Registration Dossier

Registration Dossier

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Description of key information

The structural analogue, Hordaphos CC MIS, was evaluated for possible adverse effects following repeated oral dosing for relatively limited period of time and to evaluate effects of test item on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus, parturition, and early neonatal development according to OECD 422.

The animals did not reveal any clinical signs of toxicity throughout the treatment and recovery period. No mortality or morbidity was observed throughout the experimental period. No treatment related changes in body weight, percent change in body weight, feed consumption, ophthalmoscopic examination, neurological/functional examination, haematology, clinical chemistry, urine analysis, organ weights (both absolute and relative) were observed in both the sex.

The animals (main group and recovery) did not reveal any treatment related gross as well as histopathological changes on examination.

Based on the results discussed, the No Observed Adverse Effect Level (NOAEL) of the test item Hordaphos CC MIS was found to be 1000 mg/kg body weight when administerd to the main group males for two weeks pre-mating, during mating and up to the day before sacrifice during post-  mating period (total of 35 days), the main group females treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day and the recovery group animals till first scheduled sacrifice of dam(s) (i.e. 41 days) by oral (gavage) to Sprague Dawley rats under the experimental conditions and the doses employed.

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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 July 2015 to 23 October 2015
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

Test item formulations were prepared daily before administration. The required quantity of test item was weighed into a clean glass beaker and there by adding little volume of vehicle into the beaker, mixed well with glass rod and transferred into the measuring cylinder. Again a small quantity of vehicle was added, mixed well with glass rod to rinse the beaker and transferred into the measuring cylinder. The rinsing procedure was repeated to
ensure complete transfer of the test item formulation into a measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get a desired concentration of 10, 30 and 100 mg/mL of test item for low, mid and high dose groups respectively.

VEHICLE

- Justification for use and choice of vehicle (if other than water): The test item was clearly miscible in distilled water as evidenced by the inhouse miscibility test. Hence, distilled water was selected for the test item formulation preparation.
- Concentration in vehicle: 10, 30 and 100 mg/mL of test item for low, mid and high dose groups respectively.
- Amount of vehicle (if gavage): 10 mL/kg body weight.
- Lot/batch no. (if required): Distilled Water Batch No.: 5108, Expiry Date: 06/2016; Batch No.: 5089, Expiry Date: 07/2016; Batch No.: 5103, Expiry Date: 08/2016; Batch No.: 5193., Expiry Date: 08/2016.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation analysis for dose concentration verification of Hordaphos CC MIS was done by Analytical Chemistry of Bioneeds India Private Limited. The analysis was done as per methods detailed in the Study Plan No. BIO-ANM 116 Sampling and analysis of formulations were performed on first day of treatment and during week 5 of the treatment. The samples were collected in duplicates from each concentration at volume of 5 mL (5 mLx2).
The collected samples were transferred to Analytical Chemistry of Bioneeds India Private Limited for dose concentration analysis. One set of aliquot of each formulation were analyzed. The second aliquots were stored as a back up purpose at room temperature. The second set of samples was discarded, as the analysis results of first set of samples were within the limits. Formulations were considered acceptable, as the mean results are within the range of 90 to 110% of the nominal concentration and the relative standard deviation (% RSD) is equal to or less than 10.0%.
Duration of treatment / exposure:
The test item or vehicle was administered to animals through oral (gavage) route once daily.

The main group males were treated for two weeks premating, during mating and up to the day before sacrifice during postmating period (total of 35 days of treatment).

The main group females were treated for two weeks premating period, during mating, pregnancy (gestation) and up to lactation day 4 after which the pups were sacrificed on lactation Day 4 and females (dams) were sacrificed on lactation day 5 after overnight fasting (water allowed).

The recovery group animals were treated till first scheduled sacrifice of dam(s) and were observed for 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects.

The test item/vehicle was administered by oral (gavage) route using intubation cannula attached to a disposable syringe. All the doses were administered in an equivolume of 10 mL/kg with the concentration of 10, 30 and 100 mg/mL for low, mid and high dose/high dose and recovery groups, respectively. Distilled water was administered to vehicle control/vehicle control recovery group at an equivolume of 10 mL/kg body weight. The actualdose volume for each animal was calculated based on the most recent body weight. The test item formulations were administered as soon as possible after preparation.
Frequency of treatment:
Once daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Main Group: 24 (12 Males + 12 Females) per dose
Recovery Group: 10 (5 males + 5 females) per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses of 0, 100, 300 and 1000 mg/kg body weight for vehicle control/vehicle control recovery, low dose, mid dose and high dose/ high dose recovery groups respectively were selected in consultation with the sponsor based on the results of dose range finding study of test item Hordaphos CC MIS when administered through oral (gavage) for a period of 14 consecutive days to Sprague Dawley Rats (Bioneeds Study No.: BIO-TX 1022).

These doses were selected as the test item Hordaphos CC MIS when administered orally to Sprague Dawley rats once daily for 14 consecutive days did not reveal any treatment related effects at all the tested doses (50, 300 and 800 mg/kg body weight).
Positive control:
Not Applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All the animals were observed once daily for clinical signs of toxicity and twice daily for mortality and morbidity.
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Time schedule: All the animals were subjected to detailed clinical examinations on day 1 before treatment and weekly thereafter during treatment and recovery period.

BODY WEIGHT: Yes
- Time schedule for examinations: The main group and recovery group males and females were weighed on the first day of dosing, weekly thereafter and at termination. The females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and within 24 hours of parturition (day 1 postpartum) and on day 4 postpartum during lactation period.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

OPHTHALMOLOGY:
- Yes, once before treatment for all animals, during end of the dosing period for males (Day 32 - shortly prior to scheduled sacrifice) and during lactation period for females (shortly prior to scheduled sacrifice) for vehicle control and high dose main group animals and during last week for recovery group animals (Day 53). The examination was not extended to lower dose groups as there were no treatment related changes noted in high dose group animals.

FOB:
- Neurological/Functional examination was performed for five males and five females, randomly selected from each group towards the end of the dosing period for males (Day shortly prior to scheduled sacrifice) and during lactation period for females (shortly prior to scheduled sacrifice).
The Neurological/Functional examination was performed for recovery groups during the last week of the recovery period (Day 54).

HAEMATOLOGY:
- Blood samples of both males and females were collected on the day of scheduled terminal sacrifice (for males after completion of 35 days of treatment and for females on lactation day 5).
Analysed parameters: haemoglobin, haematocrit, erythrocyte count, leucocyte count, platelet count, differential leucocytes count, prothrombin time, Activated Partial Thromboplastin Time

CLINICAL CHEMISTRY:
- Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total protein, Albumin, Total bilirubin, Glucose, Total cholesterol, Creatinine, Blood urea nitrogen, Triglycerides, Phosphorous, Calcium, Globulin, Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L)

URINALYSIS:
- Urine was collected from the five randomly selected males of each group during the last week of the treatment period for main groups and during last week of recovery period for recovery groups.
The selected animals were placed in urine collection cages overnight (approximately 12-16 hrs) and not given access to food but water was provided ad libitum during their stay in urine collection cages. The overnight urine volume (mL) collected from these animals was measured.
The volume of urine collected (mL), appearance and the color were recorded by physical evaluation.
Analysed parameters: Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, Albumin, Leucocytes

Sacrifice and pathology:
SACRIFICE
- Male animals: All animals [after completion of 35 days of treatment.]
- Maternal animals: All animals [on lactation day 5]

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY
- Testes, epididymides, liver, kidneys, adrenals, thymus, spleen, brain, heart, ovaries, prostate with seminal vesicles, organs showing macroscoipc lesions, gross lesions, spinal cord, stomach, duodenum, jejunum, ileum with Peyer´s patches, caecum, colon, rectum, thyroid, trachea, lungs, uterus, urinary bladder, mandibular lymph nodes, mesenteric lymph nodes, sciatic nerve, bone marrow

ORGAN WEIGHTS
- Testes, epididymides, liver, kidneys, adrenals, thymus, spleen, brain, heart
Statistics:
The data was subjected to various statistical analyses using SPSS software version 22.

All analyses and comparisons were evaluated at the 95% level of confidence (P<0.05), indicated by the aforementioned tests are designated by the superscripts throughout the report as stated below:

* Statistically significant (P<0.05) change than the vehicle control group.
Note: Data of nonpregnant females and females mated but not littered was excluded from statistical analysis.

The statistical analyses were followed to the parameters:
Body weight, Change in body weight, Feed consumption, Copulatory interval, Gestation length, Hematology, Urinalysis, Clinical chemistry, Absolute organ weights, Relative organ weights were analysed by Oneway ANOVA with Dunnett’s posttest for main groups and t-test for recovery groups (Parametric).

FOB ( Body temperature, Defecation, Urination, Rearing, Grip strength, Movements), Mean litter weights, Mean pup weight, Pre implantation loss, Pre natal loss, Post natal loss,Total No. of resorptions/dam, Total No. of early/late resorptions/dam were analysed by Oneway ANOVA with Dunnett’s posttest (Parametric).

Dams with live young born, with early/late resorptions, Corpora lutea/dam, Implantations/dam, No. of pups/dam, Sex ratio, Litter size, Pup weight were analysed by KruskalWallis followed by the MannWhitney test.

Pregnancy rate, No. of live pups, No. of dead pups, No. of early resorptions, No. of late resorptions, No. of litters with/without dead pups, No. of litterswith/without resorptions were analysed by Chisquare test/ Fischer's Exact Test (Cross Tabs)
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes in mean body weight and percent change in body weight with respect to day 1 at any of the doses tested in both the sex.
However, statistical significant decrease in percent change in body weight with respect to day 1 on day 7 in G2 (females) and G4 (females) and statistical significant decrease in percent change in body weight with respect to day 1 on day 7 in G4R (males) was noted when compared with vehicle control group. These changes are considered incidental due to lack of dose dependency and also the mean body weights were comparable.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes observed in haematology at any of the dose tested in both the sex. However, statistical significant increase in percent neutrophils, and statistical significant decrease in percent lymphocytes in G2 (males), statistical significant decrease in total leucocyte count in G4R (females), statistical significant decrease in prothrombin time in G4R (males), statistical significant decrease in absolute lymphocytes, monocytes, eosinophils and basophils in G4R (females) was noted when compared with vehicle control group. These changes were considered incidental as the values were within historical range or due to random biological variation
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes observed in clinical chemistry at any of the dose tested in both the sex.
However, statistical significant decrease in total protein and calcium levels in G3 (females) and statistical significant decrease in alkaline phosphatase and potassium levels in G4R (females) were noted when compared with vehicle control group. These changes were considered incidental as the values were within historical range or due to random biological variation.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes observed in the absolute and relative organ weights at any of the dose tested in both the sex.
However, statistical significant increase (slight) in relative testes weight in G4 (males) and statistical significant increase in relative brain weight in G2 (females) and G4R (males) were noted when compared with vehicle control group. All the observed changes were considered incidental and not treatment related because of the lack of dose dependency and/or there were no associated macroscopic and microscopic abnormalities noted.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to limit dose
Key result
Critical effects observed:
no
Conclusions:
Based on the results discussed, the No Observed Adverse Effect Level (NOAEL) of the test item Hordaphos CC MIS was found to be 1000 mg/kg body weight when administerd to the main group males for two weeks pre-mating, during mating and up to the day before sacrifice during post- mating period (total of 35 days), the main group females treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day and the recovery group animals till first scheduled sacrifice of dam(s) (i.e. 41 days) by oral (gavage) to Sprague Dawley rats under the experimental conditions and the doses employed.
Executive summary:

The test item, Hordaphos CC MIS, was evaluated for possible adverse effects following repeated oral dosing for relatively limited period of time and to evaluate effects of test item on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus, parturition, and early neonatal development.

A total of 116 (58 males + 58 females) Sprague Dawley rats were distributed to main and recovery groups. Each main group (G1, G2, G3and G4) consisted of 12 males and 12 females and recovery group (G1R and G4R) consisted of 5 males and 5 females. The animals in G1/G1R groups were administered with vehicle [Distilled water], the animals in G2, G3 and G4/G4R groups were administered with test item at the dose levels of 100, 300 and 1000 mg/kg body weight for low dose, mid dose and high dose/high dose recovery groups respectively. The vehicle and test item formulations were administered at the dose volume of 10 mL/kg body weight.

The main group males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 35 days of treatment). The main group females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 4 after which the pups were sacrificed on lactation Day 4 and females (dams) were sacrificed on lactation day 5 after overnight fasting (water allowed).

The recovery group animals were treated till first scheduled sacrifice of dam(s) and observed for 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects.

The test item formulations at the concentrations 10 mg/mL (low dose) and 100 mg/mL (high dose) were stable up to 6 hours at room temperature as per the in-house stability study results (Bioneeds Study No.: BIO-ANM 116). However, freshly prepared test item formulations were administered to the animals.

All animals were observed for clinical signs, mortality and morbidity, detailed clinical examination, body weight and feed consumption. Ophthalmological examination was carried out once before treatment for all animals, duringend of the dosing period for males (shortly prior to scheduled sacrifice) and during lactation period for females (shortly prior to scheduled sacrifice)for vehicle control and high dose main group animals andduring last weekfor recovery group animals. Neurological/Functional examination was performed for five males and five females, randomly selected from each group towards the end of the dosing period for males (shortly prior to scheduled sacrifice) and during lactation period for females (shortly prior to scheduled sacrifice). The Neurological/Functional examination was performed for recovery groups during the last week of the recovery period.

The clinical pathology (haematological and clinical chemistry) examinations were conducted in five males and five females from each main group (randomly selected) and for all recovery group animals. Urinalysis was performed from the five randomly selected males of each group during the last week of the treatment period for main groups and during last week of recovery period for recovery groups.

Each litter was examined after delivery (lactation day 1) and the number and sex of pups (litter size), stillbirths (dead pups born on day 1) and live births were recorded. Body weights of male and female pups were recorded separately on lactation days 1 and 4.

Gross pathology and organ weighing were performed on day 36 for main group males, on lactation day 4 for pups, lactation day 5 for dams and at end of recovery period for recovery group males and females. The number of corpora lutea, implantation sites and resorptions for dams were recorded.

Histopathological examination was conducted on all the tissues collected from the vehicle control and high dose group animals (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure).

The animals did not reveal any clinical signs of toxicity throughout the treatment and recovery period. No mortality or morbidity was observed throughout the experimental period. No treatment related changes in body weight, percent change in body weight, feed consumption, ophthalmoscopic examination, neurological/functional examination, haematology, clinical chemistry, urine analysis, organ weights (both absolute and relative) were observed in both the sex.

The dams did not reveal any treatment related changes in gestation and lactation body weight, percent change in gestation and lactation body weights, feed consumption during gestation period and lactation period, uteri observations, litter and pup weights.

The animals (main group and recovery) did not reveal any treatment related gross as well as histopathological changes on examination.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
reliable without restriction

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: publication with acceptable reporting
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
not specified
Species:
other: rat and mouse
Strain:
other: Fischer 344 rats and CD®-1 mice
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: Fischer 344 rats from Harlan Sprague-Dawley, Inc. (Indianapolis, IN); CD®-1 mice from Charles River Breeding Laboratories, Inc. (Portage, MI).
- Age at study initiation: rats: 8-12 weeks; mice: 8-10 weeks
- Weight at study initiation: male rats: 140-165 g; female rats: 112-130 g; male mice: 25-37 g; female mice: 19-26 g
- Fasting period before study: not reported
- Housing: individually in stainless steel, wire-mesh cages (15 cm x 22 cm x 18 cm for rats; 22.5 cm x 9.5 cm x 12.5 cm for mice); during exposure, animals were individually housed, separated by sex and exposure group, in stainless steel, wire-mesh cages (14.5 cm x 9.5 cm x 17.5 cm or 17.5 cm x 12 cm x 18 cm for rats, and 7.5 cm x 9.5 cm x 17.5 cm for mice).
- Diet (e.g. ad libitum): powdered food (Certified Rodent Chow 5002, Ralston Purina Co., St. Louis, MO) ad libitum
- Water (e.g. ad libitum): water (Municipal Authority of Westmoreland County, Greensburg, PA) ad libitum
- Acclimation period: animals were acclimated to the exposure chambers (air-only exposure) on 2 days prior to the initiation of the exposure regimen


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24 °C
- Humidity (%): 52-56 %
- Air changes (per hr): 13.5 per hr (in inhalation chamber)
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: no vehicle
Details on inhalation exposure:
The volume of each chamber was approximately 1330 liters, and the airflow was approximately 300 liters/minute (13.5 air changes per hour).
Temperature and relative humidity measurements were recorded at least 12 times per exposure.

Animals were acclimated to the exposure chambers (air-only exposure) for 2 days prior to the initiation of the exposure regimen. Target isopropanol vapor concentrations of 0 (control), 100, 500, 1500, and 5000 ppm were selected for this study. The rats (55 days of age) and mice (56 days of age) were exposed for 6 hours per day, 5 days per week for 13 weeks. During the 14th week, male and female rats (excluding those animals designated for neuroanatomic pathology evaluation) received 2 and 3 consecutive days of exposure, respectively. The 10 female rats of the 500, 1500, and 5000 ppm group designated for neuroanatomic pathology evaluation were exposed for 1 day during the 14th week; the male rats of the 500, 1500, and 5000 ppm group designated for neuroanatomic pathology evaluation were not exposed during the 14th week. Male and female mice received 4 and 5 consecutive days of exposure during the 14th week, respectively. Control (air-only exposure) animals were handled in an identical manner as the isopropanol-exposed animals.

Liquid isopropanol was metered from a piston pump (Fluid Metering, Inc., Oyster Bay, NY) into a glass evaporator similar in design to that described by Snellings and Dodd (1990). Piston pumps were equipped with a 1/8'' piston (G-6 FMI pump), 1/4" piston (C-6 FMI pump), 3/8" piston (C-6 FMI pump) and 1/4" piston (G-20 FMI pump) for the 100, 500, 1500 and 5000 ppm exposure chambers, respectively. The temperature in the evaporator was maintained at a level sufficient to vaporize the test substance
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations of isopropanol vapor were analyzed by flame ionization gas chromatography. At least 2 samples per hour were obtained from each exposure chamber and also the control chamber.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours per day, 5 days per week
Dose / conc.:
100 ppm (nominal)
Dose / conc.:
500 ppm (nominal)
Dose / conc.:
1 500 ppm (nominal)
Dose / conc.:
5 000 ppm (nominal)
No. of animals per sex per dose:
All exposure groups (except for the 100 ppm group) consisted of 10 rats/sex. An additional 15 rats/sex were assigned to the 0, 500, 1500, and 5000 ppm groups for assessment of neurobehavioral function.
Control animals:
yes, sham-exposed
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: during exposures, observations were recorded on a group basis. Preceding and following each exposure, animals were examined individually, and observations were recorded for each animal exhibiting overt clinical signs


BODY WEIGHT: Yes
- Time schedule for examinations: All rats and mice were weighed on Week 0 (prior to the start of exposures). These values were used as the preexposure reference weights and were subtracted from each subsequent weight to determine the change in body weight. Animals were weighed weekly during the study, at neurobehavioral evaluations, and immediately preceding sacrifice.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Weekly. For rats, food consumption was determined over a 7-day period. For mice, food consumption was measured over a 6-day period rather than a 7-day period due to excessive food spillage during the first 24 hours after the containers were filled with fresh food

WATER CONSUMPTION: Yes
- Time schedule for examinations: Weekly.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During the 6th week of the study in 10 rats/sex/group. At sacrifice in 10 rats/sex/group and 10 mice/sex/group.
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: Yes
- How many animals: 10 rats/sex/group during the 6th week; 10 rats/sex/group and 10 mice/sex/group at sacrifice

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at sacrifice
- Animals fasted: Yes
- How many animals: 10 rats/sex/group and 10 mice/sex/group

NEUROBEHAVIOURAL EXAMINATION: Yes (rats only)
- Time schedule for examinations: 10/15 rats selected were evaluated with the functional observational battery (FOB) prior to the first exposure and on the weekend following Weeks 1, 2, 4, 9, and 13. Motor activity evaluations were conducted on all 15 rats per sex selected from the 0, 500, 1500, and 5000 ppm groups prior to the first exposure and on the weekend following Weeks 4, 9, and 13.
- Dose groups that were examined: 15 rats per sex from each group

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Organ Weights: The brain, liver, lungs, kidneys, adrenals, testes (males), and ovaries (females) from all surviving animals (except animals designated for neuroanatomic pathology evaluation) were weighed at sacrifice.
Statistics:
The data for continuous, parametric variables were intercompared for the exposure and control groups by use of Levene's test for homogeneity of variances, by analysis of variance, and by t-tests. The t-tests were used, if the analysis of variance was significant, to delineate which groups differed from the control group. If Levene's test indicated homogeneous variances, the groups were compared by an analysis of variance for equal variances followed, when appropriate, by pooled variance t-tests. If Levene's test indicated heterogeneous variances, the groups were compared by an analysis of variance for unequal variance followed, when appropriate, by separate variance t-tests. Frequency data, such as microscopic diagnoses, were compared using Fisher's exact test. All statistical tests, except the frequency comparisons, were performed using BMDP Statistical Software (Dixon, 1985). The frequency data tests are described in Biometry (Sokal and Rohlf, 1969). The probability value of p < 0.05 (two-tailed) was used as the critical level of significance for all tests.
Clinical signs:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Ophthalmological findings:
effects observed, treatment-related
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Key result
Dose descriptor:
NOAEC
Effect level:
5 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Remarks on result:
other: Based on elevated liver weight and hyaline droplets in kidneys. However, these effects are unspecific and a clear NOAEC is not derivable
Key result
Critical effects observed:
not specified
Organ:
kidney
liver

- Clinical signs:

In rats, clinical signs observed following exposures included swollen periocular tissue (females) at the highest dose and perinasal encrustation (males) at 500 ppm and above.

- Behaviour:

Narcosis was observed in a few animals of both species during exposure to 5000 ppm and possibly 1500 ppm as well. However, the animals became tolerant to the narcotic effects of isopropanol after week 2. No neurobehavioral changes were observed in any parameters of the functional observational battery. However, increased motor activity was noted at week 9 of exposure in female rats of the 5000 ppm group.

- Body weight/body weight gain:

After an initial drop in body weight gain in the first week of exposure at the high dose (5000 ppm), rats in the 1500 and 5000 ppm groups had significant increases in body weight gain and/or body weight throughout most of the exposure period. But only the 5000 ppm group had greater than 10% body weight gain compared to controls. Increases in body weight and body weight gain greater than 10% were also noted in female mice in the 5000 ppm group.

- Haematology/clinical chemistry:

Consistent clinical pathology changes included an increase in mean corpuscular volume (rats; female mice) and mean corpuscular hemoglobin (male rats; female mice) at the 5000 ppm exposure level. Other changes noted include a slight anemia in rats at week 6 only and a slight dehydration in female mice at the end of the study.

- Organ weight:

Relative liver weight in rats was elevated no more than 8% in the 5000 ppm groups. However, a 10 and 21% increase in relative liver weight was observed in female mice at 1500 and 5000 ppm, respectively.

Macroscopy:

No gross lesions were observed in any organs.

- Histopathology:

The only microscopic change observed was hyaline droplets within kidneys of all male rats. This change was not clearly concentration related, although it was most pronounced in the 5000 ppm group.

Conclusions:
Based on the observed effects after inhalation exposure to 110, 500, 1500 or 5000 ppm isopropanol for 13 weeks no clear NOAEC is derivable. However, since effects on liver and kidneys of unclear relevance are reported the NOAEC might be set at 5000 ppm.
Executive summary:

Repeated exposure to isopropanol for 13 weeks produced effects at the highest concentration (5000 ppm) and a kidney change of unknown biological signifiance. Clinical signs of toxicity on the central nervous system (including ataxia, narcosis, ) are acute effects and not relevant for limit value determination for repeated dose systemic effects. Increases in absolute body weight and body weight gain, and changes in hematology parameters in animals exposed to 1500 and 5000 ppm of isopropanol, increased relative liver weight in rats exposed to 5000 ppm, as well as increased motor activity for female rats in the 5000 ppm group have been observed.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Study duration:
subchronic
Species:
rat
Quality of whole database:
reliable with restrictions
System:
hepatobiliary
Organ:
kidney
liver

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Due to the NOAEL of 1000 mg/kg bw/day in an OECD 422 repeated dose/reproduction/developmental toxicity screening test in rats with the structural analogue the registration substance does not have to be classified regarding systemic and target organ toxicity after repeated exposure according to the criteria laid down in the EU Classification Labelling and Packaging Regulation (1272/2008/EC).

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