Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Description of key information

Oral:
Rat:(OECD 422): NOAEL (systemic) = 250 mg/kg bw/d (ReachCentrum SPRL, 2010)

Read-across to CAS No. 42978-66-5

Rat: (OECD 422): NOAEL= 375 mg/kg bw/day (ReachCentrum, 2019b)

Dermal:

Read-across to CAS No. 42978-66-5

Rat: subchronic 3 months: NOAEL (systemic) = 66.66 mg/kg bw/d; LOAEL (local) = 20 mg/kg bw/d (Bio/Dynamics Inc., 1992)


Inhalation:
No data available

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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:
03 November 2009 - 02 March 2010 (experimental)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals or test system 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:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Mar - Aug 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliance
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed on August 2019
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: ReachCentrum SA
- Batch number of test material: 180003P040
- Expiration date of the lot/batch: 31. Dec 2018
- Purity/Composition: 100% (UVCB)
- Appearance: clear, colorless liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Stability under storage conditions: until 31. Dec 2018
- Stability of the test substance in the vehicle: Stability for at least 24 hours at room temperature protected from light, stability for at least 8 days in the refrigerator, and stability of 0.5 mL samples for at least 3 weeks in the freezer (≤ -15°C) is confirmed over the concentration range 1 to 200 mg/mL (solutions)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 6 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han), outbred, SPF-Quality
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source.
This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks (males), 13 weeks (females)
- Weight at study initiation: males: 251- 322 g; females: 198- 263 g
- Housing: individually (females during the post-mating phase and lactation phase with the pups) and grouping (pretest, males during the post-amting phase
- Diet: ad libitum; pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum
- Acclimation period: for 5 days prior to start of the pretest period (females) or 5 days before the commencement of dosing (males).

DETAILS OF FOOD AND WATER QUALITY: The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water is performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40-70 (daily mean relative humidity of 36 to 60%)
- Air changes (per hr): 10 or greater
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS
Test item dosing formulations (w/w) were homogenised to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 6 hours after adding the vehicle to the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 8, 25, 75 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed using a validated analytical procedure. Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory.
Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.
Duration of treatment / exposure:
males: 29 days; females: 50-62 days
The duration of treatment covered a 2-week premating period and mating in both sexes as well as entire gestation and the duration of pregnancy and at least 14 days after delivery,
up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 40-53 days.
Frequency of treatment:
daily (7d/week)
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
375 mg/kg bw/day (nominal)
No. of animals per sex per dose:
- in total 10 animals/ sex/ dose
- 5 animals /sex/ dose were selected for functional tests (males only), clinical pathology, collection of full list of organs/tissues at macroscopic examination, organ weights (full list) and histopathology (full list)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 16-day dose range finder with oral gavage administration of the test item.


Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
F0 animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Pups were observed daily for general health/mortality. The number of live and dead pups were determined on PND 1 and daily thereafter.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily (F0 and pubs)

BODY WEIGHT: Yes
- Time schedule for examinations: F0: First day of treatment (prior to dosing) and weekly thereafter (after dosing); F1: Live pups were weighed individually on PND 1, 4, 7 and 13.
- Body Weight Gains: Calculated against the body weight on Day 1 (premating, mating and lactation periods) or Day 0 (postcoitum period).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Time schedule for examinations: quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
- Relative Food Consumption Calculated against the body weight for scheduled intervals.

WATER CONSUMPTION: not quantitative
- Subjective appraisal was maintained during the study

HAEMATOLOGY: Yes, F0 animals
- Time schedule for collection of blood: day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, males only (maximum of 24 hours)
- How many animals: 5/sex/group
- Parameters checked in table 1 were examined.

COAGULATION
Blood plasma of F0 animals was analyzed for Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, males only (maximum of 24 hours)
- How many animals: 5/sex/group
- Parameters checked in table 2 were examined.

OTHER:
Functional observational battery (FOB): Males only
- Functional tests were performed on the selected 5 males (F0) during Week 4 of treatment (after dosing, after completion of clinical observations)
- Examined parameters:
• Hearing ability
• Pubillary reflex
• Static righting reflex
• Fore- and hind-limb grip strength
• Locomotor activity

Estrous cycle:
- Daily vaginal lavage was performed for all females (F0) beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus.

Female reproduction and delivery data
From the mating period onwards, the following parameters were recorded for each female (F0): male number paired with, mating date, confirmation of pregnancy and delivery day.
Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care

Thyroid hormones
- Time schedule for collection of blood: All F0 animals on scheduled necropsy, PND 14-16 and PND 4 for 2 pubs per litter
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: males only ( mximum of 24 h)
- How many animals: All F0 animals, 2 pubs per litter on PND 4 and PND 4-16
- For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no adverse changes in T4 were noted in F0- males, no adverse effects on thyroid histopathology and no treatment related changes in thyroid weight were recorded
- Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16

OTHER:
Sex was externally determined for all pups on PND 1 and 4.
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
All male pups in each litter were examined for the number of areola/nipples on PND 13.


Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 3 and 4, Organ weights)

HISTOPATHOLOGY: Yes (see table 5 for collected tissue)
The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
- Selected animals: Tissues identified in Text Table 5 (except animal identification, aorta, nasopharynx, esophagus, harderian
gland, lacrimal gland, salivary gland, larynx, optic nerve, pancreas, skin and tongue).
- Males that failed to sire (except for males which were selected), females that failed to deliver pups and females with total litter loss: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina.
- Females with total litter loss: Mammary gland.
- Remaining animals: Gross lesions/masses.
Statistics:
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level.
The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation seen after dosing among animals of all dose groups was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Further observed clinical signs affecting the skin/fur (alopecia, scabs), the eye (Chromodacryorrhoea) and breathing (rales) were observed.
Mortality:
no mortality observed
Description (incidence):
One female of the control group (no. 49) was euthanized on Lactation Day 1, as she had a total litter loss (at first litter check she had only dead pups).
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weight gain was increased in males in the second week of treatment after dosing of 125 and 375 mg/kg, and in the fifth week of treatment after dosing with 375 mg/kg. These changes in body weight gain were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment and values were within the historical control range
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The statistically significant change in relative food consumption in 40 mg/kg females during post-coitum Days 17-20 was considered to be unrelated to treatment, since no trend was apparent regarding dose and duration of treatment.
Food efficiency:
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A decreased number of reticulocytes was observed in females at 125 mg/kg, which was considered unrelated to administration of the test item due to absence of a dose-related trend response.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males: The following statistically significant increases were noted for treated males (relative changes in mean values as compared to the concurrent control group are indicated between parentheses):
-increased total protein at 375 mg/kg (6%)
-increased albumin at 375 mg/kg (6%)
-increased calcium at 375 mg/kg (4%)
-increased urea at 40 and 375 mg/kg (27% and 30%, respectively)
The changes in total protein, albumin and calcium were minimal and remained within the historical control range. No dose related trend was observed for the increase in urea and values remained within the historical control range. In addition, a slight increase in sodium was observed at 125 mg/kg (1%), which occurred in the absence of a dose related trend.

No treatment-related changes were noted in clinical chemistry parameters in females
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related higher liver weights (absolute and relative to body weights) were noted in the 375 mg/kg/day group males.
There were no other test item-related organ weight changes.
The significant relative prostate gland weight decrease and liver weight increase in the 40 mg/kg/day treated males was considered incidental and not related to treatment in absence of a dose-related trend.

for details see table 6
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related irregular surface was observed in the (fore)stomach in 2/10 males treated at 125 mg/kg/day and in 10/10 males and 2/10 females treated at 375 mg/kg/day.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Watery fluid in the uterus, found in one control female and 3 females treated with 125 mg/kg, is related to a stage in the estrous cycle and is a normal finding.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the (fore)stomach of males and females and the liver and kidneys of males.

Stomach, squamous cell hyperplasia was present in 2/5 males starting at 125 mg/kg/day up to marked degree and in females at 375 mg/kg/day up to moderate degree. This correlated with the macroscopic irregular surface.
Stomach, ulcer forestomach was present in males starting at 125 mg/kg/day at minimal degree.
Stomach, inflammation forestomach was present in males starting at 125 mg/kg/day up to moderate degree.

Liver, hepatocellular hypertrophy was present in males treated at 375 mg/kg/day at minimal degree. This correlated with the increased liver weight. Due to the absence of any indicators of cellular degeneration. The changes in the liver were not considered adverse at current severities.

Kidneys, an increased incidence and severity of hyaline droplet accumulation was present in males treated at 375 mg/kg/day up to slight degree. The increased hyaline droplet accumulation in the male kidneys was not accompanied by indicators of tubular damage and therefore this was considered to be nonadverse.

Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
- Functional observation parameters were not considered to be affected by treatment in males up to 375 mg/kg.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Forelimb and hind limb grip strength was similar between control and treated animals. Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
- Coagulation parameters (prothrombin time and activated partial thromboplastin time) of treated rats were considered not to have been affected by treatment.
-Serum levels of T4 in F0 males were increased at 125 and 375 mg/kg (35% and 37% increase in mean values compared to concurrent control, respectively). These values remained within the historical control range and the control value was on the lower limit of this range. In addition, no effect was observed in respect to thyroid weight, therefore, this effect was considered not toxicologically relevant.
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
375 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects up to and including the highest tested dose
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
40 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
no

The various analyses confirmed

- Accuracy: The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the Group 1 formulation.

- Homogeneity: The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Table 6: Mean Percent Liver Weight Differences from Control Groups

Dose level in mg/kg bw/d

Males

Females

40

125

375

40

125

375

LIVER

 

absolute

13

4

31**

-7

-4

1

Relative to bodyweight

9*

5

25**

-6

-1

0

*: P < 0.05, **: P < 0.01

Table 7: Summary Test Item-Realted Microcopic Findings-(fore)stomach

Dose level in mg/kg bw/d

Males

Females

0

40

125

375

0

40

125

375

STOMACHa

5

5

5

10

5

5

5

5

Hyperplasia squamous cell

 

Slight

-

-

-

-

-

-

-

1

Moderate

-

-

2

6

-

-

-

1

Marked

-

-

-

4

-

-

-

-

Ulcer forestomach

 

minimal

-

-

1

2

-

-

-

-

Inflammation forestomach

 

Minimal

-

-

1

6

-

-

-

-

Moderate

-

-

1

-

-

-

-

-

a = Number of tissues examined from each group

Table 8 Summary Test Item-Related Microscopic Findings – Liver and Kidneys – Males

 

Males

Dose level in mg/kg bw/d

0

40

125

375

LIVERa

5

5

5

5

Hepatocellular hypertrophy

 

minimal

-

-

-

4

KIDNEYa

5

5

5

5

Hyaline droplet accumulation

 

minimal

1

1

2

3

slight

-

-

-

2

a= Number of tissues examined from each group

Conclusions:
Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following no-observed-adverse-effect level (NOAEL) were established:
Parental local NOAEL: 40 mg/kg (based on findings in the (fore)stomach)
Parental systemic NOAEL: at least 375 mg/kg due to the absence of adverse toxicity in the study for both sexes.
Executive summary:

Wistar Han rats were treated with the test item by daily oral gavage at dose levels of 40, 125 and 375 mg/kg according to OECD 422 and in compliance with GLP. The rats of the control group received the vehicle, corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-62 days). Females that failed to deliver pups were treated for 40-53 days.

Parental toxicity was observed in the (fore)stomach of males from 125 mg/kg and in females at 375 mg/kg.

These changes consisted of ulcers and inflammation of the forestomach in males and hyperplasia squamous cells in males and females.

Other treatment-related but non-adverse changes were observed in the liver at microscopic examination. An absolute increase of 31% and a relative increase of 25% in liver weight was observed at dose 375 mg/kg. At microscopic examination, hepatocellular hypertrophy in the liver was observed at minimal severity and was in the absence of any indicators of cellular degeneration. The changes in the liver were not considered adverse at current severities. In the kidneys an increase in hyaline droplet accumulation was recorded in males which was considered to likely represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules (Alden et al., 1991). This male rat specific protein is not present in female rats nor in higher mammals, including man (Sahota et al., 2013). The increased hyaline droplet accumulation in the male kidneys at 375 mg/kg/day was not accompanied by indicators of tubular damage and therefore this was considered to be nonadverse.

Functional observations were not performed for females and therefore, possible treatment related effects on the functional parameters could not be evaluated.

No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations (males), body weight, food consumption, clinical laboratory investigations (including male T4 thyroid hormone levels), macroscopic examination and organ weights).

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, no systemic no-observed-adverse-effect level (NOAEL) were established under the conditions of this study. The Parental local NOAEL of 40 mg/kg is based on the findings in the (fore)stomach. Due to the absence of systemic toxicity up to and including the highest tested dose of 375 mg/kg bw/d no systemic NOAEL could be derived.

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
Study period:
Jun - Sep 1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD Guideline 424 (Neurotoxicity Study in Rodents)
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
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): C-178
- Physical state: liquid
- Analytical purity: 100% active ingredient
- Storage condition of test material: room temperature
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system 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
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
66.666 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
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.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
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.
- 20 mg/kg/day: erythema noted occassionally during the final 2 months; more frequently in the initial 3 weeks; exfoliation observed in approximately one-half during weeks 2 and 3 with diminishing frequency
- 66.6 mg/kg/day: erythema noted with a somewhat higher frequency than 20 mg/kg/day, frequency in females comparable to control. Exfoliation and eschar recorded for most animals by week 3.
- 200 mg/kg/day: erythema, exfoliation, and eschar seen in most animals of both sexes beginning in week 1. Atonia was observed in one males and one female, fissures present in one female and four males. A persistant fissuring was observed in one male rat from week 2 through week 7.
Males appeared somewhat more sensitive than females to erythema and eschar formation.
Mortality:
no mortality observed
Description (incidence):
All animals survived to the scheduled termination of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
None of the hematologic parameters evaluated differed significantly from control values.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Of the clinical chemical parameters which were determined at the study termination, none were affected in a manner suggestive of a treatment-effect.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Protein (2+, 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.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
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.
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.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
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 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 fibers 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.
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
LOAEL
Remarks:
skin irritation
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
dermal irritation
Dose descriptor:
NOAEL
Remarks:
for systemic toxicity
Effect level:
66.66 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Remarks:
for systemic toxicity
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects observed
Critical effects observed:
yes
Lowest effective dose / conc.:
20 mg/kg bw/day (nominal)
System:
integumentary
Organ:
skin
Treatment related:
yes
Dose response relationship:
yes
Conclusions:
The test substance did not induce neurotoxicity and no sub-chronic toxicity other than dermal irritation and decreased body weight was observed in animals, under the conditions tested. Thus, the NOAEL for local effects was considered to be 20 mg/kg/day and the NOAEL for systemic effects (based on body weight) was concluded to be 66.6 mg/kg/day.
Executive summary:

The sub-chronic dermal application of the test substance to the backs of Sprague-Dawley rats was studied to assess potential neurotoxic and other local and systemic toxic effects. Three groups of 20 rats each (10/sex/level) were treated topically with 20, 66 2/3, and 200 mg/kg/day 5 days per week for 3 months. Twenty control animals (10/sex) were treated with corn oil. Solutions at appropriate levels were prepared in corn oil and a constant dose volume (2.077 ml/kg) was applied to all animals. Treatment sites were not occluded. Dermal observations were performed pretest and 5 times/week throughout the study. Clinical laboratory studies were performed at termination. Neurological function evaluations were performed at months 1, 2, and 3. At three months, all animals were terminated. 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 were determined on all of these animals only. Histopathological evaluations of 10 organs or tissues were conducted on all formalin-fixed Group 1 and 4 animals. The remaining animals were perfused intravenously with glutaraldehyde under sodium pentobarbital aesthesia. Quantitative assessments of teased tibial nerve preparations were performed on all glutaraldehyde-perfused animals in Group 1 and 4. In addition, brain, spinal cord and sciatic nerve were evaluated microscopically (hematoxylin and eosin and Luxol-fast Blue staining) from these same animals. Erythema, eschar and exfoliation were recorded during the initial week of the study and maximum frequency of these effects were seen during Weeks 2 and 3. A dose-response was noted, with males being slightly more susceptible. In the last 2 months of the study most treated animals developed an apparent tolerance to the irritant effects of the test substance. Significantly lower body weights were recorded throughout the study for Group IV males (200 mg/kg/day). Body weights for female rats and Group II and III male rats were not significantly affected. Routine toxicologic and pharmacologic signs were unremarkable throughout the study. Month 2 neurologic evaluations showed an effect on gait in 4 of 10 male and 3 of 10 female Group IV rats. Reduced corneal reflex was also seen in some rats in this group; however, Month 3 evaluations failed to show these effects. Hematological and clinical chemistry parameters appeared unaffected by treatment with the test substance. However, there was a dose-related increase in urinary protein values in both sexes. Organ weights, gross necropsy observations and microscopic studies did not reveal any systemic toxic effects. Using tibial nerve teasing techniques, no morphometric differences were found between Group land Group IV glutaraldehyde perfused rats. Quantitative assessment of tibial nerve fiber cross-sections showed slightly increased diameters for males and slightly decreased diameters for females. These changes were not considered to be treatment-related.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
66.66 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
Study period:
Jun - Sep 1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD Guideline 424 (Neurotoxicity Study in Rodents)
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
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): C-178
- Physical state: liquid
- Analytical purity: 100% active ingredient
- Storage condition of test material: room temperature
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system 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
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
66.666 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
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.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
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.
- 20 mg/kg/day: erythema noted occassionally during the final 2 months; more frequently in the initial 3 weeks; exfoliation observed in approximately one-half during weeks 2 and 3 with diminishing frequency
- 66.6 mg/kg/day: erythema noted with a somewhat higher frequency than 20 mg/kg/day, frequency in females comparable to control. Exfoliation and eschar recorded for most animals by week 3.
- 200 mg/kg/day: erythema, exfoliation, and eschar seen in most animals of both sexes beginning in week 1. Atonia was observed in one males and one female, fissures present in one female and four males. A persistant fissuring was observed in one male rat from week 2 through week 7.
Males appeared somewhat more sensitive than females to erythema and eschar formation.
Mortality:
no mortality observed
Description (incidence):
All animals survived to the scheduled termination of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
None of the hematologic parameters evaluated differed significantly from control values.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Of the clinical chemical parameters which were determined at the study termination, none were affected in a manner suggestive of a treatment-effect.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Protein (2+, 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.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
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.
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.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
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 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 fibers 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.
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
LOAEL
Remarks:
skin irritation
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
dermal irritation
Dose descriptor:
NOAEL
Remarks:
for systemic toxicity
Effect level:
66.66 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Remarks:
for systemic toxicity
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects observed
Critical effects observed:
yes
Lowest effective dose / conc.:
20 mg/kg bw/day (nominal)
System:
integumentary
Organ:
skin
Treatment related:
yes
Dose response relationship:
yes
Conclusions:
The test substance did not induce neurotoxicity and no sub-chronic toxicity other than dermal irritation and decreased body weight was observed in animals, under the conditions tested. Thus, the NOAEL for local effects was considered to be 20 mg/kg/day and the NOAEL for systemic effects (based on body weight) was concluded to be 66.6 mg/kg/day.
Executive summary:

The sub-chronic dermal application of the test substance to the backs of Sprague-Dawley rats was studied to assess potential neurotoxic and other local and systemic toxic effects. Three groups of 20 rats each (10/sex/level) were treated topically with 20, 66 2/3, and 200 mg/kg/day 5 days per week for 3 months. Twenty control animals (10/sex) were treated with corn oil. Solutions at appropriate levels were prepared in corn oil and a constant dose volume (2.077 ml/kg) was applied to all animals. Treatment sites were not occluded. Dermal observations were performed pretest and 5 times/week throughout the study. Clinical laboratory studies were performed at termination. Neurological function evaluations were performed at months 1, 2, and 3. At three months, all animals were terminated. 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 were determined on all of these animals only. Histopathological evaluations of 10 organs or tissues were conducted on all formalin-fixed Group 1 and 4 animals. The remaining animals were perfused intravenously with glutaraldehyde under sodium pentobarbital aesthesia. Quantitative assessments of teased tibial nerve preparations were performed on all glutaraldehyde-perfused animals in Group 1 and 4. In addition, brain, spinal cord and sciatic nerve were evaluated microscopically (hematoxylin and eosin and Luxol-fast Blue staining) from these same animals. Erythema, eschar and exfoliation were recorded during the initial week of the study and maximum frequency of these effects were seen during Weeks 2 and 3. A dose-response was noted, with males being slightly more susceptible. In the last 2 months of the study most treated animals developed an apparent tolerance to the irritant effects of the test substance. Significantly lower body weights were recorded throughout the study for Group IV males (200 mg/kg/day). Body weights for female rats and Group II and III male rats were not significantly affected. Routine toxicologic and pharmacologic signs were unremarkable throughout the study. Month 2 neurologic evaluations showed an effect on gait in 4 of 10 male and 3 of 10 female Group IV rats. Reduced corneal reflex was also seen in some rats in this group; however, Month 3 evaluations failed to show these effects. Hematological and clinical chemistry parameters appeared unaffected by treatment with the test substance. However, there was a dose-related increase in urinary protein values in both sexes. Organ weights, gross necropsy observations and microscopic studies did not reveal any systemic toxic effects. Using tibial nerve teasing techniques, no morphometric differences were found between Group land Group IV glutaraldehyde perfused rats. Quantitative assessment of tibial nerve fiber cross-sections showed slightly increased diameters for males and slightly decreased diameters for females. These changes were not considered to be treatment-related.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
0.11 mg/cm²
Study duration:
subchronic
Species:
rat
Quality of whole database:
converted from LOAEL of 20 mg/kg b.w. based on standard assumptions (10% body area, 250 g average weight, 445 cm² total body surface)

Additional information

There are valid data available for the assessment of repeated dose toxicity with 1,6-hexanediol diacrylate and the read-across substance tripropylene glycol diacrylate (Cas No. 42978-66-5).

Oral:

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. 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 ReachCentrum, 2010).

 

Available data for tripropylene glycol diacrylate (TPGDA, Cas No. 42978-66-5):

Wistar Han rats were treated with the test item by daily oral gavage at dose levels of 40, 125 and 375 mg/kg according to OECD 422 and in compliance with GLP. The rats of the control group received the vehicle, corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-62 days). Females that failed to deliver pups were treated for 40-53 days. Parental toxicity was observed in the (fore)stomach of males from 125 mg/kg and in females at 375 mg/kg. These changes consisted of ulcers and inflammation of the forestomach in males and hyperplasia squamous cells in males and females. Other treatment-related but non-adverse changes were observed in the liver at microscopic examination. An absolute increase of 31% and a relative increase of 25% in liver weight was observed at dose 375 mg/kg. At microscopic examination, hepatocellular hypertrophy in the liver was observed at minimal severity and was in the absence of any indicators of cellular degeneration. The changes in the liver were not considered adverse at current severities. In the kidneys an increase in hyaline droplet accumulation was recorded in males which was considered to likely represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules (Alden et al., 1991). This male rat specific protein is not present in female rats nor in higher mammals, including man (Sahota et al., 2013). The increased hyaline droplet accumulation in the male kidneys at 375 mg/kg/day was not accompanied by indicators of tubular damage and therefore this was considered to be non-adverse. Functional observations were not performed for females and therefore, possible treatment related effects on the functional parameters could not be evaluated. No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations (males), body weight, food consumption, clinical laboratory investigations (including male T4 thyroid hormone levels), macroscopic examination and organ weights). Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, no systemic no-observed-adverse-effect level (NOAEL) were established under the conditions of this study. The Parental local NOAEL of 40 mg/kg is based on the findings in the (fore)stomach. Due to the absence of systemic toxicity up to and including the highest tested dose of 375 mg/kg bw/d no systemic NOAEL could be derived (ReachCentrum, 2019b).

Dermal:

There are only limited data available to assess the toxicity of 1,6 -hexanedioldiacrylate after repeated dermal administration. In two available studies, no systemic effects were observed in rabbits (up to 300mg/kg for 2 weeks), and in mice. Severe irritation occured in both studies. Since the focus was on dermal effects, only a limited number of additional parameters to assess systemic toxicity have been evaluated. As a consequence, it can be deduced that irritation is the leading effect after dermal exposure, but systemic effects can not be fully evaluated based on these reports (Bio/Dynamics Inc., 1979; University of Cincinnati, 1982).

Available data for tripropylene glycol diacrylate (TPGDA, Cas No. 42978-66-5:

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. 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. The test substance produced moderate levels of irritation in all dose groups 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 and hematologic parameters which were determined at the study termination, none were affected in a manner suggestive of a treatment-effect. 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. However, histopathology was comparable to controls. No information on urine volume and creatinine concentration is provided, thus the relevance of singular changes in concentration in the absence of histopathological damage is questionable. 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. Test substance administration did not alter the performance in the neurological tests conducted after 1, 2, and 3 months. 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. 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. The NOAEL for systemic effects was set at 66.66 mg/kg bw/d due to reduced body weight in male rats. Due to the local irritating effects on the skin, a LOAEL local was set at 20 mg/kg bw/d (Bio/Dynamicx Inc., 1982).

 

Inhalation:

No data available

Justification for classification or non-classification

Based on the available data for repeated dose toxicity after subacute oral (gavage) and subchronic dermal administration, the test item is no subject to classification and labelling according to Regulation (EC) No 1272/2008 (CLP).

There are currently no data available which would justify a classification of 1,6-hexanedioldiacrylate for its repeated inhalative toxicity.