Reaction mass of Phenol, dodecyl-, sulfurized, calcium salts and benzene and butene and calcium carbonate and calcium dihydroxide and sulphur trioxide

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: A GLP study conducted in accordance with OECD test guideline.

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)

Principles of method if other than guideline:

Recovery and Endocrine Assessments were included in the study design.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Each rat was uniquely identified by a Monel® metal ear tag displaying the animal number.

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: approximately 12 weeks old
- Weight at study initiation: (P) Males: 363 g to 478 g; Females: 221 g to 285 g
- Housing: Until pairing, all F0 animals were housed individually in clean, stainless steel wire mesh cages suspended above cage board. The 5 rats/sex assigned to the post-treatment phase were not paired and remained in clean, stainless steel wire mesh cages until euthanasia. The breeding phase rats (10/sex) were paired for mating in the home cage of the male. Following positive evidence of mating, the males were housed in suspended wire mesh cages until the scheduled necropsy, and the females were transferred to plastic maternity cages with nesting material, ground corncob bedding. Females with no evidence of mating or that failed to deliver were housed in plastic maternity cages until post-cohabitation or post-mating day 25.
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum except during the period of fasting prior to clinical pathology blood collection
- Water: Reverse osmosis purified (on site) drinking water, delivered by an automatic watering system ad libitum
- Acclimation period: 16 days prior to the first day of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature: 70.9 to 71.8 deg F (21.6 to 22.1 deg C)
- Humidity: 49.9 to 65.4%
- Air changes: 10 changes/hr
- Photoperiod: Fluorescent lighting provided illumination for a 12 hour light (0600 hours to 1800 hours)/12 hour dark photoperiod

IN-LIFE DATES: From: 15 June 2010 To: 23 August 2010

Route of administration:

oral: gavage

Vehicle:

other: mineral oil, USP

Details on oral exposure:

The vehicle and test item formulations were administered orally by gavage, via an appropriately sized flexible teflon®-shafted, ball-tipped dosing cannula (Natume, Japan) once daily.

PREPARATION OF DOSING SOLUTIONS: The vehicle was dispensed approximately weekly for administration to the control group (Group 1) and for preparation of the test item formulations; aliquots were prepared for daily dispensation to the control group. The vehicle was mixed throughout preparation, sampling, and dose administration procedures, and was warmed to 60°C for a minimum of 30 minutes prior to daily dispensation. The test item formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature. The test item formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures, and were warmed to 60°C for a minimum of 30 minutes prior to daily dispensation.

VEHICLE
The vehicle used in preparation of the test item formulations and for administration to the control group was mineral oil, USP.
- Concentration in vehicle: 0, 6, 20, or 200 mg/ml (corrected for % active ingredient)
- Dosage volume: 5 ml/kg
- Lot nos.: ZP0867 and YH1095, exp. dates: 15 July 2010 and 23 October 2010, respectively.
- Supplier: Spectrum Chemical Manufacturing Corporation, New Brunswick, NJ

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for homogeneity and concentration analyses were collected from the middle stratum of the control formulation and from the top, middle, and bottom strata of each test item formulation used for dose administration. One set of samples from the first, third, fifth, and last batches was subjected to the appropriate analyses. Analyses were conducted by the Analytical Chemistry Department at WIL Research using a validated high performance liquid chromatography method with ultraviolet absorbance detection.

The analyzed dosing formulations were within WIL Research’s SOP range for suspensions (85% to 115%), were homogeneous, and were stable for 10 days at room temperature.

Duration of treatment / exposure:

Males: Days 0-27 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 doses.
Females: Days 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 3) for a total of 39-44 doses.
Females: with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 25) for a total of 40-52 doses.

Frequency of treatment:

Once daily, at approximately the same time each day.

Remarks:

Doses / Concentrations:
0, 30, 100, or 1000 mg/kg/day administered at a dosage volume of 5 mL/kg.
Basis:
other: dosage formulations were corrected for % active ingredient (correction made to account for the solvent oil content within the test material)

No. of animals per sex per dose:

The low- and mid-dose groups: 10 rats/sex
control and high-dose groups: 15 rats/sex (5 animals were provided a 14-day recovery period)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosage levels were selected based on the result of previous studies and were provided by the Sponsor Representative after consultation with the Study Director.
- Rationale for animal assignment (if not random): At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements was selected for use in the computerized randomization procedure. At that time, the individual body weights and corresponding animal identification numbers were entered into WTDMS™. A printout containing the animal numbers, corresponding body weights, and individual group assignments was generated using a computer randomization procedure. The animals then were arranged into groups according to the printout. Animals not assigned to study were euthanized by carbon dioxide inhalation and discarded.
- Experimental design: Consisted of 3 test item treated groups and 1 control group composed of 10 rats/sex/group with an additional 5 rats/sex in the control and high-dose groups that were evaluated in a 14-day non-dosing post-treatment period.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Detailed physical examinations were recorded 1 week prior to initiation of test item administration and weekly thereafter (prior to test item administration during the treatment period). Each male and female was also observed for signs of toxicity approximately 1-2 hours following dose administration. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.

BODY WEIGHT:
- Male: Individual male body weights were recorded weekly, beginning 1 week prior to test item administration, on the first day of dosing, and weekly thereafter until scheduled euthanasia.
- Female: Individual female body weights were recorded weekly until evidence of copulation was observed for females selected for pairing. Weekly body weights continued to be recorded for females without evidence of mating and for those females that were not selected for pairing. 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, and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Individual food consumption was recorded on the corresponding weekly body weight days until pairing. 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 females with no evidence of mating was measured on a weekly basis until the scheduled euthanasia.

FOB ASSESSMENTS: FOB assessments were recorded for 5 animals/sex/group prior to dose administration on study day 27 (males selected for pairing) or on lactation day 4 (females). FOB testing was performed without knowledge of the animal’s group assignment. All animals were observed for the following parameters:
- Home Cage Observations: Posture, Convulsions/Tremors, Feces consistency, Biting, Palpebral (eyelid) closure.
- 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.
- Open Field Observations (2-min observation period): Mobility, Rearing, Convulsions/Tremors, Grooming, Bizarre/Stereotypic behavior, Time to first step (seconds), Gait, Arousal, Urination/Defecation, Gait score, Backing.
- Sensory Observations: Approach response, Startle response, Pupil response, Forelimb extension, Air righting reflex, Touch response, Tail pinch response, Eyeblink response, Hindlimb extension, Olfactory orientation.
- Neuromuscular Observations: Hindlimb extensor strength, Hindlimb foot splay, Grip strength hind and forelimb, Rotarod performance.
- Physiological Observations: Catalepsy, Body temperature, Body weight.

LOCOMOTOR ACTIVITY: Locomotor activity counts were recorded for 5 animals/sex/group prior to dose administration on study day 27 (males selected for pairing) and on lactation day 4 (females); the same animals evaluated for FOB were selected for locomotor activity assessment. Locomotor activity, recorded after the completion of the FOB, was measured automatically using a personal computer controlled system utilizes a series of infrared photobeams surrounding a clear plastic, rectangular cage to quantify each animal’s motor activity. Four-sided black plastic enclosures were used to surround the clear plastic boxes to decrease the potential for distraction from extraneous environmental stimuli or stimuli from biologists or adjacent animals. black enclosures rested on top of the photobeam frame and did not interfere with the path of the beams. The testing of treatment groups was done according to replicate sequence. Each animal was tested separately. 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).

CLINICAL PATHOLOGY: Blood samples for clinical pathology evaluations (hematology and serum chemistry) were collected from 5 animals/sex/group at the scheduled necropsies (study day 28 for males and lactation day 4 for females that were selected for pairing) and from 5 animals/sex in the control and high-dose groups following a 14-day non-dosing post-treatment period (study day 42 for males and study day 53 for females). The same animals selected for FOB and motor activity assessments were selected for clinical pathology assessment. All males (including those not selected for clinical pathology assessments) and the post treatment phase females were fasted overnight prior to blood collection with water available ad libitum. Blood for serum chemistry and hematology was collected from the retro orbital sinus following isoflurane anesthesia. Blood for coagulation parameters was collected from the vena cava at the time of necropsy. Blood was collected into tubes containing EDTA (hematology), sodium citrate (clotting determinations), or no anticoagulant (serum chemistry). The following parameters were evaluated:
- Hematology: Total leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Prothrombin time, Activated partial thromboplastin time, Reticulocyte count Percent / Absolute, Mean Platelet Volume, Red cell distribution width, Hemoglobin Distribution Width, Differential leukocyte count, Platelet estimate, Red cell morphology.
- Clinical Chemistry: Albumin, Total protein, Globulin [by calculation], Albumin/globulin ratio [by calculation], Total bilirubin, Urea nitrogen, Creatinine, Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma glutamyltransferase, Glucose, Total cholesterol, Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides, Bile acids.

Sacrifice and pathology:

SACRIFICE
-All F0 adults were euthanized by carbon dioxide inhalation. Males selected for pairing were euthanized following completion of the mating period. Males and females not selected for pairing were euthanized following the 14-day non-dosing post-treatment period. Females that delivered were euthanized on lactation day 4. the number 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). Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss.

GROSS NECROPSY
- Necropsy included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. The following tissues and organs were placed in 10% neutral buffered formalin (except as noted): Adrenal glands, Aorta, Bone with marrow (sternebrae), Bone marrow smear(a), Brain (Cerebrum level, Cerebrum level, Cerebellum with medulla/pons), Coagulating gland, Eyes with optic nerve(b), Gastrointestinal tract, Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys, Liver (sections of 2 lobes), Lungs (including bronchi, fixed by inflation with fixative) Lymph nodes (Axillary, Mesenteric, Mandibular), Ovaries and oviducts(c), Pancreas, Peripheral nerve (sciatic), Pituitary gland, Prostate gland, Mandibular salivary glands, Seminal vesicles, Skeletal muscle (rectus femoris), Skin with mammary gland(d), Spinal cord (cervical), Spleen, Testes with epididymides(e), Thymus gland, Thyroids [with parathyroids, if present], Trachea, Urinary bladder, Uterus (f) with cervix and vagina, All gross lesions. (a) not placed in formalin or examined; (b)Placed in Davidson’s solution; (c) Ovaries were fixed in 10% neutral-buffered formalin for approximately 48 hours before being transferred to 70% ethanol; (d) Collected from females; a corresponding section of skin was collected from the same anatomic area from males; (e) Testes and epididymides were fixed in Bouin’s solution; (f) Any uterus that was placed in 10% ammonium sulfide solution for detection of implantation sites was discarded and not preserved in 10% neutral-buffered formalin.

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

HISTOPATHOLOGY: Microscopic examination was performed on all tissues from all animals in the control and 1000 mg/kg/day groups and on gross lesions from animals in all groups at the scheduled treatment phase necropsies. The liver was identified as a target tissue and was also examined for the low- and mid-dose groups at the treatment phase necropsy and the control and high-dose groups at the recovery necropsy.

Statistics:

Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Data obtained from nongravid females were excluded from statistical analyses following the mating period. Statistical tests were performed using WTDMS™ or were conducted by BIOSTAT CONSULTANTS, INC.using SAS version 9.1 (SAS Institute, Inc., 2002 2003), or higher, software.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

clinical findings observed but no mortality

Mortality:

mortality observed, treatment-related

Description (incidence):

clinical findings observed but no mortality

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

(food consumption)

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

(minimal to mild liver changes not considered adverse)

Details on results:

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
All animals in the control, 30, 100, and 1000 mg/kg/day groups survived to the scheduled necropsies. At the weekly detailed physical examinations during the treatment period, soft stool and brown material around the anogenital area were observed for both males and females in the 1000 mg/kg/day group beginning as early as study day 4. At approximately 1-2 hours post-dosing, there was a higher incidence of soft stool for males and females in the 1000 mg/kg/day group when compared with the control group beginning on study day 7; yellow material around the anogenital area was also observed for the 1000 mg/kg/day group males beginning on study day 14. At the time of dose administration, salivation was observed for 5 of 15 females in the 1000 mg/kg/day group; this clinical finding is not considered adverse. Soft stool and yellow and brown material findings noted at 1000 mg/kg/day did not persist into the post-treatment period.
Other clinical findings observed in the test item-treated groups were observed in single animals, similarly in the control group, and/or in a manner that was not dose-dependent and were therefore not considered test item-related. Summary data - see Tables S1-S6 attached.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males: Mean body weights and body weight gains for males in the 100, 300, and 1000 mg/kg/day groups were comparable to the control group throughout the treatment and post-treatment periods. Summary data- see Tables S7-S10 attached.

Females: A significantly (p<0.05 or p<0.01) lower mean body weight gain was noted for the 1000 mg/kg/day group females during study days 0-7 and 0-13 (entire pre-mating treatment period). These decrements were due in part to 2 females in this group that lost weight (15 g or 16 g) during study days 0-7. During study days 7-13 (treatment period) and throughout the post-treatment period, mean body weight gains for the 1000 mg/kg/day group were similar to the control group. The effects on mean body weight gain were not of sufficient magnitude to substantially affect mean body weights and were not considered adverse. Mean body weights and body weight gains for the 30 and 100 mg/kg/day groups were comparable to the control group throughout the treatment and post-treatment periods. Mean body weights and body weight gains in the 30, 100, and 1000 mg/kg/day groups were comparable to those in the control group throughout gestation and lactation. Summary data- see Tables S11-S14 attached.

FOOD CONSUMPTION (PARENTAL ANIMALS)
Males: Mean food consumption in the 30, 100, and 1000 mg/kg/day group males was comparable to that in the control group throughout the pre-mating treatment and post treatment periods.
Females: Mean food consumption in the 30, 100, and 1000 mg/kg/day group females was similar to that in the control group throughout the pre-mating treatment and post treatment periods. No statistically significant differences were observed.

FUNCTIONAL OBSERVATIONAL BATTERY
FOB parameters were unaffected by test substance administration at all dosage levels.

LOCOMOTOR ACTIVITY
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).

CLINICAL PATHOLOGY
There were no test item-related alterations in hematology and coagulation parameters. However, some statistically significant (p<0.05 or p<0.01 using Dunnett's test) differences were observed when the control and test item-treated groups were compared. These findings included higher mean absolute basophil counts in teh 100 and 1000 mg/kg/day group females and a lower mean absolute eosinophil and neutrophil counts in the 1000mg/kg/day recovery group males. These group mean differences were not considered to be test item- related because the values appeared to be due to an aberration in control group values (the mean absolute basophil count for the control group females was zero) or occured only in a recovery group (lower mean absolute eosinophil and neutrophil counts in the 1000mg/kg/day group males).

There were no test item-related alterations in serum chemistry parameters.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No test item related internal findings were observed at any dosage level in females that failed to deliver or males and females at the scheduled necropsy during the treatment and post-treatment periods. The mean numbers of unaccounted-for sites and implantation sites in the 30, 100, and 100 mg/kg/day groups were comparable to the control group values.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no test item-related alterations in final body weight or organ weights at the primary or recovery necropsies. The following organ weight differences were statistically significant when compared to the control group but were not considered to be test item-related because they occurred in recovery animals and there were no similar effects in organ weights at the primary necropsy: higher mean heart to body weight ratio and lower mean absolute spleen weights and spleen to brain weight ratio for the 1000 mg/kg/day group males.


HISTOPATHOLOGY (PARENTAL ANIMALS)
In the liver there was an increased incidence of periportal hepatocellular vacuolation in males administered = 30 mg/kg/day and in the 1000 mg/kg/day group females. This was characterized microscopically as periportal hepatocytes containing minimal to mild numbers of mixed small (microvesicular) and large (macrovesicular) vacuoles in the cytoplasm. There was no evidence of accompanying inflammation, degeneration, or cellular reaction involving hepatocellular, endothelial, Kupffer, or biliary cells. The cytoplasmic vacuolation, presumed lipid, was minimal to mild and not associated with inflammatory or cellular degeneration. Serum chemistry assays helpful in assessing liver toxicity, such as alanine and aspartate aminotransferase, gamma glutamyltransferase, bilirubin, albumin, globulin, cholesterol, and tryglycerides levels, showed no test item-related alterations. Bile acid levels were slightly higher in males and females administered the test item, but these elevations were not statistically significant nor did they show a clear dose relationship. Thus, the minimal to mild periportal hepatocellular vacuolation was not considered to be adverse.

Following the 14-day nondosing period, the incidences of periportal hepatocellular vacuolation were reduced in the 1000 mg/kg/day group and/or were comparable to values in the control group. Minimal periportal hepatocellular vacuolation occurred in 2/5 and 2/5 in the 1000 mg/kg/day group males and females, respectively; but also occurred in 3/5 and 2/5 control group males and females, respectively. These data indicated recovery from the hepatocellular vacuolation identified at the primary necropsy with no persistence of the test item-related effect. Summary data- see Tables S77- S79A attached.

Dose descriptor:

NOAEL

Remarks:

Systemic Toxicity

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Clinical findings of soft stool and brown and yellow material around the anogenital area observed at 1000 mg/kg/day.

Critical effects observed:

not specified

Conclusions:

The study was conducted to evaluate the toxic potential of a tested substance EC 903-161-3 when administered oraly to rats for a period of twenty eight consecutive days at dose levels of 30, 100 and 1000 mg/kg/day. Under the condition of this stuyd, the NOAEL for systemic toxicity was considered to be 100 mg/kg/day based on the clinical findings of soft stool and brown and yellow material around the anogenital area observed at 1000 mg/kg/day.

Executive summary:

In a guideline repeated dose and reproduction / developmental toxicity screening study (OECD 422, with recovery and endocrine assessments) conducted according to Good Laboratory Practices, the potential toxic effects of a test substance EC 903 -161 -3 were evaluated when administered to rats. This study was designed to evaluate the potential toxic effects of the test item when administered to rats for 28 days and to evaluate the potential of the test item to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition, and early postnatal development.

The test item, in the vehicle, mineral oil, USP, was administered orally by gavage once daily to 3 groups of Crl:CD(SD) rats. The low- and mid-dose groups each consisted of 10 rats/sex and the high-dose group consisted of 15 rats/sex. Dosage levels were 30, 100, and 1000 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group of 15 rats/sex received the vehicle on a comparable regimen. Ten males/group selected for pairing were dosed for 14 days prior to mating through 1 day prior to euthanasia for a total of 28 doses. Ten females/group selected for pairing were dosed for 14 days prior to mating through lactation day 3 for a total of 39-44 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 40-52 doses. The extra 5 rats/sex in the control and high-dose groups were not used for mating and were treated beginning on study day 0; following 28 doses for the males and 40 doses for the females, these animals were assigned to the post-treatment period and remained on study for a 14-day non-dosing period. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. FOB and locomotor activity data were recorded for 5males/group following approximately 28 days of dose administration and for 5 females/group on lactation day 4. All F0females were allowed to deliver and rear their pups until lactation day 4. Clinical pathology evaluations (hematology and serum chemistry) were performed on 5 F0animals/sex/group at the treatment period and post-treatment period necropsies. F0males were euthanized following completion of the mating period and F0females were euthanized on lactation day 4. Spermatogenic endpoint evaluations were conducted on all males at the treatment and post-treatment period necropsies. Complete necropsies were conducted on all F0animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0animals in the control and high-dose groups that were euthanized at the treatment period necropsy.

All animals survived to the scheduled necropsy. Clinical findings of soft stool and brown and yellow material around the anogenital area were observed for F0animals in the 1000 mg/kg/day group at the weekly detailed physical examinations during the treatment period and/or at approximately 1-2 hours following dose administration. These findings did not persist into the post-treatment period. At the time of dose administration, an increased incidence of salivation was observed for the 1000 mg/kg/day group females when compared with the control group; this finding was not considered adverse. Lower mean body weight gains, in the absence of effects on food consumption, were noted for the 1000 mg/kg/day group females when compared to the control group during study days 0-7, resulting in lower mean body weight gains during study days 0-13; these decrements were not of sufficient magnitude to affect mean body weights. Mean body weights and body weight gains for the 1000 mg/kg/day group males were comparable to the control group throughout the treatment period. During the remainder of the treatment and post-treatment periods (including the gestation and lactation periods for females), mean body weights and body weight gains for the 1000 mg/kg/day group males and females were comparable to the control group. Mean body weights, body weight gains, and food consumption for the 30 and 100 mg/kg/day groups were similar to the control group throughout the study. No test item-related effects were noted during the functional observational battery and locomotor activity assessments. There were no test item-related changes in clinical pathology parameters or gross necropsy observations at any dosage level at the scheduled necropsies during the treatment and post-treatment periods. Mean organ weights in the 30,100, and 1000 mg/kg/day groups were unaffected by test item administration. In the liver, there was an increased incidence of periportal hepatocellular vacuolation in the 30 mg/kg/day group males and 1000 mg/kg/day group males and females at the end of the treatment period. This cytoplasmic vacuolation was minimal to mild and not associated with inflammatory or cellular degeneration and was not considered to be adverse. Following the 14-day non-dosing period, low incidences of periportal hepatocellular vacuolation were observed in the1000 mg/kg/day group, indicating recovery and no persistence of the test article-related effect.

Under the conditions of this screening study, the NOAEL for systemic toxicity was considered to be 100 mg/kg/day based on the clinical findings of soft stool and brown and yellow material around the anogenital area observed at 1000 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Modern GLP study conducted in accordance with OECD test guideline, Klimisch score 1.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity oral

A modern GLP study conducted in accordance with the OECD 422 (Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test ) guideline was designed to evaluate the potential toxic effects of the test item when administered to rats for up to 52 days and to evaluate the potential of the test item to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition, and early postnatal development.

For a period of up to 52 consecutive days the Crl:CD(SD) strain rats were dosed with a test material at dose levels of 30, 100 and 1000 mg/kg/day. Under the conditions of this study, the NOAEL for systemic toxicity was considered to be 100 mg/kg/day based on the clinical findings of soft stool and brown and yellow material around the anogenital area observed at 1000 mg/kg/day.

Repeated dose toxicity inhalation

In accordance with column 2 of REACH Annex XI, section 8.6, it is considered justifiable to omit the acute toxicity: inhalation study. The substance only exists in liquid form and it will not be aerosolized in its normal use pattern and is not anticipated to form small particles or droplets and will not volatilise. As such testing via the inhalation route is not considered appropriate for this substance.

Repeated dose toxicity dermal

In accordance with column 2 of REACH Annex XI, section 8.6, it is considered justifiable to omit the acute toxicity: dermal study.

The subacute (28-day) dermal toxicity study of the test substance is not required, since a study of the subacute (28-day) toxicity of the test substance via the most appropriate route of exposure (oral) is provided.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
A key study for repeat dose toxicity, oral exposure ( WIL, 2011, report number 537012), the study was conducted to the OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test ) and GLP.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
In accordance with Rule 8.6.1 of Annex VIII of the REACH Regulation, a study of the subacute (28-day) inhalation toxicity of the test substance is not required, since a study of the subacute (28- day) toxicity of the test substance via the most appropriate route of exposure (oral ) is provided. Furthermore, the potential for human exposure to the test substance via inhalation is considered negligible.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
In accordance with Rule 8.6.1 of Annex VIII of the REACH Regulation, a study of the subacute (28-day) dermal toxicity of the test substance is not required, since a study of the subacute (28-day) toxicity of the test substance via the most appropriate route of exposure (oral) is provided.

Justification for classification or non-classification

An OECD 422 oral toxicity study in rats identified a NOAEL of 100 mg/kg/day, this was based on adverse clinical effects that were observed in the 1000 mg/kg/day dose group. These effects were limited to soft stool and yellow/brown staining around the anogenital region throughout the dosing period. A reduction in bodyweight gain was also seen in 1000 mg/kg/day females through days 0 -7 in the absence of any change in food consumption, for the remainder of the study body weight and body weight changes were comparable with controls for both males and females. These effects were considered of minimal clinical importance and did not indicate significant toxicity, therefore no classification under CLP is required.

The substance has not been tested for repeat-dose toxicity via dermal or inhalation exposure.