Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 29-Apr-2014 to 16-Mar-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
adopted 21 September 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
Directive 96/54/EC, 30 September 1996
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Propylidynetrimethyl trimethacrylate
- Substance type: Organic monoconstituent substance
- Physical state: Colourless to light yellow liquid

- Storage condition of test material: At room temperature (20 ± 5 °C) protected from sunlight

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: Ca. 7 weeks
- Weight at study initiation: Body Weight Range at Acclimatization was for Males 218 - 243 g (mean: 229 g) and for Females 124 - 156 g (mean: 141 g)
- Fasting period before study: The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- Housing: In groups of up to five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (J. Rettenmaier & Söhne GmbH & Co. KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) including paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK).
- Diet: Pelleted standard Harlan Teklad 2914C (batch no. 20/14) rat / mouse maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed batches were analyzed for contaminants.
- Water: Community tap-water from Itingen was available ad libitum in water bottles. Bacteriological assay, chemical and contaminant analyses of respective samples were performed
- Acclimation period: 76 days under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.

IN-LIFE DATES: From: 06-May-2014 (start of 7-day Acclimatization) To: 05 to 07-Aug-2014 (Necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared with the test item as delivered by the Sponsor. The vehicle was pre-warmed to a temperature of approximately 40 °C. Propylidynetrimethyl trimethacrylate was weighed into a glass beaker on a tared precision balance and the warm vehicle was added (w/v). The mixture was mixed thoroughly using a homogenizer until a clear to colloid-like yellowish solution was obtained. Having obtained a homogeneous mixture, the remaining vehicle was added until the required final volume was reached. Separate formulations
were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
The dose formulations were stored for up to eight days in glass beakers at room temperature (20 ± 5 °C).

DIET PREPARATION
- Not relevant due to exposure via gavage

VEHICLE
- Justification for use and choice of vehicle: Considered non-toxic to the test animals and ability to form a homogeneous mixture with the test item
- Concentration in vehicle (nominal): 0, 20, 60 and 100 mg/mL (at 0, 100, 300 and 1000 mg/kg bw/day, respectively)
- Amount of vehicle (by gavage): 5 mL/kg body weight
- Lot/batch no.: 0240206485 and 194213993 (Source: Carl Roth GmbH + Co. KG, Expiry Dates: 30-Apr-2016 and 31-May-2016, respectively)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Representative samples were dispatched to the analytical laboratories internally (at room temperature) and stored frozen at -20 ± 5 °C until analysis.
The test item was used as the analytical standard.
Stock solutions of Propylidynetrimethyl trimethacrylate in acetonitrile were prepared for external calibration. For example, 34.77 mg of Propylidynetrimethyl trimethacrylate was weighed into a 50 mL volumetric flask and filled to about 75 % of final volume with acetonitrile. The mixture was sonicated for at least five minutes and brought to volume with acetonitrile to yield a solution with a concentration of 695.4 µg/mL. Aliquots of this stock calibration solution were diluted with acetonitrile to obtain calibration solutions with nominal concentrations ranging from 10.43 to 99.44 µg/mL. On each occasion calibration solutions derived from two stock solutions were used for calibration.
Each sample was transferred into an appropriate volumetric flask. The sample vial was successively rinsed with at least two portions of tetrahydrofuran and the rinsings were combined in the volumetric flask. The flask was filled to about 75 % of the target volume with tetrahydrofuran and dissolution was achieved by sonication for at least five minutes. The flask was filled to the mark with tetrahydrofuran. Sample solutions were further diluted with acetonitrile into the calibration range.
Gas Chromatographic Conditions:
- Computerized System: EZ Chrom Elite; Agilent Technologies
- Column: BGB 1701 - BGB; 30 m x 0.25 mm; 0.25 µm
- Carrier Gas: Helium
- Temperature Gradient:
Rate [°C/min]; Temp. [°C]; Time [min]
0.; 200; 0
10; 300; 5
- Running Time: 15 min
- Injector Temperature: 230 °C
- Injection Mode: Split ratio 5:1
- Flow: 1 mL/min (constant flow)
- Detector: FID; 250 °C (H2: 31; Air: 380; N2: 28 mL/min)
- Injection Volume: 2 µL
- Retention Time: Ca. 4.4 min
The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak areas measured and calibration solution concentrations. All calibration points used met the acceptance limit of ±20 % variation from the calibration curve derived by linear regression analysis. The coefficients of determination (R²) were higher than 0.99.
The Propylidynetrimethyl trimethacrylate peak was assigned in sample chromatograms by comparison to that of calibration solutions. In blank sample chromatograms no peak appeared at the retention time of Propylidynetrimethyl trimethacrylate and, therefore, the absence of the test item in the vehicle control samples (corn oil) was confirmed.
The Propylidynetrimethyl trimethacrylate concentrations in the dose formulations ranged from 81.5 to 92.5 % with reference to the nominal and were within the accepted range of ±20 %. The homogeneous distribution of Propylidynetrimethyl trimethacrylate in the preparations was approved because single results found did not deviate more than 1.3 % from the corresponding mean and met the specified acceptance criterion of =15 %. Accordingly the effects were assigned to the nominal concentrations.
Duration of treatment / exposure:
92/93 days
Frequency of treatment:
Daily, at approximately 24 hour intervals
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day (considered groups 1, 2, 3 and 4, respectively), corresponding nominal concentrations is vehicle were 0, 20, 60 and 200 mg/mL corn oil, respectively
Basis:
actual ingested
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected by the Sponsor.
- Rationale for animal assignment: Randomly allocated to groups by body weight.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations for viability / mortality were recorded twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Daily Observations: The animals were observed for clinical signs once before commencement of administration as well as daily on days 1 - 92/93 (twice daily during days 1 - 3) during the treatment period.
Weekly Detailed Observations: The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed once before commencement of administration and once weekly (weeks 1 to 12) thereafter (see table 1, below).

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly
Body weights were recorded weekly during acclimatization, treatment and recovery periods and before necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the Harlan Laboratories computer.
- Body weight observations checked in tables 6 and 7 were included.

FOOD CONSUMPTION: Yes
The food consumption was recorded once during the acclimatization period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the Harlan Laboratories computer.

FOOD EFFICIENCY: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During week 13
- Dose groups that were examined: All animals of the control and high dose groups
The eyes of all animals were examined using a direct ophthalmoscope once during the acclimatization period in all animals. During week 13, animals of the control and high dose groups were evaluated once in animals of the control and high dose groups for test item-related changes.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood Sampling after 13 Weeks: 05/06-Aug-2014
- Anaesthetic used for blood collection: Yes, light isoflurane anaesthesia
- Animals fasted: Yes. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- How many animals: All animals
- Parameters examined: Glucose; Urea; Creatinine; Bilirubin, total; Cholesterol, total; Triglycerides; Phospholipids; Aspartate aminotransferase; Alanine aminotransferase; Lactate dehydrogenase; Alkaline phosphatase, Gamma-glutamyl-transferase; Creatine kinase; Sodium; Potassium; Chloride; Calcium; Phosphorus; Protein, total; Albumin; Globulin; Albumin/Globulin ratio; Coagulation Prothrombin time (= Thromboplastin time); Activated partial Thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood Sampling after 13 Weeks: 05/06-Aug-2014
- Animals fasted: Yes. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- How many animals: All animals
- Parameters examined: Glucose; Urea; Creatinine; Bilirubin, total; Cholesterol, total; Triglycerides; Phospholipids; Aspartate aminotransferase; Alanine aminotransferase; Lactate dehydrogenase; Alkaline phosphatase; Gamma-glutamyl-transferase; Creatine kinase; Sodium; Potassium; Chloride; Calcium; Phosphorus; Protein, total; Albumin; Globulin; Albumin/Globulin ratio

URINALYSIS: Yes
- Time schedule for collection of urine: Urine Sampling after 13 Weeks: 05/06-Aug-2014
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- Parameters examined: Urine volume (18 hour); Specific gravity (relative density); Colour; Appearance; pH value; Nitrite; Protein; Glucose; Ketones; Urobilinogen; Bilirubin; Erythrocytes; Leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes, relevant parameters from a modified Irwin screen test were evaluated.
- Time schedule for examinations: During week 13
- Dose groups that were examined: All dose groups, all animals
- Battery of functions tested:
Grip Strength
Forelimb and hindlimb grip strength measurements were performed using a push-pull strain gauge (Mecmesin, AFG 25N). The animals were placed with the forepaws inside a triangular grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times; the means were calculated and recorded.
Locomotor Activity
Locomotor (decreased or increased) activity was measured quantitatively with AMS Föhr Medical Instruments GmbH (FMI) and DeMeTec GmbH Activity Monitor System. Animals were monitored during the fourth treatment week for a 60-minute period and the total activity of this time period was recorded. Low beams count was reported in 10-minute intervals as well as the total activity of the measuring period.
Sacrifice and pathology:
Sacrifice:
After 13 Weeks: 05/06/07-Aug-2014
All animals were weighed and necropsied. Descriptions of all macroscopical abnormalities were recorded. All animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination.

GROSS PATHOLOGY: Yes
Organ weights: The organs were weighed before fixation and recorded on the scheduled dates of necropsy. Relative organ weights were calculated on the basis of the body weight brain weight..

HISTOPATHOLOGY: Yes
Samples of the tissues and organs listed in table 1, below were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution unless indicated otherwise.

Seminology and Spermatid Count
- Time schedule: After 13 Weeks: 06-Aug-2014 (groups 1 and 4) and 07-Aug-2014 (groups 2 and 3)
- How many animals: Sperm analysis was performed in all males.
Motility: At necropsy of males, a sperm sample from the left caudal epididymis was obtained from each male. The sample was diluted with a pre-warmed (about 37 - 40 °C) physiological medium, and shortly after being obtained, one hundred sperm cells were counted in duplicates (two sperm samples) microscopically for determination of percentage of not motile, stationary motile and progressively motile sperm.
Morphology: The sperm cells in the original physiological medium for motility determination were used for morphological assessment after Eosin staining and fixation. The slides were stored at room temperature until analysis. Five hundred (500) sperm cells per sample were evaluated microscopically and classified into the following categories:
Code Description
A Normal, complete sperm
B Normal head, abnormal tail
C Normal head only, tail detached
D Abnormal head only, tail detached
E Abnormal head, normal tail
The percentages of categories A to E were determined. In the absence of treatment-related effects in the control males and high-dose males, the males of the low- and middle-dose were not assessed.
Sperm Count
The left testis and the left cauda epididymis were taken for determination of homogenization resistant sperm cells (HRS) in testes and epididymis. The testes and cauda epididymis were frozen at -20 ± 5 °C pending evaluation. For evaluation the testes and cauda epididymis were thawed and processed separately. Testes and cauda epididymis were weighed and placed in Triton-X-100 solution and homogenized with a blender (Ultra Turrax) and an ultrasonic water bath. Sperm heads were counted microscopically using a modified Neubauer chamber. Dilution factors for each sample were documented in the raw data. These evaluations were performed in the first instance only for group 1 and 4 males. In the absence of a treatment-related effect the remaining frozen tissues were not evaluated.

Oestrus Cycle Determination
Samples to determine the stage of oestrus (oestrus, prooestrus, metoestrus, dioestrus) were recorded once daily in the females of the control and test-item treated groups during the last 21 days of the study assessed were determined.

Histotechnique: All organ and tissue samples were processed, embedded and cut at an approximate thickness of 4 micrometers and stained with haematoxylin and eosin.
Histopathology: Slides of all organs and tissues collected at scheduled sacrifices from all animals of the control and high-dose groups and all gross lesions from all animals were examined by the study pathologist. In addition, test item-related findings noted in the livers and mesenteric lymph nodes of high-dose males and females and in the pituitary glands of high-dose males trigger the evaluation of these organs in the middle- and low-dose groups. Attempts were made to correlate gross observations with microscopic findings. The stage of oestrus was evaluated, as the stage of spermatogenesis and histopathology of the interstitial cell structure. A peer review was performed by W. Henderson.
Immunohistochemistry: Slides of the kidney from all male animals of the control and high-dose groups were immunostained with an anti-alpha-2-microglobulin-antibody. The slides were examined by the principal investigator for histopathology. Slides of the kidney from all male animals of the control and high-dose groups were shipped to: Stewart Jones, Propath UK Limited, Willow Court, Netherwood Road, Hereford / United Kingdom
Other examinations:
no
Statistics:
The following statistical methods were used to analyse body weight, food consumption, grip strength, locomotor activity, clinical laboratory data, ophthalmoscopy, sperm analysis, organ weights and ratios as well as macroscopic findings:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Group 4, males: reduced
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Group 4, both sexes: Elevated
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Group 4, males: Reduced
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Group 4, both sexes: Reductions of mean locomotor activity
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Group 4, both sexes: Ratios and weights
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Group 4, both sexes: Included gastric (forestomach) epithelial hyperplasia/hyperkeratosis; decreased secretory content in the prostate and liver centrilobular hepatocellular hypertrophy in females; lower extramedullary haematopoiesis; Sperm motility
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no test item-related deaths at any dose level. There were no test item-related findings in the daily or weekly observations.

BODY WEIGHT AND WEIGHT GAIN
See tables 6 and 7, below: At 1000 mg/kg bw/day, the males had lower body weights and lower body weight gain when compared to the control males. This was considered to be test-item related. The body weight development was slightly reduced in males treated with 100 mg/kg bw/day or 300 mg/kg bw/day. The differences were generally similar in both groups during the first month of treatment; males treated with 300 mg/kg bw/day gained less weight until the end of the treatment period.
There were no test item-related differences in the mean body weights or mean body weight gain of females at any dose level.

FOOD CONSUMPTION
See table 5, below: Elevated food consumption noted in males and females treated with 1000 mg/kg bw/day were considered to be related to the treatment with the test item.

FOOD EFFICIENCY
At 1000 mg/kg bw/day reduced or unchanged body weights, but increased food consumption.

OPHTHALMOSCOPIC EXAMINATION
There were no test item-related ophthalmoscopic changes.

HAEMATOLOGY / CLINICAL CHEMISTRY
Although there were statistically significant differences in the haematology and clinical biochemistry parameters to the control values, only isolated parameters exceeded the lower limits of the historical control data (such as relative eosinophil count and cholesterol activity in males at 1000 mg/kg bw/day). Such differences are generally recognized as being of no toxicological relevance, and unrelated to the treatment with the test item.

URINALYSIS
There were no test item-related differences in the urinalysis parameters of males and females at all dose levels.

NEUROBEHAVIOUR
See tables 3 and 4, below, Locomotor Activity: The mean fore- and hind limb grip strength values of the test item-treated rats compared favourably with those of the respective control rats. However, test item-related reductions in the mean locomotor activity were seen in both sexes at 1000 mg/kg bw/day.

ORGAN WEIGHTS
Differences noted in the liver and kidney weights of males and females, and in the prostate weight of males treated with 1000 mg/kg bw/day were considered to be test item-related. All remaining differences in the relative organ weights of males were considered to be body weight effects.
At 300 mg/kg bw/day and 100 mg/kg bw/day, several organ-to-body weight ratios in males showed differences that were attributed to body weight effects. The absolute and relative organ weights of the females treated with 300 mg/kg bw/day or 100 mg/kg bw/day were unaffected.

GROSS PATHOLOGY
There were no test item-related macroscopical findings at any dose level.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopical findings noted at 1000 mg/kg bw/day included gastric (forestomach) epithelial hyperplasia/hyperkeratosis (indicative of contact irritancy) in males and females (with males being more affected than females), decreased secretory content in the prostate and liver centrilobular hepatocellular hypertrophy in females. In addition, slightly lower extramedullary haematopoiesis, possibly related to the test item, was recorded at 1000 mg/kg bw/day in males and females. Slight gastric (forestomach) epithelial hyperplasia/hyperkeratosis and erosion/ulcer(s) was also recorded in 1/10 females at 300 mg/kg bw/day.
All other findings were considered to be of spontaneous origin, unrelated to the test-item.
An overall similar (low or medium) staining level were observed in the kidneys of males at 0 and 1000 mg/kg bw/day following the immunostaining with an anti alpha 2u-globulin antibody.

Seminology: The sperm count and sperm morphology of rats a t 1000 mg/kg bw/day were similar to those of the controls. The evaluation of sperm motility showed that the number of motile (progressive) sperm was lower in males treated with 1000 mg/kg bw/day when compared with controls. The reduction of sperm motility in males at 1000 mg/kg bw/day was considered to be a possible test item-related change. Males treated with 100 or 300 mg/kg bw/day were unaffected.

Oestrus Cycle Determination: There were no effects upon the duration or pattern of oestrus cycles.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: adverse effects oberved at 1000 mg/kg/d
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Adverse effects on body weight, food consumption, neurobehaviour, organ weights, non-neoplastic histopathology

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Locomotor Activity

Slight reductions seen in the mean locomotor activity of the males treated with 1000 mg/kg bw/day were considered to be test item-related. The mean locomotor activity of the males and females treated with 100 or 300 mg/ kg bw/day, as well as females treated with 1000 mg/kg bw/day were considered to be unaffected by treatment.

Table 3: Locomotor activity of male test animals

Allocation and
Dose Levels
mg/kg bw/day

Group 1
Control Males
0

Group 2
Males
100

Group 3
Males
300

Group 4
Males
1000

6 minutes

280

334 (+19.3 %)

292 (+4.3 %)

218 (-22.1 %)

12 minutes

193

228 (+18.1 %)

190 (-1.6 %)

166 (-14.0 %)

18 minutes

168

176 (+4.8 %)

152 (-9.5 %)

115 (-31.5 %)

24 minutes

154

151 (-1.9 %)

117 (-24.0 %)

79 (-48.7 %)

30 minutes

163

165 (+1.2 %)

65 (-60.1 %)

76 (-53.4 %)

Total

958

1054
(+10.0 %)

816
(-14.8 %)

653
(-31.8 %)

Bold values denote statistically significant changes.

Table 4: Locomotor activity of female test animals

Allocation and
Dose Levels
mg/kg bw/day

Group 1
Control Females
0

Group 2
Females
100

Group 3
Females
300

Group 4
Females
1000

6 minutes

403

368 (-8.7 %)

386 (-4.2 %)

300 (-25.6 %)

12 minutes

270

267 (-1.1 %)

249 (-7.8 %)

195 (-27.8 %)

18 minutes

165

190 (+15.2 %)

173 (+4.8 %)

123 (-25.5 %)

24 minutes

147

117 (-20.4 %)

118 (-19.7 %)

101 (-31.3 %)

30 minutes

85

135 (+58.8 %)

83 (-2.4 %)

88 (+3.5 %)

Total

1070

1077
(+0.7 %)

1009
(-5.7 %)

807
(-24.6 %)

Bold values denote statistically significant changes.

Food Consumption

Elevated absolute and relative food consumption values were noted in the males and females treated with 1000 mg/kg bw/day. The differences were considered to be test item-related.

Table 5: Effects on Food consumption

Allocation and
Dose Levels
mg/kg bw/day

Group 1
Control
0

Group 2

100

Group 3

300

Group 4

1000

Absolute FC males
Days 1 - 91

19.1 g

19.0 g

20.5 g

22.0 g

Absolute FC females
Days 1
 -91

12.3 g

12.7 g

12.0 g

14.7 g

Relative FC males
Days 1 - 91

47.7 g/kg/day

49.1 g/kg/day

53.6 g/kg/day

62.9 g/kg/day

Relative FC females
Days 1 - 91

58.6 g/kg/day

59.5 g/kg/day

61.0 g/kg/day

70.1 g/kg/day

With few exceptions, the mean daily food consumption values of females treated with 1000 mg/kg bw/day were higher than those of the controls; males at this dose level were also elevated from days 8 - 15 onwards.

The mean daily food consumption values of the males and females treated with 100 mg/kg bw/day and 300 mg/kg bw/day did not show test item-related differences when compared with their respective controls.

Body Weights

At 1000 mg/kg bw/day, the males had significantly lower body weights (p<0.01 except for day 8) and lower body weight gain (all p<0.01 from day 8) when compared to the controls males. This was considered to be test-item related.

The mean absolute body weights and mean body weight gain were slightly reduced in males treated with 100 mg/kg bw/day and 300 mg/kg bw/day. None of the differences attained statistical significance. The differences were evident beginning on day 15 of treatment and were similarly reduced in both groups until day 29 of treatment, at which time the males treated with 300 mg/kg bw/day were progressively lighter until the end of the treatment period.

Table 6: Male Body Weights (g) and Difference to Control (%)

Allocation and
Dose Levels
mg/kg bw/day

Group 1
Control*
0

Group 2

100

Group 3

300

Group 4

1000

Day 1

271

264 (-3 %)

267 (-1 %)

268 (-1 %)

Day 8

306

297 (-3 %)

300 (-2 %)

289 (-6 %)

Day 15

334

323 (-3 %)

325 (-3 %)

311 (-7 %)

Day 29

382

369 (-3 %)

367 (-4 %)

345 (-10 %)

Day 57

437

421 (-4 %)

413 (-5 %)

369 (-16 %)

Day 91

484

470 (-3 %)

449 (-7%)

386 (-20 %)

Bold values denote statistically significant changes.

There were no test item-related differences in the mean body weights or mean body weight gain of females at all dose levels.

Table 7: Female Body Weights (g) and Difference to Control (%)

Allocation and
Dose Levels
mg/kg bw/day

Group 1
Control*
0

Group 2

100

Group 3

300

Group 4

1000

Day 1

156

158 (+1 %)

158 (+1 %)

155 (-1 %)

Day 8

173

176 (+2 %)

177 (+2 %)

173 (±0 %)

Day 15

183

185 (+1 %)

185 (+1 %)

158 (+2 %)

Day 29

201

204 (+1 %)

199 (-1 %)

205 (+2 %)

Day 57

222

225 (+1 %)

217 (-2 %)

218 (-2 %)

Day 91

243

246 (+1 %)

236 (-3 %)

228 (-6 %)

Bold values denote statistically significant changes.

Applicant's summary and conclusion

Conclusions:
NOAEL for 90-day subchronic oral toxicity is 300 mg/kg body weight/day.
Executive summary:

The subchronic oral (gavage) 90-day repeated dose toxicity of the test item Propylidynetrimethyl trimethacrylate (CAS 3290-92-4) to Wistar rats of both sexes was investigated in a GLP-compliant dose-effect study according to the OECD TG 408 (1998) and EU B.29 (1996) protocols.

The test item was administered daily by oral gavage at dose levels of 0, 100, 300 and 1000 mg/kg body weight/day (named groups 1 to 4, respectively) using a volume of 5 mL/kg body weight. The control group was treated with the vehicle, corn oil, only. The groups comprised 10 animals per sex which were sacrificed after 92/93 days of treatment. Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during the acclimatization and treatment periods. Functional observational battery, locomotor activity and grip strength were performed during week 13. Oestrus cycling was assessed in all females for a period over 21 days during the final month of the study. At the end of the dosing period, blood samples were withdrawn for haematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Sperm samples were collected at necropsy to assess count, motility and morphology. Histological examinations were performed on organs and tissues from all control and high dose animals and all gross lesions from all animals

The administration of the test item to the test animals resulted in no test item-related deaths, no relevant findings during daily observations, weekly observations (weeks 1 - 12) or functional observational battery (week 13), no differences of toxicological relevance in the fore- and hind limb grip strength values, no test item-related differences in the ophthalmoscopy, no test item-related effects on haematology, clinical biochemistry or urine parameters. The duration and pattern of the oestrus cycles of the test item-treated females were similar to those of the control females.

At 1000 mg/kg bw/day, males showed lower mean body weights and elevated food consumption values which were considered to be test item-related. The mean locomotor activity was low in males at 1000 mg/kg bw/day. Although the mean body weights of the females were unaffected, there was a clear increase in the absolute and relative food consumption.

Differences noted in the liver and kidney weights of males and females treated with 1000 mg/kg bw/day were considered to be test item-related. There were no other differences of toxicological relevance noted in the rats treated with 300 mg/kg bw/day or 100 mg/kg bw/day.

The epithelial hyperplasia/hyperkeratosis and erosion/ulcer(s) of the non-glandular gastric mucosa was indicative of direct contact irritancy on the non-glandular mucosa (forestomach). At 1000 mg/kg bw/day, males were more affected than females, while at 300 mg/kg bw/day the finding was only present in one of 10 females. Owing to their random distribution among treated groups, the occasional erosion/ulcer(s) of the glandular mucosa was not considered to be related to the test item as such finding is occasionally seen in control animals. The centrilobular hepatocellular hypertrophy in females at 1000 mg/kg bw/day was the histological correlate of the increased liver weights recorded at necropsy. These findings were suggestive of an adaptive response to mixed function oxidase induction (Cattley et al. 2002). The decreased prostate/coagulating gland weights recorded at necropsy at 1000 mg/kg bw/day were correlated histologically with decreased secretory content. In the absence of test-item related effects in other male reproductive organs, the pathogenesis of this finding is unknown. In the spleen, the extramedullary haematopoiesis was slightly lower at 1000 mg/kg bw/day in males and females. The pathogenesis of this finding is also unknown.

In conclusion based on the results of this study, 300 mg/kg body weight/day of Propylidynetrimethyl trimethacrylate was established as the no-observed-effect-level (NOEL) and the no-observed-adverse-effect-level (NOAEL).