Registration Dossier

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:
2013-08-14 to 2013-10-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP.
Cross-reference
Reason / purpose:
reference to same study

Data source

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

Materials and methods

Test guideline
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
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tetrahydrofurfuryl methacrylate


Test animals

Species:
rat
Strain:
other: Hsd: Sprague Dawley SD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia SpA, Italy
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: 176 to 200 g (males), 151 to 175 g (females)
- Housing: up to 5 of one sex/cage, in polisulphone solid bottomed cages measuring 59.5x38x20 cm; nesting material was provided inside suitable bedding bags and changed at least twice a week
- During mating, animals were housed one male to one female in clear polycarbonate cages measuring approximately 43x27x19 cm with a stainless steel
mesh lid and floor, each cage tray held absorbent material which was inspected and changed daily
- After mating, the males were re-caged as they were before mating, the females were transferred to individual solid bottomed cages for the gestation period, birth and lactation; suitable nesting material was provided and changed as necessary
- Diet (e.g. ad libitum): laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The formulations were prepared daily.

VEHICLE
- Concentration in vehicle: 10, 24 and 60 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration and homogeneity of the low and high dose level were assessed by taking six analytical aliquots (approximately 1 g) in different positions. For the intermediate levels, only concentration was assessed by taking two different analytical aliquots (approximately 1 g).
Duration of treatment / exposure:
mailes: 29 d (2 consecutive weeks prior to pairing and thereafter through the day before necropsy)
females: 29 d (2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum or the day before sacrifice)
Frequency of treatment:
daily, 7 d / week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 120, 300 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: computerised stratified randomisation to give approximately equal initial group mean body weights

Examinations

Observations and examinations performed and frequency:
CLINICAL SINGS AND MORTALITY: Yes
- Time schedule: at least once daily, approximately 0.5-1 hour after dosing

DETAILED CLINICAL OBSERVATIONS: Yes (Functional Observation Battery Tests)
- Time schedule: once before commencement of treatment and at least once a week thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: weekly; females additionally on Days 0, 7, 14 and 20 post coitum, dams with live pups were also weighed on Days 1 and 4 post partum

FOOD CONSUMPTION AND COMPOUND INTAKE: yes
-Time scedule: weekly during the pre-mating period, Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day of necropsy
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: 5 males and 5 females randomly selected from each group
- Parameters: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count, Differential leucocyte count (Neutrophils, Lymphocites, Eosinophils, Basophils, Monocytes, Large unstained cells), Platelets, Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of necropsy
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: 5 males and 5 females randomly selected from each group
- Parameters: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Bile acids, Inorganic phosphorus, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during the study, towards the end of treatment,
- Dose groups that were examined: all groups, 5 males and 5 females
- Battery of functions tested: sensory activity / grip strength / motor activity

OTHER:
Details on reproductive parameters are given in IUCLID section "Toxicity to reproduction"
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)
Other examinations:
Examination of pups and reproductive indices are detailed in IUCLID section "Toxicity to reproduction"
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n was more than 5. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p<0.05.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study. No significant clinical signs were observed.
Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.

BODY WEIGHT AND WEIGHT GAIN
No relevant differences were found in body weight and body weight gain between male groups throughout the study and between female groups up to Day 14 of post coitum period. On Day 20 post coitum, a decrease in body weight and body weight gain (statistically significant) was evident in females dosed at 300 mg/kg bw/day respect to controls.

FOOD CONSUMPTION
Decreases in food consumption were seen in the high dose female (300 mg/kg bw/day) group when compared with controls during the post coitum and post partum periods with statistical significance on Days 7 and 14 post coitum and 4 post partum. No differences were observed in males or between the other treated female groups compared to the control group.

HAEMATOLOGY
When compared with controls, a number of treated males showed slight to moderate thrombocytopenia and leucopenia, with no dose-relation. In particular, platelets were decreased in males dosed with 50 mg/kg bw/day (15%) and 300 mg/kg bw/day (23%) and leucocytes (mainly neutrophils, lymphocytes and basophils) were decreased by 21%, 40% and 25% in those receiving 50, 120 and 300 mg/kg bw/day, respectively. Leucopenia was also recorded in females dosed with 300 mg/kg bw/day (19%). However, the decrement comprised mainly neutrophils and eosinophils. In addition, females dosed with 120 mg/kg bw/day and 300 mg/kg bw/day showed slight increase of erythrocytes, haemoglobin and haematocrit (6% to 16%) associated with reticulopenia (55%) and slight decrease of mean corpuscular haemoglobin concentration (4%) in females dosed at 300 mg/kg bw/day. Due to the low severity and the absence of consistency between sexes, changes of the red series were considered of no toxicological importance.
A statistically significant increase of prothrombin time was recorded in animals dosed with 300 mg/kg bw/day (7% in males, 17% in females).

CLINICAL CHEMISTRY
Increase of phosphorus was recorded in males receiving 300 mg/kg bw/day (14%). Due to the absence of other related findings, this change was considered of no toxicological importance.
Females receiving 300 mg/kg bw/day showed slight decrease of alanine aminotransferase (57%), aspartate aminotransferase (29%), urea (39%) and sodium (7%) and increase of glucose (48%). The findings observed included mainly liver and metabolic parameters but the magnitude and/or the direction of changes was insufficient to represent a liver injury.

NEUROBEHAVIOUR
Motor activity was unaffected by treatment. Variations recorded in the sensory reaction to stimuli measurements, performed at the end of the treatment period, were considered of no toxicological significance since they were inconsistent between sexes, low in magnitude and withouth dose relationship.

ORGAN WEIGHTS
Terminal body weight was unaffected by treatment. Absolute and relative thymus weights were slightly reduced in mid- and high dose males compared to controls. High dose females (300 mg/kg bw/day) showed a slight reduction in absolute and relative adrenals weights.

GROSS PATHOLOGY
No remarkable changes were noted at post mortem examination in treated animals, when compared with controls.

HISTOPATHOLOGY: NON-NEOPLASTIC
No treatment-related changes were noted. Periportal hepatocytic vacuolation, consistent with fatty change like associated in most instances with inflammatory cell foci and in few instances with single cell apoptosis/necrosis, was seen in a single control male and female and with an increased incidence in treated males and females. However, considering that the liver pathologyshowed a different incidence between treated males and females, without any dose-relation and that the pathological picture was similar to that observed in male and female controls, such changes could be attributed to spontaneous or incidental pathology rather than to the treatment. Lymphoid depletion in the thymus was only noted in 2 out of 10 high dose females (300 mg/kg bw/day) and was considered unrelated to treatment. The sporadic lesions reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age under our experimental conditions.

OTHER FINDINGS
Details on reproductive and developmental findings are given in IUCLID section "Toxicity to reproduction".

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no treatment-related adverse effects
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: no treatment-related adverse effects
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
120 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
female
Basis for effect level:
other: significantly increased pre-birth loss, prolonged gestation period in high dose females

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
A combined repeated toxicity study with a reproduction and developmental toxicity study was conducted in Sprague Dawley rats in order to provide information on systemic toxicity (body weight and body weight gain, clinical signs including neurotoxicity assessment, motor activity and sensory reaction to stimuli, food consumption, clinical pathology parameters, organ weights, macroscopic and microscopic examination) as well as any possible effects of the test item on male and female reproductive performance (gonadal function, mating behaviour, conception, development of the conceptus, parturition and early lactation).The dosages were 50, 120 and 300 mg/kg bw/day.
On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for systemic toxicity was considered 300 mg/kg bw/day for males and females.
The NOAEL for reproductive and developmental toxicity was considered to be 300 mg/kg bw/day for males and 120 mg/kg bw/day for females and their litters.
Executive summary:

In a combined repeated toxicity study with a reproduction and developmental toxicity study according to OECD Guideline 422 (adopted on 22 March 1996) Tetrahydrofurfuryl methacrylate was administered to 10 Sprague Dawley rats / sex / dose by gavage at dose levels of 0 (control), 50, 120 and 300 mg/kg bw/day in corn oil at a constant volume of 5 mL/kg bw.

 

Males were treated for a total of 29 days including 2 weeks prior to pairing and continuously thereafter, up to the day before necropsy. Females were dosed throughout the study including 2 weeks before pairing, thereafter during pairing, gestation and lactation periods until the day of necropsy or Day 3 post partum for females with live pups at Day 4 post partum.

The following parameters were evaluated in parental animals: body weight, clinical signs (including neurotoxicity assessment, motor activity and sensory reaction to stimuli), food consumption, oestrous cycle, mating performance, clinical pathology investigations (haematology and clinical chemistry), litter data, macroscopic observations, organ weights and histopathological examination. Pup weight, pup clinical signs and pup macroscopic observations were also performed.

 

No mortality occurred in the study. Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item. No significant clinical signs were observed.

 

Males

No relevant changes were recorded during the study and at the post mortem examinations in males at any dose level investigated. In particular no effects were seen on body weight and body weight gain,

clinical signs (including neurotoxicity assessment, motor activity and sensory reaction to stimuli), food consumption, clinical pathology investigations (haematology and clinical chemistry), macroscopic observations, organ weights and histopathological examination, including identification of the stages of

the spermatogenic cycle. The mating performance including the pre-coital interval (i.e. the number of days paired to sperm positive day) and the copulatory evidence (i.e. the presence of sperm and/or copulation plug in situ or in the cage) did not show differences between groups.

 

Females

A total of 5 females were found not pregnant at necropsy: 4 in the control group and 1 in the mid-dose (120 mg/kg bw/day) group. Unilateral implantation was observed in one low dose (50 mg/kg bw/day) female. In the high dose group (300 mg/kg bw/day), 3 females had total resorption and 7 females had total litter loss within 1 day of parturition.

Therefore, the number of females with live pups on Day 4 post partum was: 6 in the control, 10 in the low dose (50 mg/kg bw/day), 9 in the mid-dose (120 mg/kg bw/day) group and none in the high dose (300 mg/kg bw/day) group.

 

On Day 20 post coitum, a decrease in body weight and body weight gain (statistically significant) was evident in females dosed at 300 mg/kg bw/day respect to controls. Decreases in food consumption were seen in high dose females (300 mg/kg bw/day) when compared with controls during the post coitum and post partum periods with statistically significance on Days 7 and 14 post coitum and 4 post partum. Gestation length of all treated groups was higher than controls and significantly increased at statistical analysis, in the high dose group. The pre-birth loss was significantly increased at statistical analysis, in high dose females. This increase could be attributable to the prolonged gestation period which caused most probably pup suffering and the death during or shortly after the birth.

 

Offspring

An increased presence of missing or dead pups was noted in females receiving 300 mg/kg bw/day. No other treatment-related findings were noted in pups.

At necropsy no treatment-related findings were noted in pups which died or in pups sacrificed on Day 4 post partum.

No difference in sex ratios was noted between the control and treated groups.

No relevant differences in litter data were seen. Decreases in litter weights, seen in low and mid-dose groups were due to the lower number of pups in treated groups respect to control, more evident in mid-dose group in which the increased pup loss was attributed to single females.

 

On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for systemic toxicity was considered 300 mg/kg bw/day for males and females.

The NOAEL for reproductive and developmental toxicity was considered to be 300 mg/kg bw/day for males and 120 mg/kg bw/day for females and their litters.

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.