Registration Dossier

Administrative data

Description of key information

Oral:
Rat: combined 28-day repeated dose oral (gavage) toxicity study with the reproduction/developmental toxicity screening test (OECD TG 422): NOAEL (systemic) = 250 mg/kg bw/d (ReachCentrum SPRL 2010, Val. 1); Read-across to CAS No. 13048-33-4
Dermal:
Rat: subchronic 3 months: NOAEL (systemic) = 66.66 mg/kg bw/d; LOAEL (local) = 20 mg/kg bw/d (Bio/Dynamics Inc. 1992, Val. 2)
Rabbit: subacute 10 days: LOAEL = 500 mg/kg bw/d (Hazleton 1981, Val. 2)
Rabbit: subacute 10 days: LOAEL = 500 mg/kg bw/d (Bio/Dynamics Inc. 1981, Val. 2)
Rabbit: subacute 10 days: LOAEL = 250 mg/kg bw/d (Bio/Dynamics Inc. 1981, Val. 2)
Inhalation:
No data available

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
03 November 2009 - 02 March 2010 (experimental)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study conducted in compliance with GLP regulations
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: approximately 78 days old
- Weight at study initiation:
- Fasting period before study: no
- Housing: upon completion of mating single housing
- Diet (ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water (ad libitum): Reverse osmosis-purified (on site) drinking water
- Acclimation period: 16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.6 - 22.7
- Humidity (%): 37.3 - 45.3
- Air changes (per hr): a minimum of 10 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance formulations were prepared approximately twice weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature, protected from light. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.

VEHICLE
- Concentration in vehicle: 15, 50, 150 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no.: YR1134
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Quadruplicate samples for homogeneity and concentration analyses were collected from the middle of the vehicle control formulation and from the top, middle, and bottom stratum of the test substance formulations prepared for the first week of dose administration. In addition, quadruplicate samples for concentration analyses were collected from the middle stratum of the vehicle control and test substance formulations prepared for the remainder of the study. One set of duplicate samples from each collection was subjected to the appropriate analyses. The remaining set of duplicate samples was stored frozen (approximately -70°C) as back-up. All analyses were conducted by the WIL Research Laboratories, LLC Analytical Chemistry Department using a validated high performance liquid chromatography method using ultraviolet absorbance detection.
The analyzed dosing formulations were within protocol-specified range (100 % ± 5 %) and were homogeneous.
Duration of treatment / exposure:
Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses.
Females received 14 daily doses prior to pairing and were dosed through lactation day 4 for a total of 41-49 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating or post cohabitation day 25) for a total of 39-52 doses.
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
75, 250, and 750 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
The animals were paired on a 1:1 basis within each treatment group following 14 days of treatment for the males and females. For further details on the reproduction toxicity part of the study see Chapter "Toxicity to Reproduction" (IUCLID chapter 7.8).
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
Each male and female was also observed for signs of toxicity immediately following dosing and at approximately 1 hour following dose administration.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly beginning approximately 1 week prior to the initiation of dose administration


BODY WEIGHT: Yes
- Time schedule for examinations:
Males: approximately 1 week prior to the initiation of dose administration, on the first day of dose administration, and weekly thereafter until the day of scheduled euthanasia
Females: approximately 1 week prior to the initiation of dose administration, on the first day of dose administration, and weekly thereafter until the day evidence of copulation was observed. Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17, and 20, and on lactation days 0 (when possible), 1, 4, and 5.


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Individual food consumption was recorded on the corresponding weekly body weight days until pairing. Food intake was not recorded during the mating period. Once evidence of mating was observed, female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1 and 4. Following mating, food consumption for the female with no evidence of mating was measured on a weekly basis until the scheduled euthanasia


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: No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the scheduled necropsies (study day 28 for males and lactation day 5 for females)
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 6 animals/sex/group
- Parameters were examined: Total leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLATELET), Prothrombin time (PT), Activated partial thromboplastin time (APTT), Reticulocyte count, Percent (RETIC), Absolute (RETIC ABSOLUTE), Mean Platelet Volume (MPV), Red cell distribution width (RDW), Hemoglobin Distribution Width (HDW),
Differential leukocyte count: (Percent and absolute): Neutrophil (NEU), Lymphocyte (LYMPH), Monocyte (MONO), Eosinophil (EOS), Basophil (BASO), Large unstained cell (LUC),
Platelet estimatea, Red cell morphology (RBC MORPHOLOGY).


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the scheduled necropsies (study day 28 for males and lactation day 5 for females)
- Animals fasted: Yes
- How many animals: 6 animals/sex/group
- Parameters were examined: Albumin, Total protein, Globulin [by calculation], Albumin/globulin ratio (A/G Ratio) [by calculation], Total bilirubin (Total Bili), Urea nitrogen, Creatinine, Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma glutamyltransferase (GGT), Glucose, Total cholesterol (Cholesterol), Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides (Triglyceride), Bile acids.


URINALYSIS: Yes
- Time schedule for collection of urine: overnight before the scheduled necropsies (study day 28)
- Metabolism cages used for collection of urine: Yes
- How many animals: 6 males/group
- Animals fasted: Yes
- Parameters were examined: Specific gravity (SG), pH, Urobilinogen (URO), Total volume (TVOL), Color (COL), Clarity (CLA), Protein (PRO), Glucose (GLU), Ketones (KET), Bilirubin (BIL), Occult blood (BLD), Leukocytes (LEU), Nitrites (NIT), Microscopy of sediment.


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: FOB assessments were recorded for 6 animals/sex/group prior to dose administration on study day 27 (males) and lactation day 4 (females).
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity:
1. Home cage observations: Posture, Convulsions/Tremors, Feces consistency, Biting, Palpebral (eyelid) closure
2. Handling observations: Ease of removal from cage, Lacrimation/Chromodacryorrhea, Piloerection, Palpebral closure, Eye prominence, Red/Crusty deposits, Ease of handling animal in hand, Salivation, Fur appearance, Respiratory rate/character, Mucous membranes/Eye/Skin color, Muscle tone
3. Open field observations: Mobility, Rearing, Convulsions/Tremors, Grooming, Bizarre/Stereotypic behavior, Time to first step (seconds), Gait, Arousal, Urination/Defecation, Gait score, Backing
4. Sensory observations: Approach response, Startle response, Pupil response, Forelimb extension, Air righting reflex, Touch response, Tail pinch response, Eyeblink response, Hindlimb extension, Olfactory orientation
5. Neuromuscular observations: Hindlimb extensor strength, Hindlimb foot splay, Grip strength hind and forelimb, Rotarod performance
6. Physiological observations: Catalepsy, Body temperature, Body weight
7. Locomotor activity (measured automatically using a personal computer controlled system): Data were collected in 5 minute epochs and the test session duration was 60 minutes. These data were compiled as six, 10-minute subintervals for tabulation. Data for ambulatory and total motor activity were tabulated. Total motor activity was defined as a combination of fine motor skills (i.e., grooming, interruption of 1 photobeam) and ambulatory motor activity (interruption of 2 or more consecutive photobeams).
Sacrifice and pathology:
SACRIFICE: All surviving F0 adults were euthanized by carbon dioxide inhalation. Males were euthanized following completion of the mating period. Females that delivered were euthanized on lactation day 5 or within 24 hours of total litter loss; the numbers of former implantation sites and corpora lutea were recorded. Females that failed to deliver were euthanized on post mating day 25 (females with evidence of mating) or post-cohabitation day 25 (females with no evidence of mating).

GROSS PATHOLOGY: Yes
Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10 % ammonium sulfide solution for detection of early implantation loss. A gross necropsy was conducted on all animals including the female that was found dead during gestation; the numbers of corpora lutea and implantation sites were recorded and recognizable fetuses were examined externally for gross abnormalities. Necropsies included examination of the external surface, all orifices, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera.

ORGAN WEIGHTS: from F0 animals at the scheduled necropsies, the following organs were weighed: Adrenal glands, Ovaries with oviducts, Brain, Spleen, Epididymides, Testes, Heart, Thymus gland, Kidneys, Thyroids with parathyroids, Liver.

HISTOPATHOLOGY: Yes
At the time of necropsy, the following tissues and organs were placed in 10% neutral-buffered formalin: Adrenal glands (2), Lymph node (Axillary, Mesenteric, Mandibular), Aorta, Bone with marrow (sternebrae), Bone marrow smear ( not placed in formalin), Brain (Cerebrum level 1, Cerebrum level 2, Cerebellum with medulla/pons), Ovaries and oviducts (2), Pancreas, Peripheral nerve (sciatic), Pituitary gland, Coagulating glands, Prostate gland, Eyes with optic nerve (2) (in Davidson’s solution), Mandibular salivary glands (2), Gastrointestinal tract (Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum), Seminal vesicles (2), Skeletal muscle (rectus femoris), Skin with mammary gland, Spinal cord (cervical), Spleen, Testes with epididymides (2) (fixed in Bouin’s solution), Thymus gland, Thyroids [with parathyroids, if present (2)], Heart, Trachea, Kidneys (2), Urinary bladder, Liver (sections of 2 lobes), Uterus with cervix and vagina (in 10% ammonium sulfide solution), Lungs (including bronchi, fixed by inflation with fixative), All gross lesions.
Microscopic examination was performed on all tissues listed above from all animals in the control and 750 mg/kg/day groups. In addition, the liver, stomach, and all gross lesions from all animals at all dosage levels were examined microscopically.
Statistics:
Parental mating, fertility, conception, and copulation indices were analyzed using the Chi square test with Yates’ correction factor (Hollander and Wolfe, 1999). Mean parental body weights (weekly, gestation, and lactation), body weight changes, and food consumption, offspring body weights and body weight changes, gestation length, numbers of former implantation sites and corpora lutea, number of pups born, live litter size on PND 0, unaccounted-for sites, absolute and relative organ weights, clinical pathology values (except gamma glutamyltransferase), pre coital intervals, and continuous FOB data values were subjected to a parametric one way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences between the control and test substance-treated groups. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. FOB parameters that yield scalar or descriptive data and histopathological findings in the test substance-treated groups were compared to the control group using Fisher’s Exact test (Steel and Torrie, 1980). Gamma glutamyltransferase values under range were assigned a value of 0.1 (half the lower limit of quantitation) for statistical analysis and reporting. Gamma glutamyltransferase data, mean litter proportions (percent per litter) of males at birth, and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA (Kruskal and Wallis, 1952) to determine intergroup differences between the control and test substance-treated groups. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance-treated groups to the control group.
Details on results:
CLINICAL SIGNS AND MORTALITY
In the 750 mg/kg bw/day group, a single female was found dead prior to evidence of parturition on gestation day 21. This female was noted with clinical findings common to the majority of the other females in this group, including wiping mouth on the cage floor and/or walls, excessive pawing of the cage floor and/or walls, wiping mouth in bedding material following dosing, and wet and clear material around the mouth; these findings were noted at the time of and/or approximately 1 hour following dose administration. In addition, this female was gasping approximately 2 minutes following dose administration on the day of death, and was subsequently found dead approximately 11 minutes after dose administration. Upon macroscopic and microscopic examination, the cause of death of the female was undetermined. Based on these findings, the death of this female was likely attributable to the dose administration procedures and unlikely related to systemic toxicity of the test substance. With the exception of a female in the 750 mg/kg bw/day group that was euthanized on lactation day 0 due to total litter loss, all other males and females at all dosage levels survived to the scheduled necropsy.

Behaviour-related clinical findings, including wiping mouth on the cage floor and/or walls, excessive pawing of the cage floor and/or walls, and wiping mouth in bedding material following dosing (females only), were noted for the majority of the males and females in the 250 and 750 mg/kg bw/day groups throughout the treatment period. Because these findings were primarily limited to the time of dose administration and generally did not persist to approximately 1 hour following dose administration, they were attributed to the irritative properties of the test substance and were not considered adverse. Other clinical findings attributed to test substance administration included salivation-related findings (salivation prior to or at the time of dose administration and clear material around the mouth) and red material around the mouth for the majority of the animals in the 250 and 750 mg/kg bw/day groups. These findings were noted at the time of and/or approximately 1 hour following dose administration throughout the treatment period; the salivation related findings were also sporadically noted in the 75 mg/kg bw/day group animals and were likely signs of taste aversion to the test substance, which were not considered adverse.
Other clinical findings noted at the daily examinations, at the time of dose administration, or approximately 1 hour following dose administration, including hair loss on various body surfaces, occurred infrequently and/or at similar frequencies in the control group, and were not attributed to test substance administration.

BODY WEIGHT AND WEIGHT GAIN
-Males:
A test substance-related, significantly (p<0.01) lower mean body weight gain was noted in the 750 mg/kg bw/day group males following the first week of dose administration (study days 0-7) followed by a slightly (not statistically significant) lower mean body weight gain during study days 7-13. Mean body weight changes for these males were comparable to the control group during the remainder of the treatment period (study days 13-28). A mean body weight loss (13 g) was noted for the 750 mg/kg bw/day group males during study days 21-28; however, mean body weight losses were noted across all dosage groups, including the control group, as a result of being food-fasted prior to blood collection on study day 27-28. Due to the initial reductions in mean body weight gains, mean body weight gain in the 750 mg/kg bw/day group males was significantly (p<0.01) lower during the overall pre-mating period (study days 0-13) compared to the control group, and mean body weight was 6.8 % lower (not statistically significant) than the control group value on study day 28.
Mean male body weights and body weight gains in the 75 and 250 mg/kg bw/day groups were similar to the control group throughout the treatment period.

-Females:
Mean female body weights and body weight gains in the 75, 250, and 750 mg/kg bw/day groups were similar to the control group during the pre-mating period (study days 0-13). Mean body weights and body weight gains in the 75, 250, and 750 mg/kg bw/day groups were generally similar to the control group throughout gestation. During lactation, mean body weights and body weight gains in the 75, 250, and 750 mg/kg bw/day group were unaffected by test substance administration.

FOOD CONSUMPTION
-Males:
Mean male food consumption, evaluated as g/animal/day and g/kg/day, in the 750 mg/kg bw/day group was slightly reduced during the first week of dose administration (study days 0-7). The difference from the control group achieved significance (p<0.05) on a g/kg/day basis only and corresponded to a reduced mean body weight gain during this interval. Mean food consumption in this group was similar to the control group during study days 7-13. Mean male food consumption in the 75 and 250 mg/kg bw/day groups was similar to the control group during the pre-mating period (study days 0-13).

- Females:
Mean food consumption in the 75, 250, and 750 mg/kg bw/day groups was similar to the control group throughout the pre-mating period (study days 0-13).
Mean food consumption in the 75, 250, and 750 mg/kg bw/day groups was unaffected by test substance administration during gestation. Significantly (p<0.05) higher mean food consumption was noted in the 750 mg/kg bw/day group during gestation days 7-11 (g/animal/day only) and when the overall gestation period (gestation days 0-20; g/kg/day only) was evaluated. However, due to the small magnitude of these differences compared to the control group and the lack of a concurrent similar effect on mean body weight gain, the changes were not attributed to test substance administration.
Mean food consumption in the in the 75, 250, and 750 mg/kg bw/day groups was unaffected by the test substance during lactation.

HAEMATOLOGY
There were no test substance-related alterations in hematology and coagulation parameters at any dosage level.

CLINICAL CHEMISTRY
The mean urea nitrogen value was higher for the 750 mg/kg bw/day group males (43.7%) and females (16.6%) when compared with the control group; the difference was statistically significant for the 750 mg/kg bw/day group males.
The mean bile acids value was higher for the 750 mg/kg bw/day group males (218%) and females (82.3%) when compared with the control group; the difference was statistically significant for the 750 mg/kg/day group males.
The mean alanine aminotransferase (ALT) concentration was higher in the 250 and 750 mg/kg/day group females when compared with the control group (60 and 70%, respectively), and a dose relationship was present.
The mean cholesterol and triglycerides concentrations were higher for the 750 mg/kg bw/day group females when compared with the control group (81.6 and 78.7%, respectively).
The mean calcium and phosphorous concentrations were statistically significantly higher for the 750 mg/kg bw/day group females when compared with the control group (7.8 and 27.4%, respectively).
No other changes in serum chemistry parameters could be definitively attributed to test substance administration.

URINALYSIS
No test substance-related changes in urinalysis parameters were noted at any dosage level.

NEUROBEHAVIOUR
Home cage parameters, handling parameters, open field parameters, sensory parameters, neuromuscular parameters, and physiological parameters (catalepsy and body temperature) were unaffected by test substance administration at all dosage levels. Locomotor activity patterns (total activity as well as ambulatory activity counts) were unaffected by test substance administration at all dosage levels when evaluated on study day 27 (males) and lactation day 4 (females).
Values obtained from the 6 subintervals evaluated and the overall 60 minute test session values were comparable to the control group values with the following exceptions. Mean total and ambulatory activity counts for females in the 75, 250, and 750 mg/kg bw/day groups were generally lower for the 6 subintervals as well as the overall 60-minute test session; differences from the control group achieved significance (p≤0.045) for all dosage groups when the overall 60-minute test session was evaluated for ambulatory motor activity counts by a repeated measures analysis. However, no dose related trend was apparent, and females in the test substance-treated groups showed no remarkable shifts in the pattern of habituation to the test environment. In addition, 1 female in the control group was consistently noted with high total and ambulatory counts throughout the test session. Furthermore, motor activity data is generally quite variable especially in screening studies with small group sizes. The majority of the control group females had similar cumulative ambulatory motor activity counts as the females in the test substance groups. Therefore, differences from the control group were not considered indicative of a test substance-related effect on locomotor activity patterns.

ORGAN WEIGHTS
The mean final body weight was 6.8% lower for the 750 mg/kg bw/day group males when compared with the control group; the difference did not achieve statistical significance.
The mean liver weight was higher for the 750 mg/kg bw/day group males and females when compared with the control group. The difference in the absolute liver weight and liver weight relative to brain weight was significant (p<0.05 or p<0.01) for the 750 mg/kg bw/day group females, and the difference in mean liver weight relative to body weight was significant (p<0.01) for both the 750 mg/kg bw/day group males and females.
The following organ weight differences were significant (p<0.05 or p<0.01) when compared to the control group but were considered to be a result of the test substance related effect on final body weight: higher left and right testis weight relative to body weight for the 750 mg/kg bw/day group males. The mean and individual absolute testis weights and testis weights relative to body weight for the 750 mg/kg bw/day group males were all within the historical control data range for Crl:CD(SD) rats of a similar age.
There were no other test substance-related effects on organ weights.

GROSS PATHOLOGY
In the 750 mg/kg bw/day group, one female was found dead approximately 11 minutes following dose administration on gestation day 21. At necropsy, this female had 16 dead fetuses with no apparent malformations and 1 early resorption in utero; no remarkable gross findings were noted. All other animals survived to the scheduled necropsies.
At the scheduled necropsy, a pale liver was noted for one male and a thickened nonglandular portion of the stomach was noted for another male in the 750 mg/kg bw/day group. Although these findings were noted in single males, the pale liver finding was associated with swelling and fine vacuolation of periportal hepatocytes and the thickened stomach finding was associated with epithelial hyperplasia and hyperkeratosis. Therefore, these macroscopic findings in the 750 mg/kg bw/day group males were attributed to test substance administration. No other test substance related internal findings were observed at any dosage level in females that failed to deliver, the female with total litter loss, or males and females at the scheduled necropsy.
HISTOPATHOLOGY: NON-NEOPLASTIC
All of the 750 mg/kg bw/day group males and females exhibited mild to moderate epithelial hyperplasia and mild to severe hyperkeratosis in the non-glandular portion of the stomach. This consisted of thickening of the squamous epithelial lining with multiple superficial layers of keratin. This change was also observed in a single 250 mg/kg/day group male and 2 females in the 250 mg/kg bw/day group. It was not observed in any of the 75 mg/kg bw/day group or control group animals.
Nine of 12 males from the 750 mg/kg bw/day group had diffuse vacuolation of periportal hepatocytes. These hepatocytes were swollen with abundant fine well-delineated microvesicular cytoplasmic vacuoles. This change was also observed in 9 of the 250 mg/kg bw/day group males, 4 of the 75 mg/kg bw/day group males, and a single control group male. Although the change was present in 1 control group male, a dose-response relationship was present among the test substance-treated males; therefore, the change was considered to be test substance-related. Eight of 12 of the 750 mg/kg bw/day group females exhibited periportal to midzonal hepatocellular vacuolation. This differed from the periportal vacuolation observed in the 750 mg/kg bw/day group males in that the vacuoles ranged from fine (microvesicular) to large with displacement of the nucleus (macrovesicular), and the hepatocytes did not appear swollen. This change was also observed in 8 of the 250 mg/kg bw/day females and 3 of the 75 mg/kg bw/day females, as well as a single 75 mg/kg bw/day male. Although the change was also observed in a single control group male and a single control group female, a dose response relationship was present among the test substance-treated females; therefore, the change was considered to be test substance-related. There were no other test substance-related histologic changes.
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight; clinical chemistry; liver weights
Critical effects observed:
not specified

Toxicologically Relevant Final Body Weight And Organ Weight Changes

Parameter

Direction and magnitude of change

Dosage level (mg/kg bw/day)

Sex

 

 

 

 

Final Body Weight

↓ 6.8%

750

M

 

 

 

 

Liver

 

 

 

  Absolute

↑ 7.7%, ↑19.6%**

750

M, F

  Relative to body weight

↑ 15.5%**, ↑15.9%**

750

M, F

  Relative to brain weight

↑ 9.2%, ↑18.8%*

750

M, F

 

 

 

 

* =   Significantly different from the control group at p<0.05 using Dunnett's test

** = Significantly different from the control group at p<0.01 using Dunnett's test

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented and acceptable for assessment.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Principles of method if other than guideline:
The subchronic dermal application of the test substance to the backs of Sprague-Dawley rats was studied to assess potential neurotoxic and other local and systemic effects.
GLP compliance:
not specified
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: 41 days (age: 28 days at receipt)
- Weight at study initiation: week 0 males: 157-163 g; week 0 females: 133-139 g
- Housing: individually in elevated stainless steel cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
other: not occluded
Vehicle:
corn oil
Details on exposure:
TEST SITE
- Area of exposure: the back of the rats
- % coverage: no data
- Type of wrap if used: not occluded no further data
- Time intervals for shavings or clipplings: all animals were clipped ca. 23 h prior to initial dose. The animals were reclipped when necessary.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2.077 ml/kg
- Concentration (if solution): 1.0, 3.33 and 10.0 %
- Constant volume or concentration used: yes

VEHICLE
- Justification for use and choice of vehicle (if other than water): immiscible with water
- Amount(s) applied (volume or weight with unit): 2.077 ml 7kg of the test substance in corn oil
- Concentration (if solution): 1.0, 3.33 and 10.0 % of the test substance in corn oil

USE OF RESTRAINERS FOR PREVENTING INGESTION: no data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
the remaining samples of weekly dosing solutions for each dose were returned to the sponsor for analysis, no further data
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily, 5 days/week
Remarks:
Doses / Concentrations:
0, 20, 66.66, 200 mg/kg bw
Basis:
other: nominal conc.
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: for mortality and gross signs of toxicologic or pharmacologic effects

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly including signs of local or systemic toxicity, pharmacologic effects and palpation for tissue masses

BODY WEIGHT: Yes
- Time schedule for examinations: twice pretest, weekly during treatment and terminally (after fasting)


OTHER:
- blood was obtained from over night fasted rats via venipuncture of the orbital sinus under light ether anestehsia, the same animals were used that were intended for formalin-fixation (5/sex/dose).
- hematology upon termination: hemoglobin, hematocrit, erythrocytes, clotting time, total and differential, leukocytes, erythrocytes morphology
- clinical chemistry upon termination: serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, blood urea nitrogen, fasting glucose, total protein, total bilirubin, sodium, potassium, calcium, inorganic phosphorus
- urinanalysis 6 days before termination: gross appearance, specific gravity, pH, protein, glucose, occult blood
Sacrifice and pathology:
One-half of the animals were sacrificed by exsanguination under light ether anesthesia, and selected organs and tissues were fixed
in formalin. Organ and organ-body weight ratios (adrenals, brain, liver, kidney, heart, spleen, testes with epididymides, ovaries) were determined on all of these animals only. Histopathological evaluations of 10 organs or tissues (liver, kidney, lung, heart, stomach, adrenal, pituitary, testes, ovaries,
spleen and skeletal muscle) were conducted on all formalin-fixed control animals and the high dose animals. The remaining animals were perfused
intravenously with glutaraldehyde under sodium pentobarbital anesthesia . Quantitative assessments of teased tibial nerve preparations were
performed on all glutaraldehyde-perfused animals in control animals and the high dose animals. In addition, brain, spinal cord and sciatic nerve were evaluated microscopically (hematoxylin and eosin and Luxol-fast blue staining) from these same animals.
Other examinations:
- neurologic functions were evaluated monthly
- Parameters examined according to a scoring system: posture, gait, muscular tone, reflexes (corneal), righting and toe-pinch
- no further data
Statistics:
Body weight, hematology and clinical chemistry parameters, organ weights and organ/body weight ratios were analyzed. Mean values of
all dose groups were compared to control at each time interval.
Details on results:
CLINICAL SIGNS AND MORTALITY
All animals survived to the scheduled termination of the study; weekly physical examinations of all animals failed to indicate any toxic effects of the test material other than irritation at the application site. Alopecia observed on the forepaws and legs was the most common finding in both sexes; however, the incidence was spread over all test groups, including control. The test substance produced moderate levels of irritation, in a dose-related manner, beginning the first week of the study . Males were generally more susceptible than females to the dermal effects of the test substance
throughout the study. Dermal effects (erythema and eschar formation) were only scored as present or absent therefore, the number of times per week the effects were noted was used as a general indication of the severity of the dermal observations.

DERMAL OBSERVATIONS
In the controls dermal effects were absent except for one female rat which had one exfoliation score in Week 8, and one other female rat which
exhibited eschar and exfoliation at various times during the initial 2 months of the study.
In the low dose group, erythema was only noted occasionally during the final 2 months of the study. In the initial 3 weeks, erythema was recorded more frequently (approximately 12-13 % of total observations) than later in the study. Exfoliation was recorded with gradually diminishing frequency in approximately one-haft of the low dose group animals (both sexes) during weeks 2 and 3. During the last 5 weeks of the study, only two female rats and one male rat were observed with this effect. Edema, atonia, and fissuring were not observed in any low dose group animals.
In mid dose group male rats, erythema was noted with a same what higher frequency than was seen for low dose group male rats. Erythema frequency for female mid dose group rats was comparable to low dose group. After week 3 of the study, a somewhat lower frequency of erythema was recorded in both sexes. Exfoliation and eschar were recorded for most animals in the mid dose group by week 3 of the study, with diminishing frequency thereafter. As in the low dose group rats, edema, atonia and fissuring were not observed.
In the high dose group, erythema, exfoliation and eschar were seen in most animals of both sexes beginning in week 1. The highest frequency was
noted in week 2 (both sexes), with diminishing frequency thereafter. Atonia was observed in one male and one female during weeks 3-4 and 5-6,
respectively. Fissures were present in one female rat (on one day during week 2 only). Four male rats showed fissures on week 2 of the study.
A persistant fissuring in one of these rats was observed from week 2 through week 7 of the study. Male rats appeared somewhat more sensitive than females to erythema and eschar formation.

BODY WEIGHT AND WEIGHT GAIN
Significantly reduced body weights in high dose male rats (versus control male rats) were noted from weeks 3 through 12, except during weeks 10
and 11. Male rat weights of the low and mid dose groups were also reduced in dose-related fashion, but the differences were not statistically
significant in comparison to control values. No statistically significant effects on weight were seen in treated female rats, however, high dose female animals never exceeded 95% of the mean weights of control females after 2 weeks on test.

BIOCHEMISTRY
Of the clinical chemical parameters which were determined at the study termination, none were affected in a manner suggestive of a treatment-effect.

HEMATOLOGY
None of the hematologic parameters evaluated differed significantly from control values.

URINALYSIS
Protein (100 mg/dl) was confirmed to be present in the urine of one mid-dose male and female, in two high-dose males and one high-dose
female. A large amount of occult blood was also present in the urine of this one high-dose female. The specific gravity of this high-dose female and one mid-dose female was also high (>1.090). These findings suggested a possible effect of the test substance on the kidneys, but no alterations were observed in the histopathological examination to support this conclusion. Additionally, without relation to urinary volumes and creatinine, the relevance of in urinary protein concentration is questionable. All in all the singular changes are not considered adverse by the registrant.

TERMINAL ORGAN and BODY WEIGHT AND ORGAN/BODY WEIGHT RATIOS
There was a significant downward trend in male liver weights; however, this was not evident in the liver/body weight ratio and is therefore of doubtful significance. Other organ weights and organ/body weight ratios were comparable across all groups.

NEUROBEHAVIOUR
Month 1 neurological function tests showed two high-dose males with slightly reduced corneal resporse. All other evaluations were normal.
Month 2 neurological function tests were unremarkable in control, low and mid dose animals (both sexes). Four high dose males and three high dose females showed slightly abnormal gait described as "stilted". A slight decreasad corneal reflex was observed in four males and one female. A
moderately decreased toe pinch response (hindtoes only) was also present in one male rat.
Month 3 neurological function tests showed a slightly stilted gait and altered righting reflex in one male control rat and a slightly relaxed body tone in one mid dose male rat. One male high dose animal continued to exhibit a moderately decreased toe-pinch response (hindtoes only) . All
neurological observations were normal in both the control and treated female rats at Month 3.

GROSS PATHOLOGY/HISTOPATHOLOGY
Formalin-fixed rats:
No changes, gross or microscopic, were evident which could be attributed to a systemic toxic effect of the test substance. The most common
spontaneous gross necropsy findings, occurring across all groups, were inflammations around the ear tags, and slight hair loss on the extremities. No unusual microscopic pathological findings were evident which could be attributed to the topical administration of the test substance.

NEUROPATHOLOGY
Glutaraldehyde-perfused rats:
Histopathological examinations of hematoxylin and eosin and Luxol-fast Blue stained slides of brain, spinal cord and sciatic nerve from ten rats
(5/sex) treated with the high dose of the test substance failed to reveal any treatment-related lesions when these tissues were compared to similar
ones from ten control rats (5/sex). In addition, microscopic examinetion of 50 teased nerve fibers from the tibial nerve of ten high-dose animals
(5/sex) were canparable to those of the controls. When quantitative measurements were taken of myelinated nerve tibers in cross-section of the distal sciatic nerve, a slight shift to larger diameters could be detected in high-dose males when compared to the controls. However, there was also a slight decrease in fiber diameters in treated females. The relatively large standard deviation in data from both males and females suggest that these slight changes in fiber diameters are not significant. Moreover, the absence of lesions by more conventional histopathological examinations substantiate this conclusion.
Dose descriptor:
NOAEL
Remarks:
skin irritation
Effect level:
20 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: skin irritation
Dose descriptor:
NOAEL
Remarks:
for systemic toxicity
Effect level:
66.66 mg/kg bw/day
Sex:
male
Basis for effect level:
other: significantly reduced body weights in high dose males
Dose descriptor:
NOAEL
Remarks:
for systemic toxicity
Effect level:
200 mg/kg bw/day
Sex:
female
Critical effects observed:
not specified
Endpoint conclusion
Dose descriptor:
NOAEL
66.7 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented and acceptable for assessment.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Principles of method if other than guideline:
The subchronic dermal application of the test substance to the backs of Sprague-Dawley rats was studied to assess potential neurotoxic and other local and systemic effects.
GLP compliance:
not specified
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: 41 days (age: 28 days at receipt)
- Weight at study initiation: week 0 males: 157-163 g; week 0 females: 133-139 g
- Housing: individually in elevated stainless steel cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
other: not occluded
Vehicle:
corn oil
Details on exposure:
TEST SITE
- Area of exposure: the back of the rats
- % coverage: no data
- Type of wrap if used: not occluded no further data
- Time intervals for shavings or clipplings: all animals were clipped ca. 23 h prior to initial dose. The animals were reclipped when necessary.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2.077 ml/kg
- Concentration (if solution): 1.0, 3.33 and 10.0 %
- Constant volume or concentration used: yes

VEHICLE
- Justification for use and choice of vehicle (if other than water): immiscible with water
- Amount(s) applied (volume or weight with unit): 2.077 ml 7kg of the test substance in corn oil
- Concentration (if solution): 1.0, 3.33 and 10.0 % of the test substance in corn oil

USE OF RESTRAINERS FOR PREVENTING INGESTION: no data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
the remaining samples of weekly dosing solutions for each dose were returned to the sponsor for analysis, no further data
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily, 5 days/week
Remarks:
Doses / Concentrations:
0, 20, 66.66, 200 mg/kg bw
Basis:
other: nominal conc.
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: for mortality and gross signs of toxicologic or pharmacologic effects

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly including signs of local or systemic toxicity, pharmacologic effects and palpation for tissue masses

BODY WEIGHT: Yes
- Time schedule for examinations: twice pretest, weekly during treatment and terminally (after fasting)


OTHER:
- blood was obtained from over night fasted rats via venipuncture of the orbital sinus under light ether anestehsia, the same animals were used that were intended for formalin-fixation (5/sex/dose).
- hematology upon termination: hemoglobin, hematocrit, erythrocytes, clotting time, total and differential, leukocytes, erythrocytes morphology
- clinical chemistry upon termination: serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, blood urea nitrogen, fasting glucose, total protein, total bilirubin, sodium, potassium, calcium, inorganic phosphorus
- urinanalysis 6 days before termination: gross appearance, specific gravity, pH, protein, glucose, occult blood
Sacrifice and pathology:
One-half of the animals were sacrificed by exsanguination under light ether anesthesia, and selected organs and tissues were fixed
in formalin. Organ and organ-body weight ratios (adrenals, brain, liver, kidney, heart, spleen, testes with epididymides, ovaries) were determined on all of these animals only. Histopathological evaluations of 10 organs or tissues (liver, kidney, lung, heart, stomach, adrenal, pituitary, testes, ovaries,
spleen and skeletal muscle) were conducted on all formalin-fixed control animals and the high dose animals. The remaining animals were perfused
intravenously with glutaraldehyde under sodium pentobarbital anesthesia . Quantitative assessments of teased tibial nerve preparations were
performed on all glutaraldehyde-perfused animals in control animals and the high dose animals. In addition, brain, spinal cord and sciatic nerve were evaluated microscopically (hematoxylin and eosin and Luxol-fast blue staining) from these same animals.
Other examinations:
- neurologic functions were evaluated monthly
- Parameters examined according to a scoring system: posture, gait, muscular tone, reflexes (corneal), righting and toe-pinch
- no further data
Statistics:
Body weight, hematology and clinical chemistry parameters, organ weights and organ/body weight ratios were analyzed. Mean values of
all dose groups were compared to control at each time interval.
Details on results:
CLINICAL SIGNS AND MORTALITY
All animals survived to the scheduled termination of the study; weekly physical examinations of all animals failed to indicate any toxic effects of the test material other than irritation at the application site. Alopecia observed on the forepaws and legs was the most common finding in both sexes; however, the incidence was spread over all test groups, including control. The test substance produced moderate levels of irritation, in a dose-related manner, beginning the first week of the study . Males were generally more susceptible than females to the dermal effects of the test substance
throughout the study. Dermal effects (erythema and eschar formation) were only scored as present or absent therefore, the number of times per week the effects were noted was used as a general indication of the severity of the dermal observations.

DERMAL OBSERVATIONS
In the controls dermal effects were absent except for one female rat which had one exfoliation score in Week 8, and one other female rat which
exhibited eschar and exfoliation at various times during the initial 2 months of the study.
In the low dose group, erythema was only noted occasionally during the final 2 months of the study. In the initial 3 weeks, erythema was recorded more frequently (approximately 12-13 % of total observations) than later in the study. Exfoliation was recorded with gradually diminishing frequency in approximately one-haft of the low dose group animals (both sexes) during weeks 2 and 3. During the last 5 weeks of the study, only two female rats and one male rat were observed with this effect. Edema, atonia, and fissuring were not observed in any low dose group animals.
In mid dose group male rats, erythema was noted with a same what higher frequency than was seen for low dose group male rats. Erythema frequency for female mid dose group rats was comparable to low dose group. After week 3 of the study, a somewhat lower frequency of erythema was recorded in both sexes. Exfoliation and eschar were recorded for most animals in the mid dose group by week 3 of the study, with diminishing frequency thereafter. As in the low dose group rats, edema, atonia and fissuring were not observed.
In the high dose group, erythema, exfoliation and eschar were seen in most animals of both sexes beginning in week 1. The highest frequency was
noted in week 2 (both sexes), with diminishing frequency thereafter. Atonia was observed in one male and one female during weeks 3-4 and 5-6,
respectively. Fissures were present in one female rat (on one day during week 2 only). Four male rats showed fissures on week 2 of the study.
A persistant fissuring in one of these rats was observed from week 2 through week 7 of the study. Male rats appeared somewhat more sensitive than females to erythema and eschar formation.

BODY WEIGHT AND WEIGHT GAIN
Significantly reduced body weights in high dose male rats (versus control male rats) were noted from weeks 3 through 12, except during weeks 10
and 11. Male rat weights of the low and mid dose groups were also reduced in dose-related fashion, but the differences were not statistically
significant in comparison to control values. No statistically significant effects on weight were seen in treated female rats, however, high dose female animals never exceeded 95% of the mean weights of control females after 2 weeks on test.

BIOCHEMISTRY
Of the clinical chemical parameters which were determined at the study termination, none were affected in a manner suggestive of a treatment-effect.

HEMATOLOGY
None of the hematologic parameters evaluated differed significantly from control values.

URINALYSIS
Protein (100 mg/dl) was confirmed to be present in the urine of one mid-dose male and female, in two high-dose males and one high-dose
female. A large amount of occult blood was also present in the urine of this one high-dose female. The specific gravity of this high-dose female and one mid-dose female was also high (>1.090). These findings suggested a possible effect of the test substance on the kidneys, but no alterations were observed in the histopathological examination to support this conclusion. Additionally, without relation to urinary volumes and creatinine, the relevance of in urinary protein concentration is questionable. All in all the singular changes are not considered adverse by the registrant.

TERMINAL ORGAN and BODY WEIGHT AND ORGAN/BODY WEIGHT RATIOS
There was a significant downward trend in male liver weights; however, this was not evident in the liver/body weight ratio and is therefore of doubtful significance. Other organ weights and organ/body weight ratios were comparable across all groups.

NEUROBEHAVIOUR
Month 1 neurological function tests showed two high-dose males with slightly reduced corneal resporse. All other evaluations were normal.
Month 2 neurological function tests were unremarkable in control, low and mid dose animals (both sexes). Four high dose males and three high dose females showed slightly abnormal gait described as "stilted". A slight decreasad corneal reflex was observed in four males and one female. A
moderately decreased toe pinch response (hindtoes only) was also present in one male rat.
Month 3 neurological function tests showed a slightly stilted gait and altered righting reflex in one male control rat and a slightly relaxed body tone in one mid dose male rat. One male high dose animal continued to exhibit a moderately decreased toe-pinch response (hindtoes only) . All
neurological observations were normal in both the control and treated female rats at Month 3.

GROSS PATHOLOGY/HISTOPATHOLOGY
Formalin-fixed rats:
No changes, gross or microscopic, were evident which could be attributed to a systemic toxic effect of the test substance. The most common
spontaneous gross necropsy findings, occurring across all groups, were inflammations around the ear tags, and slight hair loss on the extremities. No unusual microscopic pathological findings were evident which could be attributed to the topical administration of the test substance.

NEUROPATHOLOGY
Glutaraldehyde-perfused rats:
Histopathological examinations of hematoxylin and eosin and Luxol-fast Blue stained slides of brain, spinal cord and sciatic nerve from ten rats
(5/sex) treated with the high dose of the test substance failed to reveal any treatment-related lesions when these tissues were compared to similar
ones from ten control rats (5/sex). In addition, microscopic examinetion of 50 teased nerve fibers from the tibial nerve of ten high-dose animals
(5/sex) were canparable to those of the controls. When quantitative measurements were taken of myelinated nerve tibers in cross-section of the distal sciatic nerve, a slight shift to larger diameters could be detected in high-dose males when compared to the controls. However, there was also a slight decrease in fiber diameters in treated females. The relatively large standard deviation in data from both males and females suggest that these slight changes in fiber diameters are not significant. Moreover, the absence of lesions by more conventional histopathological examinations substantiate this conclusion.
Dose descriptor:
NOAEL
Remarks:
skin irritation
Effect level:
20 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: skin irritation
Dose descriptor:
NOAEL
Remarks:
for systemic toxicity
Effect level:
66.66 mg/kg bw/day
Sex:
male
Basis for effect level:
other: significantly reduced body weights in high dose males
Dose descriptor:
NOAEL
Remarks:
for systemic toxicity
Effect level:
200 mg/kg bw/day
Sex:
female
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
200 µg/cm²
Study duration:
subchronic
Species:
rat
Quality of whole database:
Assuming an average b.w. of 200g and an exposed area of 20cm²

Additional information

Dermal:

There are valid data available to assess the toxicity of tripropylene glycol diacrylate after repeated dose administration.

 

The subchronic dermal application of the test material tripropylene glycol diacrylate (TPGDA) to the backs of Sprague-Dawley rats was studied to assess potential neurotoxic and other local and systemic effects (Bio/Dynamics Inc. 1982, Val. 2). Ten male and female Sprague-Dawley rats per dose were exposed on their backs to doses of 0, 20, 66.66, 200 mg/kg bw/d, 5 days a week for 90 days. The animals were observed for mortality and gross signs of toxicologic or pharmacologic effects, and the body weight was determined weekly. Blood was obtained, hematology, clinical chemistry and urinalysis were conducted. Neurologic functions were evaluated monthly (posture, gait, muscular tone, corneal reflexes, righting and toe-pinch). One-half of the animals were sacrificed by exsanguination under light ether anesthesia, and selected organs and tissues were fixed in formalin. Organ and organ-body weight ratios (adrenals, brain, liver, kidney, heart, spleen, testes with epididymides, ovaries) were determined on all of these animals only. Histopathological evaluations of 10 organs or tissues (liver, kidney, lung, heart, stomach, adrenal, pituitary, testes, ovaries, spleen and skeletal muscle) were conducted on all formalin-fixed control animals and the high dose animals. The remaining animals were perfused intravenously with glutaraldehyde under sodium pentobarbital anesthesia . Quantitative assessments of teased tibial nerve preparations were performed on all glutaraldehyde-perfused animals in control animals and the high dose animals. In addition, brain, spinal cord and sciatic nerve were evaluated microscopically (hematoxylin and eosin and Luxol-fast blue staining) from these same animals.

All animals survived to the scheduled termination of the study; weekly physical examinations of all animals failed to indicate any toxic effects of the test material other than irritation at the application site. Alopecia observed on the forepaws and legs was the most common finding in both sexes; however, the incidence was spread over all test groups, including control. The test substance produced moderate levels of irritation, in a dose-related manner, beginning the first week of the study . Males were generally more susceptible than females to the dermal effects of the test substance throughout the study.

Significantly reduced body weights in high dose male rats (versus control male rats) were noted from weeks 3 through 12, except during weeks 10 and 11. Male rat weights of the low and mid dose groups were also reduced in dose-related fashion, but the differences were not statistically significant in comparison to control values. No statistically significant effects on weight were seen in treated female rats, however, high dose female animals never exceeded 95% of the mean weights of control females after 2 weeks on test.

Of the clinical chemical parameters which were determined at the study termination, none were affected in a manner suggestive of a treatment-effect. In low dose animals, erythema were only occasionally noted and with decreasing frequency towards the end of the study. Thus this level was considered the no effect concentration for local effects.

None of the hematologic parameters evaluated differed significantly from control values. Protein (100 mg/dl) was confirmed to be present in the urine of one mid-dose male and female, in two high-dose males and one high-dose female. A large amount of occult blood was also present in the urine of this one high-dose female. The specific gravity of this high-dose female and one mid-dose female was also high (>1.090). These findings suggested a possible effect of the test substance on the kidneys, but no alterations were observed in the histopathological examination to support this conclusion. Additionally, without relation to urinary volumes and creatinine, the relevance of in urinary protein concentration is questionable. All in all the singular changes are not considered adverse by the registrant.

There was a significant downward trend in male liver weights; however, this was not evident in the liver/body weight ratio and is therefore of doubtful significance. Other organ weights and organ/body weight ratios were comparable across all groups.

Month 1 neurological function tests showed two high-dose males with slightly reduced corneal response. All other evaluations were normal. Month 2 neurological function tests were unremarkable in control, low and mid dose animals (both sexes). Four high dose males and three high dose females showed slightly abnormal gait described as "stilted". A slight decreased corneal reflex was observed in four males and one female. A moderately decreased toe pinch response (hindtoes only) was also present in one male rat. Neurological function tests showed a slightly stilted gait and altered righting reflex in one male control rat and a slightly relaxed body tone in one mid dose male rat. One male high dose animal continued to exhibit a moderately decreased toe-pinch response (hindtoes only). All neurological observations were normal in both the control and treated female rats at Month 3.

No changes, gross or microscopic, were evident which could be attributed to a systemic toxic effect of the test substance in formalin-fixed rats: The most common spontaneous gross necropsy findings, occurring across all groups, were inflammations around the ear tags, and slight hair loss on the extremities. No unusual microscopic pathological findings were evident which could be attributed to the topical administration of the test substance.

Histopathological examinations of hematoxylin and eosin and Luxol-fast Blue stained slides of brain, spinal cord and sciatic nerve from ten glutaraldehyde-perfused rats (5/sex) treated with the high dose of the test substance failed to reveal any treatment-related lesions when these tissues were compared to similar ones from ten control rats (5/sex). In addition, microscopic examination of 50 teased nerve fibers from the tibial nerve of ten high-dose animals (5/sex) were comparable to those of the controls. When quantitative measurements were taken of myelinated nerve tibers in cross-section of the distal sciatic nerve, a slight shift to larger diameters could be detected in high-dose males when compared to the controls. However, there was also a slight decrease in fiber diameters in treated females. The relatively large standard deviation in data from both males and females suggest that these slight changes in fiber diameters are not significant. Moreover, the absence of lesions by more conventional histopathological examinations substantiate this conclusion.

The NOAEL for systemic effects in males was set here at 66.66 mg/kg bw/d due to reduced body weight only, and for females to 200mg/kg, the highest dose tested. Due to the local irritating effects observed on skin, a LOAEL local was set at 20 mg/kg bw/d for both sexes.

 

Oral:

There is no repeated-dose study by the oral route available for tripropylene glycol diacrylate, but data from the structural analogue 1,6-Hexamethylene Diacrylate can be adopted by read-across. A detailled justification is included in IUCLID chapter 13.

1,6-Hexamethylene Diacrylate (HDDA) was tested in a Combined 28-Day Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test according to OECD TG 422 and in compliance with GLP regulations by WIL Research Laboratories for ReachCentrum (2010). The test substance, in the vehicle corn oil, was administered orally by gavage once daily to 3 groups of Crl:CD(SD) rats, each group consisting of 12 males and 12 females. Dosage levels were 75, 250, and 750 mg/kg bw/day administered at a dosage volume of 5 mL/kg bw. A concurrent control group of 12 rats/sex received the vehicle on a comparable regimen. Males and females were approximately 11 weeks of age at the beginning of test substance administration. Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 4 for a total of 41-49 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating or post‑cohabitation day 25) for a total of 39-52 doses.

All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals.and locomotor activity data were recorded for 6 males/group following approximately 28 days of dose administration and for 6 females/group on lactation day 4. All F0 females were allowed to deliver and rear their pups until lactation day 4. F1 clinical observations and body weights were recorded on PND 1 and 4. Pups were euthanized and discarded on PND 4. Clinical pathology evaluations (haematology, serum chemistry, and urinalysis [males only]) were performed on 6 F0 animals/sex/group at necropsy. F0 males were euthanized following completion of the mating period. F0 females were euthanized on lactation day 5 for females that delivered and post-mating or post-cohabitation day 25 for females that failed to deliver. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0 animals in the control and high-dose groups; the liver, stomach, and gross lesions from all animals in all dosage groups were also examined microscopically.

No incidences of mortality or moribundity were attributed to systemic toxicity of the test substance. In the 750 mg/kg bw/day group, a single female was found dead following dose administration on gestation day 21. However, due to the lack of evidence of test substance-related toxicity in this female, as well as the time of mortality relative to dose administration (11 minutes following dosing), this single mortality was not attributed to systemic toxicity of the test substance. All other animals in all dosage groups survived to the scheduled necropsies. Test substance-related clinical findings were noted in the 250 and 750 mg/kg bw/day group males and females and included wiping mouth on cage floor and/or walls, excessive pawing of cage floor and/or walls, wiping mouth in bedding material following dosing (females only), salivation-related findings, and red material around the mouth. The salivation-related findings were also occasionally noted in the 75 mg/kg bw/day group animals. Because the aforementioned clinical findings were noted at the time of dosing and/or approximately 1 hour following dose administration, they were attributed to the irritative properties of the test substance and not considered adverse.

In the 750 mg/kg bw/day group males, test substance-related lower mean body weight gain and food consumption were noted during the pre-mating period, resulting in mean male body weight that was 6.8% lower than the control group on study day 28. Mean body weights, body weight changes, and food consumption were unaffected by test substance administration in the 75 and 250 mg/kg bw/day group males throughout the study and in the 75, 250, and 750 mg/kg bw/day group females during the pre-mating, gestation, and lactation periods.

No test substance-related effects were noted during the FOB or locomotor activity evaluations at any dosage level.

Test substance administration was associated with micro- to macrovesicular vacuolar change within the liver at 75, 250, and 750 mg/kg bw/day. This vacuolar change was also present within the liver of 3 control group animals (2 males and 1 female). The change in the control group animals and all test substance-treated animals was minimal to mild and there was no evidence of cellular or tissue damage; therefore, the change was not considered to be an adverse effect. At 750 mg/kg bw/day, higher liver weights, higher serum bile acid values, and higher urea nitrogen values were noted in both males and females, a higher total bilirubin value was noted in males, and higher ALT, cholesterol, triglycerides, calcium, and phosphorous values were noted in females. At 250 mg/kg bw/day, a higher ALT value was noted for females; however, this difference was not statistically significant and was not considered to be an adverse effect. Test substance administration at dosage levels of 250 and 750 mg/kg bw/day to males and females was associated with squamous epithelial hyperplasia and hyperkeratosis in the non‑glandular stomach. This was a manifestation of local irritation rather than a systemic effect; therefore, it was not considered to be systemically adverse.

Based on reduced mean body weights and body weight gains in the 750 mg/kg bw/day group males and adverse changes in serum chemistry parameters associated with increased liver weights in the 750 mg/kg bw/day group males and females, the NOAEL for systemic toxicity was considered to be 250 mg/kg bw/day.

  

Inhalation:

No data available


Justification for classification or non-classification

Based on the available data for repeated dose toxicity no classification according to CLP/GHS is warranted.