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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-10-19 to 2004-12-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted on 22 March 1996
Deviations:
no
Qualifier:
according to
Guideline:
other: US EPA guideline OPPTS 870.3650, July 2000
Deviations:
no
Principles of method if other than guideline:
The study was conducted as a combined repeated dose toxicity study with Reproduction / Developmental Toxicity Screening Test. In this part all parameters concerning systemic toxicity were considered.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L’Arbresle, France
- Age at study initiation: males were 8 weeks old and the females were 10 weeks old
- Weight at study initiation: 303 g (range: 280 g to 332 g) for the males and 229 g (range: 199 g to 273 g) for the females
- Fasting period before study: no
- Housing: The animals were housed individually (except during mating) in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm). A metal tray, containing autoclaved sawdust 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 autoclaved sawdust and wood shavings as nesting material (SICSA, Alfortville, France).
- Diet: The animals had free access to SSNIFF R/M-H pelleted maintenance diet, Batch Nos. 2764132 and 4764439 (SNIFF Spezialdiäten GmbH, Soest, Germany) distributed weekly.
- Water: The animals had free access to bottles containing tap water (filtered with a 0.22 μm filter).
- Acclimation period: 7 days

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

IN-LIFE DATES: From: 2004-10-12 To: 2004-12-07

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Vehicle
The vehicle was corn oil, Batch No. 103K0107, supplied by Sigma (Saint-Quentin-Fallavier, France).

Dosage formulation preparation
The test item dosage formulations were prepared by suspending Allyl Methacrylate (AMA) in corn oil in order to achieve the concentrations of 0.6, 3 and 12 mg/mL and were stored at +4°C or up to 9 days protected from light.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analysis of the dosage formulations
Homogeneity
Two dosage formulations were prepared at 0.2 and 15 mg/mL of AMA (in corn oil) in order to cover all the concentrations intended for use in this study. From both dosage formulations, duplicate samples were taken at three different levels of the container (top, middle, bottom) and analyzed for concentration of the test item just after preparation (day 0) and after 9 days of storage at +4°C (protected from light).
Stability
The two dosage formulations prepared at 0.2 and 15 mg/mL of AMA in corn oil for homogeneity analysis were sampled after 0 (just after preparation), 4 and 9 days storage at +4°C (protected from light). The aliquots (n = 2) sampled on day 4 were stored frozen at -20°C pending analysis on the last sampling occasion, when all samples were assayed. The mean concentrations measured on days 0 and 9 for homogeneity were taken as the initial and final values for the stability test.
Concentration
The concentration of samples taken from each dosage formulation (including the control) prepared for use in weeks 1, 2, 4 and 7 was determined.

Results
A satisfactory agreement was observed between the nominal and actual concentrations of the test item in the dosage formulations analyzed since the deviations from nominal concentration remained in an acceptable range of ± 10%. Furthermore, there was a satisfactory correspondence between the nominal and the measured concentrations of the test item in the vehicle.
Duration of treatment / exposure:
Males: 15 days before mating, through mating and post-mating periods through sacrifice (minimum 4 weeks);
Females: 15 days before mating, through mating period, pregnancy and lactation, until day 5 post-partum inclusive (or until sacrifice, for unmated females);
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
3 mg/kg bw/day (actual dose received)
Remarks:
analytical concentration
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Remarks:
analytical concentration
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Remarks:
analytical concentration
No. of animals per sex per dose:
10
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. 28198 TSR in which rats were dosed (gavage) withAllyl Methacrylate (AMA), Batch. 00216X, at 0, 25, 75 and 150 mg/kg/day for 10 consecutive days. In this study, 150 mg/kg/day resulted in morbidity and mortality, both sexes in the 25 and 75 mg/kg/day groups showed signs of hypersalivation during the dosing period, males showed signs of piloerection at 75 mg/kg/day, and the females in the 75 mg/kg/day group had increased liver weight.
Taking into account that the duration of the dosing period for this study was nearly 6 weeks in females, the high dose-level was set at 60 mg/kg/day. The intermediate and low dose-levels were selected in order to identify a No Effect Level.
Positive control:
not required

Examinations

Observations and examinations performed and frequency:
- CLINICAL OBSERVATIONS: Clinical observations were conducted daily throughout the study period.

- DETAILE CLINICAL OBSERVATIONS (DCO): Conducted on all rats pre-exposure and weekly throughout the study. The DCOs included cage-side, handheld and open-field observations that were recorded categorically or using explicitly defined scales.

- FUNCTIONAL TESTS: The functional tests included reactivity to manipulation or different stimuli and motor activity.

BODY WEIGHT: Yes
- Time schedule for examinations: Male body weights were recorded weekly throughout the study. Female body weights were performed weekly for the premating and mating periods. Maternal body weights were recorded on gd 0, 7, 14, 17 and 20. Females that delivered were weighed on ld 1 and 5. Females that failed to mate or deliver were weighed on a weekly basis for the study duration.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was determined weekly for males and females during the pre-mating period. Due to co-housing, food consumption was not measured during mating. During gestation, food consumption for the females was measured on gd 0, 7, 10, 14, 17 and 20. After parturition, food consumption was measured on ld 1 and 5.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At necropsy, blood samples were removed from the orbital sinus.
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: all
- Parameters: erythrocytes, hemoglobin, packed cell volume, thrombocytes, leucocytes (WBC), differential WBC count with morphology, mean cell hemoglobin (MCH), mean cell volume (MCV), and mean cell hemoglobin concentration (MCHC); Coagulation Assay: prothrombin time, activated partial thromboplastin time and fibrinogen;

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy, blood samples were removed from the orbital sinus.
- Animals fasted: Yes
- How many animals: all
- Parameters: Enzyme activities of alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, triglyceride, albumin, cholesterol, creatinine, sodium, potassium, inorganic phosphorous, chloride, calcium, glucose, total bilirubin, albumin/globulin ratio, total protein, bile acids and urea

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected from the first 5 surviving male rats in each dose group during the week prior to necropsy.
- Metabolism cages used for collection of urine: Yes
- Parameters: Color, appearance, specific gravity, urine volume, pH, bilirubin, glucose, proteins, ketones, blood, nitrates and urobilinogen;

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
A complete necropsy of all animals was performed. Male rats were sacrificed at the end of the mating period (total treatment was at least 40 days). The females and their pups were sacrificed on ld 6. The necropsy included an examination of: the external tissues and all orifices, the cranial, nasal, thoracic and abdominal cavities, and all viscera. Selected tissues of the viscera were incised. On the day prior to necropsy, all animals were fasted overnight. At necropsy, animals were anesthetized and blood samples were removed from the orbital sinus.
In five males and five females from each group, the following tissues were harvested and preserved in appropriate materials: macroscopic lesions, adrenals, brain, cecum, colon, duodenum, epididymis, heart, ileum, jejunum, kidneys, liver, lungs with bronchi, lymph nodes, ovaries, prostate, rectum, sciatic nerve, seminal vesicles, spinal cord, spleen, sternum with bone marrow, stomach with forestomach, testes, thymus, thyroids, trachea, urinary bladder, and uterus. Weights were also recorded for the adrenals, brain, epididymis, heart, kidneys, liver, spleen, testes and thymus.
Statistics:
The following sequence was used for the statistical analyses of body weight, food consumption, hematology, blood biochemistry, urinalysis and organ weight data. If there were 3 or less animals/sex/group remaining, no statistical analysis was performed. If there were 5 or less animals/sex/group remaining for 3 or more groups, the data were analyzed using the Dunn test. If there were 5 or less animals/sex/group remaining for less than 3 groups, the data were analyzed using the Mann-Whitney/Wilcoxon test. If there were 5 or more animals/sex/group, the data were analyzed by Kolmogorov-Lillefors test to test for the normality of the distribution of the data. If it was not normal, the values were logarithmically transformed and retested for normality. If it still did not meet the criteria for normality, the original values were analyzed by the Dunn test. If the data were normal, and there were 3 or more groups, the data were assessed for homogeneity of variance between groups with the Bartlett test. If not homogeneous, the data were analyzed by the Dunn test; if homogeneous, the data were assessed by the Dunnett test. If the data were normal, and there were less than 3 groups, the data were assessed for homogeneity of variance between groups with the Fisher test. If not homogeneous, the data were assessed using the Mann-Whitney/Wilcoxon test; if homogeneous, the data were analyzed by the Student test. The statistical comparison of results of motor activity (horizontal and rearing movements recorded from five males and five females per group) was performed, according to the following steps: step 1: normality and homogeneity of variances (Kolmogorov-Smirnov and Bartlett test respectively), step 2: parametric (ANOVA plus Dunnett test if necessary) or non-parametric (Kruskal-Wallis plus Dunn test if necessary) tests was conducted based on the results obtained at step 1. These statistics were performed using the software SAS Enterprise Guide V2 (2.0.0.417, SAS Institute Inc).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Hypersalivation was observed in a dose-related manner in males and females given 15 or 60 mg/kg/day during the premating period (from week 2) and then in females during gestation and lactation. One female given 60 mg/kg/day experienced hypotonia and half closed eyes (on day 5 p.p.);
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect of treatment on group mean male body weight gain during the premating phase. The marginal reduction in body weight gain observed at 15 and 60 mg/kg/day (p<0.05)
during the mating period was not considered to be biologically significant based on lack of clear dose response and lack of effects at other time periods.
All female groups given Allyl Methacrylate gained less weight than the controls during the premating phase. During gestation, no clear effect was observed on the body weight gain, although females given the lowest dose-level continued to have low body weight gain. Females
given 60 mg/kg/day had lower body weight gain than controls during the lactation period. But none of these variations reached statistical significance when compared to control mean values.
Based on the lack of dose response and consistency across periods, none of these differences was considered to be related to treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was no effect of treatment on group mean male food consumption during the study.
All female groups given Allyl Methacrylate had marginally lower food consumption during the premating period. These differences reached statistically significance at 3 and 15 mg/kg/day only
although all values were similar in the three treated groups. Food consumption was comparable with the controls during gestation and marginally reduced during lactation. Based on the lack of
dose response, the small magnitude of the changes, and lack of consistency across periods, none of these differences was considered to be related to treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect of treatment on any male or female hematological parameters. There was a lower value of white cell counts recorded in males given 15 or 60 mg/kg/day (13% and 19% lower, respectively) that did not attain statistical significance. When compared to the mean control values, increased values of fibrinogen plasmatic levels were noted in all male-treated groups (3.31, p<0.05, 3.15 and 3.28 g/L at 3, 15 and 60 mg/kg/day respectively, vs. 2.62 g/l in controls). However, there was no dose-response.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There was no effect of treatment on any male or female hematological parameters.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There was no effect of treatment on pH, specific gravity or volume or urine produced by the males in any group.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Reactivity to manipulation or to different stimuli: There was no evidence of disturbance of either autonomal or physiological functions at any dose level.
Motor Activity: When compared to the control values, a significant increase in the mean total number of rearing movements was recorded in the female group given 60 mg/kg/day. No other differences in the measured motor activity were noted in any group.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
details see below
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Yellowish areas were noted in the liver of 2/5 females given 60 mg/kg/day. This was considered to be treatment-related and correlated with microscopic findings. All other necropsy findings reported at the end of treatment period were those which commonly occur in the rat of this strain and age when kept under laboratory conditions, and were considered to be of no toxicological importance.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At histopathology, several foci of degenerated/necrotic hepatocytes, together with slight periportal fibrosis, slight biliary proliferation, and greenish-pigment-laden macrophages, were noted in 3/5 females given 60 mg/kg/day. There were no other treatment-related histopathological changes for either sex at any dose.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Blood chemistry:
Decreased triglyceride concentration was noted in all male test item-treated groups, but without clear dose-relationship or statistical significance (0.37, 0.39 and 0.31 mmol/L at 3, 15 and 60 mg/kg/day respectively, vs. 0.57 mmol/L in controls). Therefore, these differences were not
considered to be treatment-related.
In females, the differences recorded in blood biochemistry parameters were considered not to be toxicologically relevant since they were slight and/or without clear dose-relationship and great inter-animal variability was noted or no correlation could be clearly made with other parameters (e.g. increased in Alanine aminotransferase activity in the high dose group females). However, the total bilirubin concentration in female G21411 (10 µmol/L, vs. 3 to 4 µmol/L
in controls) and the high concentration of biliary acids recorded in female G21415 (402 µmol/L, vs. 56.6 µmol/L in controls) were considered to be related to the treatment with the test item at 60 mg/kg/day, as these changes correlated with histopathological findings in the liver.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: liver effects
Key result
Dose descriptor:
LOAEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: hyperrsalivation
Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: parental toxicity

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Salient Male Clinical Observations
# Animals Affected Dose Level (mg/kg/day)
0 3 15 60
Hypersalivation (Premating)  0 1 9 10
Salient Female Clinical Observations
# Animals Affected Dose Level (mg/kg/day)
0 3 15 60
Hypersalivation (Premating)  0 0 8 10
Hypersalivation (Gestation) 0 0 8 10
Hypersalivation (Lactation) 0 0 8 10
Food Consumption in the Pre-mating period
Sex Males Females
Dose (mg/kg/day) 0 3 15 60 0 3 15 60
Pre-mating period (g/rat/d)
Days 1-8 26 26 26 26 20 18* 18* 18
Days 8-15 26 26 26 27 20 18* 17** 18
* = p < 0.05; ** = p< 0.01
Motor Activity
Sex Males Females
Dose (mg/kg/day) 0 3 15 60 0 3 15 60
Horizontal
Movements
(mean +/- SD)
808
105
628
121
683
93
577
221
303
116
377
95
359
88
415
73
Rearing
(mean +/- SD)
314
68
252
70
294
99
214
91
82
35
107
29
155
52
157*
36
SD: Standard Deviation
*: p<0.05 (Dunnett test, comparison with the mean control value)
-Organ Weights:
Sex Males Females
Dose (mg/kg/day) 3 15 60 3 15 60
Adrenals  
       absolute +4 +6 -10 +1 -2 -12
       relative +1 +2 -15 +6 -1 -8
Thymus  
       absolute +19 +24* +28* -1 -1 -17
       relative +15 +20 +20 +2 -1 -14
*: p<0.05 (Dunnett test, comparison with the mean control value)

Applicant's summary and conclusion

Conclusions:
In this study, the NOAEL was found to be 15 mg/kg/day parental to liver damage in the female rats.
Executive summary:

In an OECD 422 study, three groups of 10 male and 10 female Sprague-Dawley rats received the test item, allyl methacylate (AMA), 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 3, 15 and 60 mg/kg bw/day. Another group of 10 males and 10 females received the vehicle, corn oil, alone, under the same experimental conditions and served as a control group. The dosing volume was 5 mL/kg.

Clinical signs and mortality were checked daily. Body weight and food consumption were recorded at designated intervals throughout the study. Detailed clinical observations, reactivity to different stimuli (Functional Observational Battery, (FOB)) and motor activity were also recorded. Blood was taken from five males and five females for hematological and blood biochemical investigations at terminal sacrifice (i.e. in week 5 for males and week 7 for females). At the same time, urine was collected from five males for analysis. Males were sacrificed approximately one week after the end of the mating period, females on day 6 p.p. The animals were sacrificed and subjected to a macroscopic examination of the principal thoracic and abdominal organs. Designated organs were weighed and examined microscopically. In addition, the numbers of corpora lutea and implantation sites were recorded for each female. Due to potentially treatment-related microscopic findings observed in the livers of some animals in the 60 mg/kg bw/day treatment group, the retained livers of the males and females in the low- and intermediate-dose groups also were examined microscopically. The pups were observed daily for clinical signs, sexed and weighed on days 1 and 5 p.p.. After sacrifice on day 6 p.p., the pups were examined for gross abnormalities and discarded.

Hypersalivation was observed in a dose-related manner in males and females given 15 or 60 mg/kg bw/day during the premating period (from week 2) and then in females during gestation and lactation. This clinical sign was not considered to be adverse as it likely represents a reaction to the dosing procedure and not a direct effect of AMA toxicity. At 60 mg/kg bw/day, one female experienced hypotonia and half-closed eyes on day 5 p.p.. There was no disturbance of autonomal or physiological functions at any dose-levels and the motor activity of the animals was considered to be unaffected by treatment. There was no effect of treatment on group mean male body weight gain during the premating phase and only a marginal reduction in body weight gain was observed at 15 and 60 mg/kg bw/day (p<0.05) during the mating period. There were no changes in hematological parameters that could be considered toxicologically relevant.

For blood biochemistry investigation, one female dosed with 60 mg/kg bw/day had increased total bilirubin concentration and another one had increased biliary acid concentration, these changes were correlated with histopathological findings in the liver. There were no effects of treatment on pH, specific gravity or volume of urine produced by the males in any group. The necropsy of the adult males and females revealed the presence of yellowish areas in the liver of 2/5 females in the 60 mg/kg bw/day dose group. At histopathology, several foci of degenerated/necrotic hepatocytes, together with slight periportal fibrosis, slight biliary proliferation, and greenish-pigment-laden macrophages, were noted in 3/5 females given 60 mg/kg bw/day. There were no other treatment-related histopathological changes for either sex at any dose.

Conclusion

The test item, Allyl Methacrylate (AMA; CAS RN 96-05-9), was administered to male and female Sprague-Dawley rats by oral route (gavage), under the above detailed experimental

conditions, at the dose-levels of 3, 15 or 60 mg/kg/day.

The following observations were made in the dose groups:

60 mg/kg/day group:

Hypersalivation (although not considered to be related to AMA toxicity) was recorded in all the males and females and hypotonia and half-closed eyes were observed in one female. A marginally lower body weight gain was recorded in males during the mating period. Females gained less weight during the premating phase, which coincided with reduced food consumption during this period and lactation. Male and female pups gained less weight than controls, and the number of pups cold to the touch was higher than in the control litters. At clinical pathology, a higher plasma concentration of total bilirubin (1/5) and biliary acids (1/5) in females given 60 mg/kg/day were recorded and correlated with histopathological findings in the liver. At necropsy, 2/5 females had yellowish areas in the liver. Histopathology revealed the presence of foci of degenerated/necrotic hepatocytes (associated with periportal fibrosis, biliary proliferation and greenish-pigment-laden macrophages) in 3/5 females.

15 mg/kg/day group:

The majority of males and females had hypersalivation throughout the study. A slight reduction in body weight gain was recorded for males during the mating period. Females gained less weight than the controls during the premating phase associated with a slight reduction in food consumption. During lactation, food consumption was marginally reduced.

Male and female pups gained less weight than controls. None of these changes was considered to be related to AMA toxicity.

3 mg/kg/day:

Body weight gain and food consumption of females were lower than controls during the

premating phase. Food consumption was marginally reduced during the lactation period. None of these changes was considered to be related to AMA toxicity.

There were no substance-induced effects on the male and female reproductive performance, or on the progeny of the parental rats at any dose-level.

Based on the results of this study, the dose-level of 15 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL) for parental toxicity.