about 13% below controls. Overall, the food consumption of the low-dose does (50 mg/kg bw/d) did not show test substance-related impairments. The reduced food consumption at the 150 and 450 mg/kg bw/d levels is considered to be related to the treatment.

Food efficiency:

not specified

Ophthalmological findings:

not specified

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:

no effects observed

Description (incidence and severity):

The mean gravid uterus weights of all test groups did not show staatistically significant differences in comparison to control.

Gross pathological findings:

no effects observed

Description (incidence and severity):

At necropsy, only spontaneous findings were seen in single females of every test group. No test substance-related findings were observed in the doses.

Neuropathological findings:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

There were no test item-related and/or biologically relevant differences between the control and the treated groups in conception rate, the mean number of corpora lutea, the mean number of implantation sites and/ or the calculated values for the pre- and postimplanation loss, the number of resorptions and viable fetuses. Gestational parameters were within the normal range for animals of this strain and age.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no test item-related and/or biologically relevant differences between the control and the treated groups in conception rate, the mean number of corpora lutea, the mean number of implantation sites and/ or the calculated values for the pre- and postimplanation loss, the number of resorptions and viable fetuses. Gestational parameters were within the normal range for animals of this strain and age.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There was no total litter loss by resoption obsevered in any of the does.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no test item-related and/or biologically relevant differences between the control and the treated groups in conception rate, the mean number of corpora lutea, the mean number of implantation sites and/ or the calculated values for the pre- and postimplanation loss, the number of resorptions and viable fetuses. Gestational parameters were within the normal range for animals of this strain and age.

Dead fetuses:

no effects observed

Description (incidence and severity):

All fetuses were alive after euthanasia of the does.

Changes in pregnancy duration:

not examined

Description (incidence and severity):

In an OECD 414 study, a Caesarian section is performed.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The conception rate reached 96% in test groups 1 and 3 (50 and 450 mg/kg bw/d) and 100% in test groups 0 and 2 (0 and 150 mg/kg bw/d). Importantly, a sufficient number of pregnant females was available for the purpose of the study, as 24-25 pregnant rabbits per group had implantation sites in the uterus, at terminal sacrifice. There were no test substance-related and/or biologically relevant differences between the control and all dosed groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. Gestational parameters were within the normal range for animals of this strain and age.

Dose descriptor:

NOAEL

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed

Dose descriptor:

NOEL

Remarks:

food consumption

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

food consumption and compound intake

Remarks on result:

other: actual dose: 41 mg/kg/d

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal weights of all treated groups were not influenced by the test substance. Neither female nor male weights showed statistically significant or biologically relevant differences between the test substance-treated groups and the controls.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no dead fetuses in any of the dose groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (50; 150 and 450 mg/kg bw/d) was comparable to the control fetuses. Observable differences were without biological relevance.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The mean fetal weight of all treated groups and the litter size were unaffected by the test item.

Changes in postnatal survival:

not examined

Description (incidence and severity):

In an OECD 414 study, the fetuses were euthanised immediately after the does' death.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One sole external malformation (unilateral microphthalmia) was recorded for two fetuses from 2 litters in the high-dose group (450 mg/kg bw/d). This malformation is present in the historical control data. Thus an association of these individual findings to the treatment is not assumed. The total incidences of external malformations were comparable to the historical control data.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Malformations of the fetal skeletons were noted in fetuses of test groups 0, 2 and 3 (0; 150 and 450 mg/kg bw/d). Neither statistically significant differences between treated groups and the control were calculated nor a dose-response relationship was observed. All individual skeletal malformations were present in the historical control data at a comparable frequency.
- Fetal skeletal variations: For all test groups, variations in different skeletal structures were detected with or without effects on the corresponding cartilages. The observed skeletal variations were related to various parts of the fetal skeletons and were statistically significant higher in the low- and the high-dose groups on a fetus per litter basis. Several specific skeletal variations were statistically significant higher than the concurrent control in the dosed groups (on a fetus per litter basis). These findings are delays or minor disturbances of ossification which are reversible or do not considerably affect the integrity of the underlying structures. Such slight changes of the ossification process occur very frequently in gestation day 29 rabbit fetuses of this strain and all observed incidences were within the historical control data. Thus an association of these findings to the treatment is not assumed.
- Fetal skeletal unclassified cartilage observations: Additionally, isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all groups including the control. The observed unclassified cartilage findings did not show a relation to dosing and were comparable to historical control data and, therefore, regarded to be spontaneous in nature.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- Fetal soft tissue malformations: The examination of the soft tissues revealed a variety of malformations in fetuses of all test groups including the controls (0; 50; 150 and 450 mg/kg bw/d). With the exception of a lateral pouch in the tongue of 2 fetuses all individual soft tissue malformations were present in the historical control data at comparable frequencies. No statistically significant differences between the test groups and the control were observed. The total incidences of external malformations were comparable to the historical control data. No malformation pattern was evident. Thus an association of these findings to the treatment is not assumed.
- Fetal soft tissue variations: A number of soft tissue variations, such as absent lung lobe (lobus inferior medialis) and malpositioned carotid branch, was detected in each test group including the controls. Incidences were without a relation to dosing. Neither statistically significant differences between the test groups nor differences to the historical control data were noted.
- Fetal soft tissue unclassified observations: Unclassified soft tissue observations, such as infarct of liver, hemorrhagic thymus or ovary and blood coagulum around urinary bladder, were recorded for some fetuses of test groups 0, 1, 2 and 3 (0; 50; 150 and 450 mg/kg bw/d). A relation to dosing is not present if normal biological variation is taken into account. Therefore, a test substance induced effect is not assumed.

Details on embryotoxic / teratogenic effects:

- Abstract of all classified fetal external, soft tissue and skeletal observations: Various external, soft tissue and skeletal malformations occurred throughout all test groups including the control. All individual malformations are present in the historical control data, with the exception of lateral pouches in the tongue of 2 fetuses. They did neither show a consistent pattern since a number of morphological structures of different ontogenic origin were affected nor a clear dose-response relationship. The overall incidence of malformations was comparable to the historical control data. One external (paw hyperflexion), two soft tissue (absent lobus inferior medialis and malpositioned carotid branch) and a broad range of skeletal variations occurred in all test groups including the controls. All fetal and litter incidences for these variations and the corresponding mean percentages of affected fetuses/litter were not significantly different from the concurrent control and their frequency is comparable to the historical control data. Therefore, they were not considered to be related to the treatment. A spontaneous origin is also assumed for external, soft tissue and unclassified skeletal cartilage observations which were observed in several fetuses of all test groups including controls (0, 50; 150 and 450 mg/kg bw/d). Distribution and type of these findings do not suggest relation to treatment.

Key result

Dose descriptor:

NOAEL

Remarks:

prenatal developmental toxicity

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Abnormalities:

no effects observed

Developmental effects observed:

no

Table with the requested information is attached to the IUCLID entry.

Table: Occurence of statistically significantly increased fetal skeletal variation (expressed as mean percentage of affected fetuses/litter)

Finding	Group 0 0 mg/kg/d	Group 1 50 mg/kg/d	Group 2 150 mg/kg/d	Group 3 450 mg/kg/d	HCD (range)
Incomplete ossification of parietal; unchanged cartilage	0.0	0.0	1.9*	2.1*	0.4 (0.0 – 2.6)
Incomplete ossification of hyoid; cartilage present	11.2	11.4	19.1	20.4*	9.8 (0.0 – 21.6)
Splitting of skull bone	0.4	3.3*	3.3	2.3	2.9 (0.0 – 7.7)
Incomplete ossification of cervical centrum; unchanged cartilage	2.5	2.2	3.6	7.3*	2.5 (0.0 – 9.3)
Supemumerary 13th rib; cartilage not present	2.5	9.8	6.1	9.9*	6.6 (0.0 – 17.5)
Total fetal skeletal variations	46.3	63.7*	59.3	71.6**	63.5 (46.3 – 81.9)

HCD = Historical control data; * = p ≤ 0.05, ** = p ≤ 0.01 (Wilcoxon-Test [one-sided])

 

 

Table: Total fetal malformations

		Group 0 0 mg/kg/d	Group 1 100 mg/kg/d	Group 2 300 mg/kg/d	Group 3 1000 mg/kg/d
Litter Fetuses	N N	25 171	24 154	25 157	24 158
Fetal incidence	N (%)	4 (2.3%)	2 (1.3%)	6 (3.8%)	9 (5.7%)
Litter incidence	N (%)	4 (16%)	1 (4.2%)	4 (16%)	7 (29%)
Affected fetuses/litter	Mean%	2.3	1.2	3.6	6.2

 

 

Table: Total fetal variations

		Group 0 0 mg/kg/d	Group 1 100 mg/kg/d	Group 2 300 mg/kg/d	Group 3 1000 mg/kg/d
Litter Fetuses	N N	25 171	24 154	25 157	24 158
Fetal incidence	N (%)	106 (62%)	106 (69%)	106 (68%)	122 (77%)
Litter incidence	N (%)	21 (84%)	24 (100%)	24 (96%)	23 (96%)
Affected fetuses/litter	Mean%	59.9	69.8	64.3	74.2

 

Conclusions:

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 450 mg/kg bw/d and the no observed effect level (NOEL) for maternal toxicity is 50 mg/kg bw/d based on effects on food consumption. The NOAEL for prenatal developmental toxicity is 450 mg/kg bw/d. No adverse fetal findings of toxicological relevance were evident at any dose.

Executive summary:

The study was performed according to OECD TG 414 in compliance with GLP.

Methyl Methacrylate was tested for its prenatal developmental toxicity in Himalayan rabbits. The test substance was administered as an aqueous preparation to 25 inseminated female Himalayan rabbits by stomach tube at doses of 50; 150 and 450 mg/kg body weight/day on gestation days (GD) 6 through GD 28. The control group, consisting of 25 females, was dosed with the vehicle (1% Carboxymethylcellulose CB 30.000 in drinking water and a few drops Cremophor EL and one drop hydrochloric acid [1% CMC]) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 29, 24-25 females per group had implantation sites.

The following test substance-related adverse effects/findings were noted:

Test group 3 (450 mg/kg body weight/day):

-        Reduced food consumption (-18%) and body weight gain (-31%)

-        No test substance-related adverse effects on gestational parameters or fetuses

 

Test group 2 (150 mg/kg body weight/day):

-        Reduced food consumption (-13%) and body weight gain (-27%)

-        No test substance-related adverse effects on gestational parameters or fetuses

 

Test group 1 (50 mg/kg body weight/day):

-        No test substance-related adverse effects on does, gestational parameters or fetuses

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is nominal 450 mg/kg bw/d (actual 406 mg/kg bw/d), the highest dose tested. The no observed effect level (NOEL) for maternal toxicity is nominal 50 mg/kg bw/d (effective 41 mg/kg bw/d) based on effects on food consumption being a consequence of reduced appetite observed at the LOEL (Lowest Observed Effect Level) of 150 mg/kg bw/d (actual 132 mg/kg bw/d).

The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is nominal 450 mg/kg bw/d (actual 406 mg/kg bw/d). No adverse fetal findings of toxicological relevance were evident at any dose.

Reference 5

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

9th April 1991 until 22nd August 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

before 27th June 2018

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

TEST MATERIAL:
- Name of test material: d-Camphor
- CAS No. 464-49-3

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Source: Hazelton Research Products, Inc., Denver, PA
- Weight at study initiation: 2490-4400 g
- Housing: individually in stainless steel cages with mash flooring
- Diet: Purina Certified Rabbit (R) Chow (#5322) pellets, ad libitum
- Water: deionised/ filtered water, ad libitum
- Acclimatisation period: 2 weeks

ENVIRONMENTAL CONDITIONS:
- Temperature (°F): 65-66
- Humidity (%): 58-64
- Air changes (per hr): not mentioned
- Photoperiod (hrs light/ hrs dark): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Substance was administered by gavage in a volume of 2 mg/kg bw on Gestation Day 6 to 19. The actual dose delivered was adjusted daily, according to the weight taken prior to dosing. Pre-dosing analysis of CAM were performed and the formulations were within 95-104% of their theoretical concentrations. Treatment and examination of animals were performed by staff without knowing the dose level.

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

Females were artifically inseminated after the 14 days quarantine-period.

Duration of treatment / exposure:

From Gestation Day 6 to 19

Frequency of treatment:

once daily

Duration of test:

All does and pups were euthanised on GD 30.

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

26

Control animals:

yes

Details on study design:

Dose selection rationale: The top dose of 400 mg/kg/day was based on a preliminary dose range finding study which found 500 mg/kg/day CAM caused 60% maternal mortality.

Maternal examinations:

CAGE SIDE OBSERVATIONS:
Yes, animals were observed daily before (GD 0-5), during (GD 6-19) and after (GD 20-30) dosing with the test item.

DETAILED CLINICAL OBSERVATIONS:
Yes, animals were observed daily before (GD 0-5), during (GD 6-19) and after (GD 20-30) dosing with the test item.

BODY WEIGHT:
Yes , does were weighted on GD 0, 6 through 19, 25 and 30

FOOD CONSUMPTION:
Yes. Food was weighted on GD 0, 3, 6, 9, 12, 15, 18, 19, 22, 25, 28 and 30.


POST-MORTEM EXAMINATIONS:
Yes . At necropsy, the maternal body, liver and intact uterus were weighted and corpora lutea were counted.

Ovaries and uterine content:

The uterine of each doe was examined and the number of implantation sites, resorptions, dead fetuses and live fetuses was determined. Uteri with no visible implantation sites were stained to detect very early resorptions.

Fetal examinations:

Live fetuses were dissected from the uterus and also euthanised. Afterwards, their body weigt was determined and they were examined for external morphological and visceral abnormalities. All fetal carcasses were stained and examined for skeletal malformations.

Statistics:

"General linear Models (GLM) procedures were applied for the analyses of variances (ANOVA) of maternal and fetal parameters. Prior to GLM analysis, an arcsine-square root transformation was performed on all litter-derived percentage data and Bartlett's test for homegeneitiy of variance was performed on all data to be analyzed by ANOVA. GLM analysis determined the significance of dose-response relationships and the significance of dose effects, replicate effects and dose x replicate interactions.
When ANOVA revelaed a significant (p<0.05) dose effect. William's and Dunnett's Multiple Comparison Test compared CAM-exposed to control groups. One-tailed tests were used for all pair-wise comparisons except maternal body weight and percent males per litter. Non-significant (p>0.05) dose x replicate effects on selected fetal parametric measures were considered justification for pooling data across replicates for nonparametrc analysis on related measures. When significant (p<0.05) dose x replicate interactions occured, the data for that endpoint and for any related nominal scale data were analysed separtely for dose effects weithin each replicate in the study, as well as for all replicates combined. Nominal scale measures were analysed by a X2 test for independence and by a test for linear trend on proportions. When a X2 test showed significant group differences, a one-tailed Fisher's exact probability test was used for pairwise comparisons of CAM and control groups." (cited from the report)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Detailed clinical information on does were given in the report. The report is attached to the IUCLID entry.

Mortality:

no mortality observed

Description (incidence):

There were no CAM-related deaths observed. Due Does were removed from the highest dose group due to dosing errors.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Maternal body weights in all CAM-treated does, before, during and after the treatment period were always comparable to the control group. The maternal weight gain was reduced 13, 5 and 59% in the 50, 200 and 400 mg/kg bw dose groups, respectively.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No effects on food consumption were observed in any of the treated groups.

Food efficiency:

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:

no effects observed

Description (incidence and severity):

The maternal liver weights were unaffected by the treatment with the test item.

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

The percentage of resorptions per litter, late fetal deaths per litter, nonlive implants per litter and adversely affected implants per litter is comparable to the control group. The proportion of litters with one or more resoprtion, late fetal deaths, nonlive implants or adversely affected implants was not affected by the treatment.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The percentage of resorptions per litter, late fetal deaths per litter, nonlive implants per litter and adversely affected implants per litter is comparable to the control group. The proportion of litters with one or more resoprtion, late fetal deaths, nonlive implants or adversely affected implants was not affected by the treatment.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Total litter loss by resorption was not observed in the study.

Early or late resorptions:

no effects observed

Description (incidence and severity):

The percent of litter with on or more late death is comparable to control.

Dead fetuses:

no effects observed

Description (incidence and severity):

LIve litter size was unaffected by the treatment.

Changes in pregnancy duration:

not specified

Description (incidence and severity):

In OECD 414 studies, a Caesarian section is performed.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The number of pregnant does is comparable to the control group.

Details on maternal toxic effects:

Examination of the uterine contents revealed that CAM had no effect on fetal growth, viability, or morphological development.

Dose descriptor:

NOAEL

Effect level:

> 400 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The fetal body weight was unaffected by the treatment.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The number of live fetuses per litter is comparable to control group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The percentage of male fetuses was unaffected by the treatment.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The litter size and the fetal body weight per litter were comparable to control.

Changes in postnatal survival:

not examined

Description (incidence and severity):

The fetuses were euthanised immediately after the doe's death.

External malformations:

no effects observed

Description (incidence and severity):

The percentage of fetues with external malformation is comparable to control.

Skeletal malformations:

no effects observed

Description (incidence and severity):

The percentage of fetues with skeletal malformation is comparable to control.

Visceral malformations:

no effects observed

Description (incidence and severity):

The percentage of fetues with visceral malformation is comparable to control.

Details on embryotoxic / teratogenic effects:

Examination of the uterine contents revealed that CAM had no effect on fetal growth, viability, or morphological development.

Dose descriptor:

NOAEL

Remarks:

fetotoxicity

Effect level:

> 400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

> 400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

The results from this study indicate that CAM is neither developmentally toxic nor toxic to the doe at doses as high as 400 mg/kg/day.

Executive summary:

NTP: "

Camphor is a very common constituent of over-the-counter medications used to relieve muscle aches, nasal congestion, and discomfort from cold sores. Ingestion ofcan cause severe toxicity in humans. Because of the large population at risk for CAM exposure, and since it readily crosses the placenta, CAM was evaluated as a potential developmental toxicant. CAM (0, 50, 200, or 400 mg/kg/day) was administered in corn oil by gavage to pregnant rabbits during the major period of organogenesis (6-19). The top dose of 400 mg/kg/day was based on a preliminary dose range finding study which found 500 mg/kg/day CAM caused 60% maternal mortality. Does were monitored for physical and clinical signs of toxicity. On gd 30, fetuses were examined for effects of CAM on growth, viability, and morphological development.

There was no maternal mortality in any of the experimental groups. Maternal body weights in the CAM-treated groups were comparable to controls at all gestational ages. However, maternal weight gain tended to decrease with increasing dose of CAM. Maternal food consumption in all three CAM groups was similar to controls throughout gestation. Examination of the uterine contents revealed that CAM had no effect on fetal growth, viability, or morphological development. The results form this study indicate that CAM is neither developmentally toxic nor toxic to the doe at doses as high as 400 mg/kg/d."

Reference 6

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1991-02-05 until 1991-05-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

before 27th June 2018

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

TEST MATERIAL:
- Name of test material: d-Camphor MRI
- CAS: 464-49-3

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboaratories, Inc., Raleigh, NC
- Age at study initiation: 8-10 weeks upon reciept
- Weight at study initiation: 213-275 g (on GD 0)
- Fasting period before study: no
- Housing: sperm-positive females were housed individually in solid-bottom polycarbonate cages with stainless steel wire lids and Ab-Sorb-Dri cage litter
- Diet: ad libitum, ground Purina Certified Rodent Chow (#5002)
- Water: ad libitum. deionised/ filtered water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 67-75 (average 72)
- Humidity (%): 55
- Air changes (per hr): not mentioned
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Substance was administered by gavage in a volume of 5 ml/kg on Gestation Day 6-15. The acutal dose delivered was adjusted daily, according to a weight taken prior to dosing. Pre-dosing analysis of the CAM dose formulations for each replicate were within 95-105% of their theoretical concentrations. Treatment and examination of animals was performed without the knowledge of dose levels.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The chemical/ vehicle mixture was shown to be stable for 4 weeks when kept at room temperature in amber glass jars, as it was stored in the current study.

Details on mating procedure:

After the 7 day quaratine period, the individual breeding partner were cohabited overnight. On the following morning, females were examined for the presence of sperm by vaginal smear. The day of detection of sperm was designated as Gestation Day 0.

Duration of treatment / exposure:

From Gestation Day 6 to 15

Frequency of treatment:

once daily

Duration of test:

All dams and pups were euthanised on GD 20.

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

Dose / conc.:

800 mg/kg bw/day (nominal)

No. of animals per sex per dose:

26-29 sperm-positive female rats

Control animals:

yes

Details on study design:

- Dose selection rationale: The dose levels selected for this study were based on preliminary data furnished by NTP, 1990. The preliminary data revealed that 1250 and 2500 mg/kg bw/d were highly toxic.

Maternal examinations:

CAGE SIDE OBSERVATIONS:
Yes; animals were observed before dosing (GD 0-5) and daily during dosing (GD 6-15) and after dosing (GD 16-20).

DETAILED CLINICAL OBSERVATIONS:
Yes; animals were observed before dosing (GD 0-5) and daily during dosing (GD 6-15) and after dosing (GD 16-20).

BODY WEIGHT:
Yes; dams were weighed on GD 0, 3, 6 through 15, 18 and 20.

FOOD CONSUMPTION:
Yes; Food measurements were taken on GD 0, 3, 6, 9, 12, 15, 18 and 20.

WATER CONSUMPTION:
Yes; Water measurements were taken on GD 0, 3, 6, 9, 12, 15, 18 and 20.

POST-MORTEM EXAMINATIONS:
Yes; Animal were sacrified on GD 20; at necrosy, the maternal body weight, liver and intact uterus were weighed and corpora lutea were counted.

Ovaries and uterine content:

The uterus of each dam was examined and the number of implantation sites, resorptions, dead fetuses and live fetuses was determined. Uteri with no visible implantation sites were stained to detect very early resorptions.

Fetal examinations:

Live fetuses were dissected from the uterus and also euthanised. Afterwards, their body weight was determined and they were examined for external morphological and visceral abnormalities. All fetal carcasses were stained and examined for skeletal malformations.

Statistics:

"General linear Models (GLM) procedures were applied for the analyses of variances (ANOVA) of maternal and fetal parameters. Prior to GLM analysis, an arcsine-square root transformation was performed on all litter-derived percentage data and Bartlett's test for homegeneitiy of variance was performed on all data to be analyzed by ANOVA. GLM analysis determined the significance of dose-response relationships and the significance of dose effects, replicate effects and dose x replicate interactions.
When ANOVA revelaed a significant (p<0.05) dose effect. William's and Dunnett's Multiple Comparison Test compared CAM-exposed to control groups. One-tailed tests were used for all pair-wise comparisons except maternal body weight and percent males per litter. Non-significant (p>0.05) dose x replicate effects on selected fetal parametric measures were considered justification for pooling data across replicates for nonparametrc analysis on related measures. When significant (p<0.05) dose x replicate interactions occured, the data for that endpoint and for any related nominal scale data were analysed separtely for dose effects weithin each replicate in the study, as well as for all replicates combined. Nominal scale measures were analysed by a X2 test for independence and by a test for linear trend on proportions. When a X2 test showed significant group differences, a one-tailed Fisher's exact probability test was used for pairwise comparisons of CAM and control groups." (cited from the report)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs were infrequent and generally confined to the 800 mg/kg bw/d group. The most common clinical sign in the highest dose group was hypoactivity, which was confined to the first % two days of dosing. The incidence for hypoactivity in the 800 mg(kg bw/d group fell from 27% (7/26) on GD 6, to 8% (2/26) on GD 7, and finally 0% on GD 8.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There was no mortality observed in any of the dose groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Maternl body weight, corrected body weight and maternal body weight change from GD 0 to 20 were unaffected by treatment. However, maternal body weight change during the dosing period (GD 6 to15) displayed a significant dose-dependent decrease. Weight gain was 9% lower than controls in the 400 mg/kg bd/d group and 16% lower in the 800 mg/kg bw/d group (individually significant).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was initially suppressed by both 400 and 800 mg/kg/day. but recovered to control levels by the end of the treatment period. No effect on food consumption was seen at 100 mg/kg/day.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Both 400 and 800 mg/kg bw/d CAm produced comparable and consistent increases in maternal water intake. Relative water intake in the mentioned groups went from 115% of controls on GD 6 to 9 to 130-136% at the end of dosing period. Water consumption in these groups remained 18-32% above controls through GD 18 to 20. Smaller increases in relative maternal water intake were noted for 100 mg/kg bw/d CAM, but significant effect was observed only on GD 6 to 9.

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

Description (incidence and severity):

Absolute and relative liver weights exhibited a significant dose-related increase, but did not exceed 10% of control values in any individual group.

Neuropathological findings:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

There was an Caesarian section performed on GD 20.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The rate of pregnancy in the CAM-treated rats was comparable to control.

control: 23/26 (88%)
100 mg/kg bw/d: 21/26 (84%) (1 dam was removed from study, because it exceeded the maximum allowable weight on GD 0).
400 mg/kg bw/d: 23/29 (85%) (2 dams were removed from study, because one exceeded the maximum allowable weight on GD 0 and one dam was adminstered the incorrect dose on GD 15).
800 mg/kg bw/d: 25/26 (96%) (1 dam was removed from study, because it exceeded the maximum allowable weight on GD 0).

Other effects:

no effects observed

Description (incidence and severity):

The number of corpora lutea and implantation sites were unaffected by CAM-treatment.

Details on maternal toxic effects:

Examination of the uterine contents revealed that CAM, even at 800 mg/kg/day, had no adverse effect on fetal growth, viability, or morphological development.

Dose descriptor:

LOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

water consumption and compound intake

Remarks on result:

other: effects not considered as adverse

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The average fetal body weight was unaffected by CAM-treatment of the dams.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The CAM-treatment had no influence on the number of live fetuses. There was just one dead fetus in the control group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The number of male pups per litter was unaffected by treatment.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The number of live fetuses and average fetal body weight were unaffected by treatment.

Changes in postnatal survival:

not examined

Description (incidence and severity):

The live pups were euthanised shortly after the dam.

External malformations:

no effects observed

Description (incidence and severity):

There was no effect observed on the incidence of external, visceral or skeletal malformations. The number of variations was also unaffected by CAM-treatment of the dams.

Skeletal malformations:

no effects observed

Description (incidence and severity):

There was no effect observed on the incidence of external, visceral or skeletal malformations. The number of variations was also unaffected by CAM-treatment of the dams.

Visceral malformations:

no effects observed

Description (incidence and severity):

There was no effect observed on the incidence of external, visceral or skeletal malformations. The number of variations was also unaffected by CAM-treatment of the dams.

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

Examination of the uterine contents revealed that CAM, even at 800 mg/kg/day, had no adverse effect on fetal growth, viability, or morphological development.

Dose descriptor:

NOAEL

Remarks:

fetotoxicity

Effect level:

> 800 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effects observed

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

> 800 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effects observed

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

In summary, CAM does not affect fetal growth, viability, or morphological development at doses causing minor maternal toxicity

Executive summary:

NTP: "Camphor is widely used as a constituent of over-the-counter medicaments marketed for the relief of muscle aches, nasal congestion, and discomfort from cold sores. Ingestion of camphor can cause severe toxicity in humans. Because of the large population at risk forexposure during the manufacture or use of such products and because it readily crosses the placenta. CAM was evaluated as a possible developmental toxicant. Accordingly, CAM (0, 100, 400, or 800 mg/kg) was admistered daily by gavage to pregnant rats during the major period of organogenesis (gd 6-15). CAM, 800 mg/kg, was selected as the high dose in our study, since previous reports indicated that 1250 mg/kg/day CAM causes 90% maternal mortality. Dams were monitored for physical and clinical signs of toxicity, and the effect of CAM on fetal growth, viability, and external, visceral and skeletal malformations was assessed on gd 20.

There was no maternal mortality during the study. Absolute and relative liver weights exhibited a significant dose-related increase, but did not exceed 10% of control values in any individual group. Maternal toxicity consisted of alterations in food and water consumption (absolute and relative) and decreased weight gain during the treatment period. Food consumption was initially suppressed by both 400 and 800 mg/kg/day CAM. but recovered to control levels by the end of the treatment period. No effect on food consumption was seen at 100 mg/kg/day CAM. Unlike the dose-dependent effects of CAM on food intake, all three doses of CAM increased maternal water consumption during one or more of the following measurement periods (gd 6 to 9; gd 12 to 15; gd 15 to 18). Although maternal body weights in all treatment groups were within 5% of control values at all gestational ages, maternal weight gain during the treatment period in the 800 mg/kg/day group was significantly reduced.

Examination of the uterine contents revealed that CAM, even at 800 mg/kg/day, had no adverse effect on fetal growth, viability, or morphological development. In summary, CAM does not affect fetal growth, viability, or morphological development at doses causing minor maternal toxicity."

Reference 7

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29-05-1990 to 26-06-1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

before 27th June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.4350 (Inhalation Developmental Toxicity Screen)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

TEST MATERIAL:
-Name of test material: Methyl methacrylate
- CAS No 80-62-6
- Supplier: Rohm and Haas Company
- colorless, volatile liquid

Species:

rat

Strain:

other: Crl:CDBR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- 225 nulliparous female rats
- Source: Charles River Breeding Laboratories Inc., Kingston, NY
- Age at study initiation: 81 -88 days
- AGE AT TIME OF MATING: 88-95 days.
- Weight at study initiation: 183-240 grams upon arrival
- Fasting period before study:
- Housing: Animals were housed individually, except during mating, in suspended stainless-steel cages (7" x 8" x 13.5"). During exposures, females were housed individually in suspended stainless-steel, wire mesh cages (6" x 7" x 11"). 
- Diet (e.g. ad libitum): Food (Certified Purina Rodent Chow #5002 ad libitum except during exposures.
- Water (e.g. ad libitum): filtered tap water ad libitum except during exposures.
Neither feed nor water was available during the 6 hour exposure period.
- Acclimation period: 7 days prior to mating

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2°C
- Humidity (%): ranged from 40-60% during cohabitation and 63-80% during the exposure and post-exposure periods.
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light was maintained

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

The test material exposure concentrations were generated by metering the test material with calibrated Fluid Metering Pumps (Fluid Matering Inc., Oyster Bay, NY) into 500 mL three-necked round bottom flasks (Lab Glass Inc., Vineland, NJ).
Exposures were whole body and were conducted in 2000 L stainless steel, glass and Plexiglas® chambers. Cage positions within the chamber were rotated daily. The temperature and relative humidity within the chambers during exposure were 22-24°C and 55-67%, respective.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test substance in the chambers was determined by the use of a Miran gas analyzer attached to a strip chart recorder. A probe was placed into the center of the chamber and the chamber atmosphere was drawn into the Miran A1 gas analyzer at a rate of 9.5 L/min. Each chamber was analyzed initially within 40 min. of the end of the t99 to insure that each chamber was within the accepted target range. Subsequently, each chamber was sampled every 
120 min. A range of plus or minus 10% of the target chamber concentration was maintained by making minor adjustments on the generator pump delivery rates whenever necessary.


Target concentrations [ppm] Analytical concentrations [ppm]
mean +/- SD
--------------------------------------------------------------------------------
100 98.8 ± 3.4
300 304.4 ± 9.1
1200 1178.1 ± 69.1
2000 2028.2 ±107.3
--------------------------------------------------------------------------------

Details on mating procedure:

Females were mated with males overnight (one male:one female) and the presence of sperm in the vaginal smear was considered gestation day 0. Mated females were exposed via inhalation to the test material for 6 hrs/day on gestation days 6 through 15 and then sacrificed on day 20.  

Duration of treatment / exposure:

6 - 15 day of gestation

Frequency of treatment:

6 hours/day

Duration of test:

20 d (dams were euthanized on gestation day 20)

Dose / conc.:

99 ppm

Remarks:

corresponding to 412 mg/m3 or 0.41 mg/L

Dose / conc.:

304 ppm

Remarks:

corresponding to1285 mg/m3 or 1.29 mg/L

Dose / conc.:

1 178 ppm

Remarks:

corresponding to 4900 mg/m3 or 4.9 mg/L

Dose / conc.:

2 028 ppm

Remarks:

corresponding to 8436 mg/m3 or 8.44 mg/L

No. of animals per sex per dose:

27 animals per group exposed; 22-25 pregnant females per exposure group.

Control animals:

yes, sham-exposed

Details on study design:

- Other: The strain was selected because background development toxicity data exists as Rohm and Haas Company on this rat strain. The test material was given by inhalation since the respiratory route is a potential route of human exposure.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed daily for behavioral changes.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily GD 0 - 20

BODY WEIGHT: Yes
- Time schedule for examinations: recorded on GD 0, 6, 8, 10, 13, 16 and 20.

 Food consumption was measured for GD intervals 0-6, 6-10, 10-16 and 16-20. 

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
A limited necropsy was performed on all adult female animals. During the necropsy, animals were examined for evidence of pregnancy
and gross lesions of the abdominal and thoracic cavities. Each uterus was weighted.

Ovaries and uterine content:

On GD 20, all dams were asphyxiated with carbon dioxide, the thoracic and abdominal cavities were examined and the uterus was removed and weighed, and corpora lutea, implantation sites and resorptions were counted. The number of fetuses per litter was counted and position inside the uterus recorded. The uteri of apparently non pregnant rats were stained with a 10% ammonium sulfide solution to detect very early resorptions. All fetuses were weighed, examined for external alterations and the sex of each fetus was determined. 

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [half per litter ]

One half of the fetuses from each litter were examined for visceral alterations using the Staples' technique. Head alterations were recorded for these fetuses examined for soft tissue alterations using the technique of Barrow and Taylor (1969, J. Morphol., 127: 291-306). The carcasses of all fetuses were stained with alizarin red S and examined for skeletal alterations. 

Statistics:

For the purpose of statistical evaluation, the litter was considered the experimental unit for fetal parameters. Pregnancy rate, clinical signs, maternal deaths, gross necropsy findings and litters with total resorptions were statistically analyzed using the  Fisher's exact test. Maternal body weight data and feed consumption values were statistically analyzed using Dunnett's test when the one-way ANOVA was significant. The number of implantations, live fetuses, resorptions, corpora lutea, mean fetal body weight/litter, and incidence of fetal alterations were statistically analyzed using the Mann-Whitney U test. When more than 75% ties occurred, then Fisher's exact test was used in place of the Mann-Whitney U test to detect significant differences between groups.

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were observed bevore, during, or after the exposures at any exposure levels tested.
The only clinical sign noted was a minimal (only noted once or twice in 6 of 7 affected females) increase in the incidence of scant feces at 2028 ppm.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related or spontaneous mortalities in any group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related decreases on maternal body weight and feed consumption were noted at all exposure levels. The decreases in maternal body weight at 99 and 304 ppm were minimal and transient since they occurred only during the first 2 days of exposure and returned to control values by the next weighing period. In addition, maternal body weight gain over the entire treatment period (days 6-16 G) and corrected maternal body weight gain at 99 and 304 ppm were similar to the control values. The body weight and feed consumption values returned to control values for all groups during the post exposure period (GD 16-20). At 1178 and 2028 ppm, treatment-related effects included losses in maternal body and/or decreased body weight gain throughout the exposure period (GD 6 - 16) and decreased corrected maternal body weight gain (body weight gain minus gravid uterine weight).

Body weight during gestation in rats inhaling Methyl methacrylate on days 6 to 15 of gestation and euthanized on day 20:
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Concentrations No of dams Mean Body weight [g] ± SD
[ppm], 6 h/day on GD 0 on GD 6 on GD 8 on GD 10 on GD 13 on GD 16 on GD 20
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
0 25 244.7 ±3.13 285.6 ± 3.93 295.4 ±4.33 307.1 ±4.20 327.3 ±4.68 350.6 ±5.05 414.2 ±6.84
99 23 246.3 ±2.93 286.1 ± 3.56 289.9 ±4.01 304.8 ±3.99 325.4 ±4.31 351.8 ±4.72 416.6 ±6.87
304 22 248.7 ±2.62 286.2 ± 3.11 288.9 ±3.60 301.3 ±3.91 321.1 ±4.09 347.1 ±4.58 413.9 ±6.09
1178 23 244.9 ± 2.77 282.2 ± 3.53 278.1a ±3.96 289.0a ±3.94 308.3a ±4.04 334.6 ±4.88 400.4 ±6.80
2028 23 246.7 ± 2.85 286.4 ± 3.03 275.2a ±2.68 280.9a ±3.40 306.2a ±3.49 332.7a ±4.27 401.3 ±5.56
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
a Denote significant differences from the control (0 ppm), p < 0.05, respectively.


Change in weight during gestation in rats inhaling Methyl methacrylate on days 6 to 15 of gestation and euthanized on day 20:
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Concentrations No of dams Mean body weight gain ± SD on GD [g]
[ppm], 6 h/day on GD 0 - 6 on GD 6 - 8 on GD 8 - 10 on GD 10-13 on GD 13-16 on GD 6-16 on GD 16-20
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
0 25 41.0 ±1.86 9.8 ±0.83 11.6 ±0.58 20.3 ±0.94 23.2 ±1.76 65.0 ±2.33 63.6 ±2.75
99 23 39.7 ±2.36 3.8a ±1.39 15.0 ±1.90 20.5 ±1.28 26.4 ±1.52 65.7 ±2.83 64.8 ±3.81
304 22 37.5 ±2.05 2.7a ±1.09 12.4 ±1.09 19.8 ±1.04 26.0 ±1.30 60.9 ±2.62 66.8 ±2.54
1178 23 37.3 ±1.91 -4.1a ±-1.41 10.9 ±0.91 19.3 ±1.09 26.3 ±1.76 52.4a ±2.56 65.8 ±3.15
2028 23 39.7 ±1.08 -11.2a ±0.88 5.7a ±2.24 25.4a ±1.62 26.5 ±1.99 46.4a ±2.35 68.6 ±2.12
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
a Denote significant differences from the control (0 ppm), p < 0.05, respectively.


Summary of Uterine and Net body weights in rats inhaling Methyl methacrylate on days 6 to 15 of gestation and euthanized on day 20:
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Concentrations No of dams Gravid Uterus weight ± SD [g] Corrected Mean body weight ± SD on GD 20 [g] Net weight change ± SD from GD 6 [g]
[ppm], 6 h/day
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
0 25 82.7 ±3.81 331.5 ±4.77 45.8 ±2.21
99 23 82.7±5.76 333.9 ±5.08 47.9 ±2.76
304 22 86.4 ±3.75 327.5 ±4.71 41.3 ±2.73
1178 23 79.9 ±5.04 320.5 ±5.04 38.3 ±3.04
2028 23 84.5 ±3.62 316.8 ±3.60 30.4a ±2.25
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
a Denote significant differences from the control (0 ppm), p < 0.05, respectively.

Corrected body weight: terminal body weight at GD 20 minus Uterine weight
Net weight change from GD 6: Corrected body weight minus day 6 body weight

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Treatment related decrease were noted on feed consumption between days 6-10 G at all exposure levels teasted. Decreases in feed consumption continued between days 6-10 G at 304, 1178, and 2028 ppm. Feed consumption of all treated groups returned to control value during post-treatment period (days 16-20 G).

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 specified

Immunological findings:

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 treatment-related changes detected during the gross postmortem examination.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

There were no abortions observed in any of the treated dose groups.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The pre- and postimplantation loss was unaffected by treatment.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

In the study, there was one dam high-dose dam with total litter loss. This observation is considered as not treatment-related.

Early or late resorptions:

no effects observed

Description (incidence and severity):

In the study, there were no early or late resorptions observed.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no dead fetuses found in the uterine.

Changes in pregnancy duration:

not specified

Description (incidence and severity):

There was a Caesarian section performed on GD 20.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There

Details on maternal toxic effects:

Details on maternal toxic effects:
No animals died and no treatment-related clinical signs were noted for the dams in the 99, 304 or 1178 ppm groups. Scant feces was noted in the 2028 ppm group throughout the exposure period (GD 6-15). Treatment-related decreases on maternal body weight and feed consumption were noted at all exposure levels. The decreases in maternal body weight at 99 and 304 ppm were minimal and transient since they occurred only during the first 2 days of exposure and returned to control values by the  next weighing period. The body weight and feed consumption values returned to control values for all groups during the post exposure period  (GD 16-20). At 1178 and 2028 ppm, treatment-related effects included losses in maternal body and/or decreased body weight gain throughout the exposure period (GD 6 - 16) and decreased corrected maternal body weight gain. The gross necropsy evaluations did not indicate any treatment-related effects and there were no treatment-related differences between the control and treated groups in any reproductive parameter.  

Dose descriptor:

NOAEC

Remarks:

maternal toxicity

Effect level:

8.44 mg/L air (analytical)

Based on:

test mat.

Remarks on result:

other: no adverse effects observed

Dose descriptor:

LOEC

Remarks:

maternal toxicity

Effect level:

ca. 0.41 mg/L air (analytical)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on fetal body weight in males, females and both sexes combined at any dose level tested.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Reproductive parameters in rats inhaling Methyl methacrylate on days 6 to 15 of gestation and euthanized on day 20:
--------------------------------------------------------------------------------------------------------------------------------------------
Concentrations No of litters No. of live fetuses per litter
[ppm], 6 h/day
-------------------------------------------------------------------------------------------------------------------------------------------
0 25 13.88
99 23 13.87
304 22 14.86
1178 23 13.70
2028 23 14.65
-------------------------------------------------------------------------------------------------------------------------------------------

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in the sex ratio observed at any dose group.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The mean number of pups per litter and litter weight were unaffected by treatment.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

The pups were euthanised shortly after the Caesarian section.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related increase were detected in the type of external, visceral, or skeletal malformations.
Spontaneous malformations detected in the study included duplicated hypothalamus in a control fetus, hydrocephaly in a fetus at 304 ppm and an omphalocele in one fetus and enlarged adrenal glands in two fetuses at 1178 ppm was not considered to be treatment related as at 2028 ppm no increase was observed.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The significant increase in skeletal developmental variations at 1178 ppm was not detected at 2028 ppm, the highest dose tested. The slightly, not statistically significant number of fetuses with partial or unossified sternebra at 304 ppm was not considered a result of treatment since no dose response was demonstrated.

Visceral malformations:

no effects observed

Description (incidence and severity):

There were no visceral malformations observed in any of the dose groups.

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
 Fetal body weight was not affected by exposure to MMA vapors. The fetal external, visceral and skeletal examinations did not show any treatment related effects.

Dose descriptor:

NOAEC

Remarks:

fetotoxicity

Effect level:

8.44 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: corresponding to 2028 ppm; no substance related effects observed

Key result

Dose descriptor:

NOAEC

Remarks:

teratogenicity

Effect level:

8.44 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: corresponding to 2028 ppm; no substance related effects observed

Abnormalities:

no effects observed

Developmental effects observed:

no

Mean measured concentrations (± SD) within the chambers for the 0, 100, 300, 1200 and 2000 ppm groups were 98.8 (±3.4), 304.4 (±9.1), 1178.1  (±69.1) and 2028.2 (±107.3) ppm, respectively.

Conclusions:

Exposure by inhalation to methyl methacrylate concentrations up to 8.44 mg/L (2028 ppm) resulted in no embryo or fetal toxicity or malformations even at exposure levels that resulted in maternal toxicity.

Executive summary:

In a developmental toxicity study (1991) acc. OECD 414 by inhalation on CRl: CD Br rats pregnant rats were exposed to methyl methacrylate at concentrations of 0 (control), 99, 304, 1178 and 2028 ppm on days 6-15 of gestation. A maternal no observed level was not demonstrated since losses in maternal body weight or decreases in maternal body weight gain and decreases in maternal feed consumption were noted at all exposure levels tested. No embryo or fetal toxicity was evodent and no increase in the incidence of malformations or variations was noted at exposure levels up to and including 2028 ppm. Therefore toxicity to the conceptus was not evident even at expsoure levels that resulted in overt maternal toxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

Developmental toxicity of IBOMA has been investigated in OECD 421 combined reproductive/ developmental toxicity study on a screening level.

IBOMA is rapidly hydrolized by non-specific carboxylesterases to MMA and IBO (see chapter “Toxicokinetics”) so that the systemic toxicity of the parent ester can be assessed with information from the metabolites and their donor substances (i.e. MMA for MAA and IBOAc for IBO).

Here, thedevelopmentaltoxicity of the primary metabolite MAA is assessed byreliable studies in rodents (rat, inhalation, with MMA itself) and non-rodents (rabbit, oral, with the metabolite donor substance MMA). Thedevelopmentaltoxicity of the primary metabolite IBO is assessed by reliable oral studies in rodents and non-rodents with IBOAc, the metabolite donor substance and CAM, the metabolite of IBO.

EFSA evaluated Camphor for the use in flavourings and other food ingredients with flavouring properties and concluded that “there was no evidence of reproductive and developmental toxicity after oral administration to rats and rabbits” (EFSA, 2008).

 

IBOMA

In a reproduction/developmental toxicity screening study according to OECD 421 and GLP requirements, Sprague-Dawley rats (10 male and 10 females per dose group) received 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 (based on liver and kidney findings) and the reproductive/ developmental NOAEL was 500 mg/kg bw/day as there were no findings on reproductive/ developmental toxicity.

 

MAA

The OECD SIAR concluded that: “Methacrylic acid (MAA), the common metabolite for all the esters, also was tested in groups of 19-25 pregnant female rats (whole-body inhalation exposure for 6 hr/day, during days 6 to 20 of gestation), at 0, 50, 100, 200, and 300 ppm (0, 179, 358, 716 and 1076 mg/m³) and produced no embryo- or fetal lethality, nor fetal malformations after exposure with MAA at any concentration, despite overt maternal toxicity (decreased body weight and feed consumption) at 300 ppm (1076 mg/m³). The NOAEL for developmental toxicity was considered 300 ppm (1076 mg/m³) MAA (Saillenfait et al., 1999).”

 

MMA (metabolite donor substance for MAA)

Methyl methacrylate has been tested in a series of developmental toxicity studies on rats and rabbits. In a developmental toxicity study according to OECD 414, MMA was administered by inhalation exposure to 99, 304, 1178, and 2028 ppm (412, 1285, 4900, 8436 mg/m³ (Solomon et al., 1991; currently not selected for the IUCLID dataset). No relevant maternal, treatment-related effects were noted at any concentration tested. No embryo of foetal toxicity was evident and no increase in the incidence in the malformations or variations was noted at exposure levels up to and including 2028 ppm. In this study the NOAEC for developmental toxicity was 2028 ppm (8436 mg/m³).

Since the ESR and the OECD review another study with MMA has been performed, an oral OECD 414 study in rabbits at 50, 150, and 405 mg/kg/d. The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 450 mg/kg bw/d. No adverse foetal findings of toxicological relevance were evident at any dose, even in the presence of maternal toxicity(i.e. NOEL of 50 mg/kg bw/ d based on reduced food consumption;BASF, 2009b).

IBOAc (metabolite donor substance of IBO)

IBOAc was studied in an oral limit test with rats according to OECD 414 and GLP requirements for its developmental toxicity properties. Neither maternal nor embryonic/fetal toxicity was found at oral doses of 1000 mg/kg bw/day IBA in rats. An indication of teratogenic effect could not be noticed. The no observed adverse effect levels (NOAEL) for maternal and prenatal developmental toxicity, respectively is 1000 mg/kg bw/d in this study.

 

CAM (metabolite of IBO)

In two NTP studies, D-Camphor as one stereoisomer of CAM was administered daily by gavage to pregnant rats (NTP 1992a) and pregnant rabbits (NTP 1992b), respectively.

 

Rats were dosed with 100, 400 and 800 mg/kg bw/day, from gestational day (GD) 6 through 15. According to NTP, “there was no maternal mortality during the study. Absolute and relative liver weights exhibited a significant dose-related increase, but did not exceed 10% of control values in any individual group. Maternal toxicity consisted of alterations in food and water consumption (absolute and relative) and decreased weight gain during the treatment period. Food consumption was initially suppressed by both 400 and 800 mg/kg/day CAM. but recovered to control levels by the end of the treatment period. No effect on food consumption was seen at 100 mg/kg/day CAM. Unlike the dose-dependent effects of CAM on food intake, all three doses of CAM increased maternal water consumption during one or more of the following measurement periods (gd 6 to 9; gd 12 to 15; gd 15 to 18). Although maternal body weights in all treatment groups were within 5% of control values at all gestational ages, maternal weight gain during the treatment period in the 800 mg/kg/day group was significantly reduced. Examination of the uterine contents revealed that CAM, even at 800 mg/kg/day, had no adverse effect on fetal growth, viability, or morphological development.”Based on the increased water consumption in the lowest dose without further findings, a LOEL for maternal toxicity was 100 mg/kg bw/day in this study. The NOAELs for fetotoxicity and developmental toxicity were 800 mg/kg bw/day.

 

In the study with rabbits, CAM was administered at doses of 50, 200 and 400 mg/kg/day, from GD 6 through 19. NTP states that “there was no maternal mortality in any of the experimental groups. Maternal body weights in the CAM-treated groups were comparable to controls at all gestational ages. However, maternal weight gain tended to decrease with increasing dose of CAM. Maternal food consumption in all three CAM groups was similar to controls throughout gestation. Examination of the uterine contents revealed that CAM had no effect on fetal growth, viability, or morphological development.” The NOAEL for maternal, fetal and developmental toxicity was determined to be 400 mg/kg bw/day.

Accordingly, EFSA evaluated Camphor for the use in flavourings and other food ingredients with flavouring properties and concluded that “there was no evidence of reproductive and developmental toxicity after oral administration to rats and rabbits” (EFSA, 2008).

 

Read across assessment according to ECHA’s Read Across Assessment Framework (RAAF)

Developmental toxicity of IBOMA has been investigated in an OECD 421 combined reproductive/ developmental toxicity study.Existing data gaps are covered using the read across approach mentioned in chapter1considering the ECHA guidance on Read Across (RAAF, ECHA 2017a). For this endpoint, the common metabolic pathway of the read across substances (rapid hydrolysis of IBOMA by ester cleavage, leading to the primary metabolites MAA and IBO) is considered as the most relevant aspect of the read across approach. The relatively short half-life shown for IBOMA as parent ester leads to the conclusion that systemic effects after repeated exposure are mainly driven by the products of ester hydrolysis, namely MAA and IBO. Local effects after inhalation are determined by local release of MAA. MMAand IBOAc are known to hydrolyse also rapidly so that these substances serve as donor substances for the primary metabolites MAA and IBO.

Qualitatively, this aspect can be categorized as “(Bio) transformation to common compound(s)” (RAAF scenario #1). The read across approach is done with a high level of confidence.

Justification for classification or non-classification

No classification according to directive 1272/2008/EC criteria.