Silicic acid, ethyl ester

Endpoint summary

Description of key information

Repeated dose toxicity: Oral

Based on the data available, the No Observed Adverse Effect Level (NOAEL) for the test chemical was considered to be in range of 50 -200 mg/Kg bw using male and female rats. Hence the test chemical is not likely to be toxic upon repeated exposure by oral route as per the criteria mentioned in CLP regulation.

Repeated dose toxicity: inhalation

Based on the data available, the No Observed Adverse Effect concentration (NOAEC) for the test chemical was considered to be in range of 147-200000 mg/m³using male rats or mice . Hence the test chemical is not likely to be toxic upon repeated exposure by inhalation route as per the criteria mentioned in CLP regulation.

Repeated dose toxicity: dermal

The acute toxicity value for test chemical (as provided in section 7.2.3) is >2000 mg/kg body weight. Also, given the use of the chemical; repeated exposure by the dermal route is unlikely since the use of gloves is common practice in industries. Thus, it is expected that test chemical shall not exhibit 28 day repeated dose toxicity by the dermal route. In addition, there is no data available that suggests that test chemical shall exhibit repeated dose toxicity by the dermal route. Hence this end point was considered for waiver.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Weight of evidence approach based on the available information from various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

other: As mentioned below

Principles of method if other than guideline:

WoE report is based on repeated dose toxicity studies on rats

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: 2.Sprague-Dawley 3.Crl:CD (SD)BR

Details on species / strain selection:

No data available

Sex:

male/female

Details on test animals or test system and environmental conditions:

Details on test animal
TEST ANIMALS
- Age at study initiation: seven weeks old
- Weight at study initiation: 213 to 279 grams
for the males and from 150 to 207 grams for the females.

Route of administration:

oral: gavage

Details on route of administration:

No data available

Vehicle:

other: 2.corn oil 3.peanut oil

Details on oral exposure:

2.Details on exposure
PREPARATION OF DOSING SOLUTIONS:
Test chemical dissolved in corn oil.

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Test chemical dissolved in corn oil.
- Concentration in vehicle: 0,10, 50 and 100 mg/kg bw/day
3.Details on oral exposure
PREPARATION OF DOSING SOLUTIONS: The test chemical in the vehicle, peanut oil sparged with nitrogen,
DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test chemical was dissolved in peanut oil

- Concentration in vehicle: 0, 70, 200 and 600 mg/kg/day
- Amount of vehicle (if gavage): 10 mL/kg.
- Lot/batch no. (if required): No data
- Purity: No data

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

2.28 days in males; up to 53 days in females
3.91 or 92 consecutive days

Frequency of treatment:

Daily

Remarks:

Study 2.
0,10, 50 and 100 mg/kg bw/day
Study 3.
0, 70, 200, and 600 mg/kg/day

No. of animals per sex per dose:

2.Total :80
0 mg/kg /day: 10males and 10 females
10 mg/kg /day: 10males and 10 females
50mg/kg /day:10males and 10 females
100 mg/kg /day:10males and 10 females
3.Total:120
0 mg/kg/day:15 male and 15 female
70 mg/kg/day:15 male and 15 female
200 mg/kg/day:15 male and 15 female
600 mg/kg/day:15 male and 15 female

Control animals:

yes, concurrent vehicle

Details on study design:

No data available

Positive control:

No data available

Observations and examinations performed and frequency:

2.Parental animals observation and examinations
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight were recorded at designated intervals
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): food consumption were recorded at
designated intervals
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

OTHER:
Detailed clinical observations and
neurobehavioral tests were conducted at designated intervals to assess behavior, main functions and reflexes (landing foot splay, rectal temperature and forelimb grip strength) and to measure motor activity over a 1- and 10-minute periods. Blood samples were collected from all males of the principal groups and from all females of the toxicity groups at the end of the treatment period.
3.Observations and examinations performed & frequency

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were observed twice daily for
mortality and moribundity
- Cage side observations checked in table [No.?] were included. No data

DETAILED CLINICAL OBSERVATIONS: Clinical exams were performed
daily and detailed physical examinations were performed
weekly.
BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were
recorded weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Individual food consumption wererecorded weekly.

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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 data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: No data

OPHTHALMOSCOPIC EXAMINATION: yes
- Time schedule for examinations: No data
- Dose groups that were examined: No data

Clinical pathology parameters (hematology and serum chemistry) were evaluated on all animals during week 4 and at study termination (week 13). The animals were fasted overnight prior to the collection of blood samples. Blood samples for general clinical pathology evaluations were collected from a lateral tail vein at both time points. Blood samples for assessment of coagulation parameters were collected from the vena cava at the time of necropsy. The
following hematology parameters were evaluated: total leukocyte count (white cell), erythrocyte count (red cells), hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, prothrombin time, activated partial thromboplastin time (APTT; terminal evaluation only), reticulocyte count (percent and absolute),differential leukocyte count (percent and absolute:
neutrophil, lymphocyte, monocyte, eosinophil and basophil). The following serum chemistry parameters were evaluated: alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, blood urea nitrogen, total protein, total bilirubin, creatinine, Ca, Na, K, Cl, P, glucose, albumin, globulin, albumin/globulin ratio, and total cholesterol. Urine samples were collectedvia metabolism chambers following the eighth exposure of female rats and following the seventh exposure of male rats. Urine volume was measured using calibrated test tubes, and urine color and turbidity were visually assessed.Urinalysis parameters were urine osmolality, pH, protein, glucose, ketone, bilirubin, blood and urobilinogen. Vaginal smears for determination of the stage of estrus were obtained from all surviving females once daily beginning 21 days prior to the scheduled necropsy. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P]) beginning 21 days prior to
the scheduled necropsy. The final vaginal smear for each female was collected on the day of necropsy.

Sacrifice and pathology:

2.At final sacrifice of the parents, specified organs were weighed and a complete macroscopic post-mortem examination was performed. A microscopic examination was performed on a range of sampled tissue and organs for all males of the principal groups and females of the toxicity groups; a microscopic examination was not performed on the principal group females.
3.A complete necropsy was conducted on all animals. Animals euthanized in extremis or at the scheduled necropsy were euthanized by carbon dioxide asphyxiation followed by exsanguinations. The necropsy included, but was not limited to, examination of the external surface, all orifices and the cranial,abdominal and pelvic cavities and their viscera. At the time of necropsy, the following tissues and organs were collected and preserved in 10% neutral buffered formalin: adrenals (2), aorta, bone with marrow (femur, sternebrae), bone marrow smear (from femur), brain (forebrain, midbrain, hindbrain), coagulating gland, eyes with optic nerve (2; preserved in Davidson's solution), gastrointestinal tract (esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum), heart, kidneys (2), liver (sections of two lobes), lungs (including bronchi, fixed by inflation with fixation), lymph node (mesenteric, submandibular), mammary gland (females only), ovaries with oviducts (2), pancreas, peripheral nerve (sciatic), pituitary, prostate, salivary glands (submaxillary, 2), seminal vesicles (2), skeletal muscle (vastus medialis), skin, spinal cord (cervical, midthoracic, lumbar), spleen, testis with epididymis (1) and vas deferens, thymus, thyroid (with parathyroids if present(2)), trachea, urinary bladder, uterus with vagina and cervix, and all gross lesions (when possible). Bone marrow smears were obtained from all animals not found dead, but were not placed in 10% neutral buffered formalin. The right testis/epididymis from all males at the scheduled necropsy and both testes/epididymides from those males founddead were preserved in Bouin's solution and prepared for microscopic examination using PAS/hematoxylin staining. The left testis/epididymis from all males euthanized at the scheduled necropsy were prepared for sperm analysis as described below. The following organs from animals euthanized at the scheduled necropsy were weighed: adrenals, brain, epididymides (weighed separately; total and cauda), kidneys, liver, ovaries (with oviducts), pituitary, prostate, seminal vesicles with coagulating glands (with accessory fluids), testes (weighed separately), and thyroid (fixed weight). Organ to final body weight and organ to brain weight ratios were calculated. After fixation, specified tissues were trimmed according to standard operating procedures. Trimmed tissues were processed into paraffin blocks, sectioned at five to eight microns, mounted on clean glass microscope slides and stained with
hematoxylin and eosin. The tissues noted above from all animals found dead or euthanized in extremis and from all animals in the control and 600 mg/kg/day groups euthanizedat the scheduled necropsy, as well as the lungs, liver, and kidneys from all animals in the 70 and 200 mg/kg/day groups were examined microscopically

Statistics:

3.All analyses were conducted using two-tailed tests for minimum significance levels of 1% and 5% comparing the test article-treated groups to the control group by sex. All means were presented with standard deviations (S.D.) and the number of sampling units (N) used to calculate the means. Statistical analyses were not conducted if the number of animals was two or less. All statistical tests were performed using appropriate computing devices or programs. Body weight, body weight change, food consumption, clinical pathology, absolute and relative organ weight data and epididymal and testicular sperm numbers and sperm production rates were subjected to a one-way analysis of variance (ANOVA), followed by Dunnett's test if the ANOVA revealed statistical significance (p<0.05). The percentage of motile spermatozoa and the percentage of sperm with normal morphology were analyzed by the Kruskal-Wallis nonparametric ANOVA test to determine intergrouup differences, followed by the Mann-Whitney U-Test comparing the control and test article-treated groups if the ANOVA revealed statistical significance (p<0.05). Clinical laboratory values for leukocytes that occur at a low incidence (i.e., monocytes, eosinophils and basophils) were not subjected to statistical analysis.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

3.Test chemical -related clinical signs were generally limited to the 600 mg/kg/day group males and females. The predominant clinical sign was rales. Sporadic occurrences of rales for individual animals were noted as early as the first week of dosing, with as many as one-half of the animals having sporadic observations of rales noted by the third week of dosing, and continuing at a similar incidence at the observations prior to and following dosing through the end of the study. Additional treatment-related clinical signs in the 600 mg/kg/day group included wet and/or dried material (red, yellow, brown and/or clear) on various body surfaces and abnormal excreta (feces smaller than normal, soft and/or mucoid feces and decreased defecation). These additional clinical signs also occurred at the lower dose levels; however, the incidence was generally sporadic. No other treatment-related clinical signs were noted. All other findings occurred similarly in the control group, were limited in incidence (typicallysingle occurrences in one or two animals/group) and/or were common findings in laboratory rats.

Mortality:

mortality observed, treatment-related

Description (incidence):

3.One control group male (day 28), one 200 mg/kg/day group female (day 65) and two 600 mg/kg/day groups males (day 18) were found dead. Macroscopic and/or microscopic findings indicated that none of these deaths was treatment related; these deaths were attributed to dsoing error. Test article-related lethality was limited to the 600 mg/kg/day group. One male and eight females in this group were found dead or euthanized in extremis. Just prior (one or two days) to death,

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

2.Induced a transient decrease in body weight gain during lactation at 100 mg/kg/day. Slight transient decrease in body weight gain was also noted in females of the toxicity group at 100 mg/kg/day but no change in body weight was noted in males of the principal group at any dose-level.
3.Body weight gains were unaffected by test article administration at all dose levels

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

3.food consumption were unaffected by test article administration at all dose levels.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

3.No oculopathic changes indicative of a test chemical -related effect were observed at any dose level.

Haematological findings:

no effects observed

Description (incidence and severity):

3.No test chemical related changes were present in hematology parameters.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

2.slightly lower sodium, potassium and glucose levels in males given 100 mg/kg/day,
3.No test chemical related changes were present in mean aspartate aminotransferase values for the males at week 13 and in mean alanine aminotransferase values for the males and females at both evaluations during the study (weeks 4 and 13).

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

3.Test chemical related organ weight changes were seen in the liver of the 600 mg/kg/day males. In this treatment group, although the mean absolute liver weight was not increased, the mean liver-to-brain weight was increased (not statistically significantly), and the mean liver-to-body weight was statistically increased when compared to controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

3.Gaseous distention in the intestinal tract (primarily in the cecum) was observed macroscopically at necropsy for most males and females, including those that died prior to the scheduled necropsy

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

2.Tubular nephropathy was observed among some treated males (principal group) at 50 and 100 mg/kg/day and females (toxicity group) at 100 mg/kg/day. This was associated with slightly lower plasma levels of sodium, potassium and glucose. No signs of substance-induced maternal or paternal toxicity occurred at the low dose-level (10 mg/kg/day).
3.Test chemical related microscopic changes were limited to the males, and consisted of increased severity of centrilobular to generalized hepatocellular vacuolation. No other test article-related microscopic changes were observed.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

3.No test chemical related changes were present in estrous cycle data or spermatogenic endpoints.

Dose descriptor:

NOAEL

Effect level:

> 50 - <= 200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical signs

gross pathology

histopathology: non-neoplastic

mortality

Remarks on result:

other: No toxic effects were observed

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Conclusions:

No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be in range of 50-200 mg/kg/day. When male and female rats were treated with test chemical orally.

Executive summary:

Data available for the test chemical was reviewed to determine the toxic nature of test chemical .The studies are as mentioned below:

Study 2

The combined repeated reprductive and developmental toxicity study of test chemical was performed on male and female Sprague-Dawley rats. The test chemical was dissolved in corn oil and administered via oral gavage route throughout the pre-mating period (15 days), during the mating and post-mating periods until final sacrifice for the males (at least 4 weeks in total)and throughout pre-mating (15 days) and mating period, during pregnancy and lactation, until day 4 post-partum inclusive (or until sacrifice for un-mated females) for the females. Three groups of 10 males and 10 females received the test chemical by oral gavage once a day at 10, 50 or 100 mg/kg/day while A group of 10 males and 10 females was given the vehicle (corn oil) under the same experimental conditions and acted as a control group.

Mortality and clinical signs were checked daily in all animals. Body weight and food consumption were recorded at designated intervals. Detailed clinical observations and neurobehavioral tests were conducted at designated intervals to assess behavior, main functions and reflexes (landing
foot splay, rectal temperature and forelimb grip strength) and to measure motor activity over a 1- and 10-minute periods. Blood samples were collected from all males of the principal groups and from all females of the toxicity groups at the end of the treatment period. At final sacrifice, specified organs were weighed and a complete macroscopic post-mortem examination was performed. A microscopic examination was performed on a range of sampled tissue and organs for all males of the principal groups and females of the toxicity group. slightly lower sodium, potassium and glucose levels in males given 100 mg/kg/day, treatment-related degenerative/necrotic nephropathy in 9/10 males,while  slightly lower sodium, potassium and glucose levels in males given 50 mg/kg/day,minimal degenerative/necrotic nephropathy in 4/10 males at 50 mg/kg/day. In female slight not significant transient decrease in body weight, slight degenerative/necrotic nephropathy in 3/10 females, no other substance-induced toxicity.While  no substance-induced toxicity at these two dose-levels i.e. 10, 50 mg/kg/day. Under the conditions of this combined repeat-dose toxicity study with the reproduction/developmental toxicity screening test, the oral administration of test chemical to male and female Sprague-Dawley rats, from the pre-mating period, during mating and until sacrifice (males) or during gestation and lactation until day 4 post-partum (females), induced a transient decrease in body weight gain during lactation at 100 mg/kg/day. Slight transient decrease in body weight gain was also noted in females of the toxicity group at 100 mg/kg/day but no change in body weight was noted in males of the principal group at any dose-level. Tubular nephropathy was observed among some treated males (principal group) at 50 and 100 mg/kg/day and females (toxicity group) at 100 mg/kg/day. This was associated with slightly lower plasma levels of sodium, potassium and glucose. No signs of substance-induced maternal or paternal toxicity occurred at the low dose-level (10 mg/kg/day). Based on these results, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 50 mg/kg/day for the females and 10 mg/kg/day for the males

Study 3

90 Day oral toxicity study in ratswas performed to determine the toxic nature of the test chemical. The test chemical was dissolved inpeanut oil in dose concentration 0, 70, 200, and 600 mg/kg/day to each dose a group of 15 male and 15 female via oral gavage 91 or 92 consecutive days. A concurrent control group received the vehicle on a comparable regimen. The animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity. Clinical observations were performed on all animals prior to and approximately 1-2 hours following dosing. All significant findings were recorded. Detailed physical examinations were conducted weekly during the study period and just prior to the scheduled necropsy. Mean body weight changes were calculated for each week. A final (fasted) body weight was recorded for each animal on the day of scheduled necropsy. Individual food consumption was measured weekly beginning one week prior to randomization (week 1). Food intake was calculated as g/animal/day for the corresponding body weight intervals. Clinical pathology parameters (hematology and serum chemistry) were evaluated on all animals during week 4 and at study termination (week 13). The animals were fasted overnight prior to the collection of blood samples. Blood samples for general clinical pathology evaluations were collected from a lateral tail vein at both time points. Blood samples for assessment of coagulation parameters were collected from the vena cava at the time of necropsy, Urinalysis parameters were urine osmolality, pH, protein, glucose, ketone, bilirubin, blood and urobilinogen. Vaginal smears for determination of the stage of estrus were obtained from all surviving females once daily beginning 21 days prior to the scheduled necropsy. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P]) beginning 21 days prior to the scheduled necropsy. The final vaginal smear for each female was collected on the day of necropsy. A complete necropsy was conducted on all animals. Animals euthanized in extremis or at the scheduled necropsy were euthanized by carbon dioxide asphyxiation followed by exsanguinations. The necropsy included, but was not limited to, examination of the external surface, all orifices and the cranial, abdominal and pelvic cavities and their viscera.

One control group male (day 28), one 200 mg/kg/day group female (day 65) and two 600 mg/kg/day groups males (day 18) were found dead. Macroscopic and/or microscopic findings indicated that none of these deaths was treatment related; these deaths were attributed to dsoing error. Test article-related lethality was limited to the 600 mg/kg/day group. One male and eight females in this group were found dead or euthanized in extremis. Just prior (one or two days) to death, Test chemical -related clinical signs were generally limited to the 600 mg/kg/day group males and females. The predominant clinical sign was rales. Sporadic occurrences of rales for individual animals were noted as early as the first week of dosing, with as many as one-half of the animals having sporadic observations of rales noted by the third week of dosing, and continuing at a similar incidence at the observations prior to and following dosing through the end of the study. Additional treatment-related clinical signs in the 600 mg/kg/day group included wet and/or dried material (red, yellow, brown and/or clear) on various body surfaces and abnormal excreta (feces smaller than normal, soft and/or mucoid feces and decreased defecation). These additional clinical signs also occurred at the lower dose levels; however, the incidence was generally sporadic. No other treatment-related clinical signs were noted. All other findings occurred similarly in the control group, were limited in incidence (typicallysingle occurrences in one or two animals/group) and/or were common findings in laboratory rats. Body weight gains and food consumption were unaffected by test article administration at all dose levels. No oculopathic changes indicative of a test chemical –related effect were observed at any dose level. No test chemical related changes were present in hematology parameters, estrous cycle data or spermatogenic endpoints. No test chemical related changes were present in mean aspartate aminotransferase values for the males at week 13 and in mean alanine aminotransferase values for the males and females at both evaluations during the study (weeks 4 and 13).Test chemical related organ weight changes were seen in the liver of the 600 mg/kg/day males. In this treatment group, although the mean absolute liver weight was not increased, the mean liver-to-brain weight was increased (not statistically significantly), and the mean liver-to-body weight was statistically increased when compared to controls. Gaseous distention in the intestinal tract (primarily in the cecum) was observed macroscopically at necropsy for most males and females, including those that died prior to the scheduled necropsy. Test chemicalrelated microscopic changes were limited to the males, and consisted of increased severity of centrilobular to generalized hepatocellular vacuolation. No other test article-related microscopic changes were observed. Although several of these test article-related findings (clinical signs, gaseous intestinal distension, hepatocellular vacuolation) also occurred at 0, 70 and/or 200 mg/kg/day, all were limited or sporadic in incidence.HenceThe No observed Adverse Effect level (NOAEL) for the test chemical using male and female rats was considered to be 200 mg/Kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Data is Klimicsh 2 and from authoritative database

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Weight of evidence approach based on the available information from various test chemicals.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

other: As mentioned below

Principles of method if other than guideline:

WoE report is based on repeated dose toxicity studies on rats or mice

GLP compliance:

not specified

Limit test:

no

Species:

other: 2.rat 3.mice

Strain:

other: 2.Fischer 344 3.ICR

Sex:

male

Details on test animals or test system and environmental conditions:

2.Details on test animal
- Age at study initiation:
101 to 153 grams
3.Details on test animal
TEST ANIMALS
- Age at study initiation: Four week-old
- Weight at study initiation:
- Fasting period before study:
- Housing: ive mice each were housed in plastic cages in filtrated air-ventilated housing for small animal breeding.
- Diet (e.g. ad libitum): fed pelleted rodent chow (CRF-1, Oriental Kobo) ad libitum.
- Water (e.g. ad libitum): water ad libitum.
- Acclimation period: 1 week.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24±1°C
- Humidity (%):60±5%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12-h light, 12-h dark cycle

Route of administration:

inhalation: aerosol

Vehicle:

air

Details on inhalation exposure:

2.The inhalation chambers used in the study were constructed of stainless steel with glass windows for animal observations. Chamber volume was 1330 liters and the airflow was approximately 300 L/min (13.5 air changes per hour). Chamber temperature and relative humidity were determined at least twelve times per exposure. The animals were acclimated to the chamber (air-only exposure) for 2 days prior to the initiation of the exposure regimen. The position of the cages was rotated daily within each chamber. Target concentrations of 0 and 150 mg/m3 were selected for this study. A 2% test substance hydrolysate solution was
prepared daily and used to generate the aerosol atmosphere in the inhalation chamber. Chamber concentrations of test substance hydrolysate were determined by gravimetric methods. The nominal concentration was calculated daily by dividing the total amount of material delivered to the chamber by the total airflow rate. The particle size distribution was measured once a day for the first 16 exposure days of the study. The data collected were analyzed by probit analysis (Finney, 1964) to obtain the mass median aerodynamic diameter (MMAD) and the geometric standard deviation.
3.The test chemical was bubbled with dry air. The test chemical vapor was diluted with filtered room air, and introduced into a 550-1 stainless steel chamber with a dynamic flow rate of 30 l/min. Groups of ten mice in stainless steel wire cages were exposed to 200 ppm test chemical 6 h/day, 5 days/week, for 2 or 4 weeks in the subchronic inhalation study. Control mice were exposed to filtered room air using the same type of exposure chamber. The air sample was collected from the center of the chamber, and the test chemical concentration was monitored at approximately
20-min intervals in the subchronic inhalation study using a gas chromatograph (163, Hitachi). At the end of daily exposure, the chamber was quickly ventilated by filtrated room air at flow rate of 150 l/min

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

2.6 hours per day for a total of 19 exposures over 4 weeks
3.for 6 h/day, 5 days/week, for 2 or 4 weeks

Frequency of treatment:

2.Five days/week for three weeks and four consecutive days during the fourth week

Remarks:

Study 2.
0 and 147 mg/m3
Study 3.
0 and 200000mg/m3

No. of animals per sex per dose:

2.Total:30
0 mg/m3:15 male
147 mg/m3:15 male
3.Total:120
0 mg/m3:15 male
200000 mg/m3:15 male

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

2.Clinical observations were performed daily. All animals were weighed prior to study initiation, weekly during the study, and immediately prior to study termination.
3.The behavior and external appearance of mice were checked everyday and body weight was measured before starting test chemical exposure, during the inhalation period and in the morning during the observation period.

Sacrifice and pathology:

2.GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

2.Ten animals of the control and treated groups were subjected to a complete necropsy and the following tissues were fixed in 10% neutral buffered formalin for histologic evaluation: gross lesions, larynx, lungs, trachea, nasal turbinates and kidneys. For the satellite groups, the larynges of three control animals and five test substance-treated animals were taken and immersion-fixed in 2% glutaraldehyde for possible electron microscopic examination. The remaining two rats from thecontrol group were subjected to a complete necropsy and perfusion-fixed with 5% methanol-free EM grade fomaldehyde. The larynges from these two rats were then further immersion-fixed in 2% glutaraldehyde. Other organs (brain, spinal cord, and peripheral nerves) were taken from these control animals and processed for light microscopic evaluation.
3.Animals were sacrificed 2 days after the final exposure. Blood was collected prior to termination of the study for evaluation of some hematology and clinical chemistry parameters including blood urea nitrogen (BUN), serum creatinine (CRNN), aspartate aminotransferase (AST), alanine aminotransferase (ALT),total serum bilirubin (t-BIL), direct serum bilirubin(d-BIL), red blood cell count (RBC), hemoglobinconcentration (Hb), packed cell volume (Ht), and total and differential white blood cell counts (WBC). Urine sampleswere collected using metabolic cages for evaluation of NAG and LDH in urine. Body weights were recorded at initiationand termination of the study, and 3 times/week during the study. Their organs were examined grossly and the cornea,nasal cavity, respiratory tract, lungs, liver, kidneys,spleen, pancreas, thymus, thyroid and bone marrow were examined histopathologically.

Statistics:

2.The data for continuous, parametric variables were intercompared for the exposure and control groups by use of Levene's test for homogeneity of variances and by t-tests. If Levene's test indicated homogeneous variances, the groups were compared by pooled variance t-tests. If Levene's test indicated heterogeneous
variances, the groups were compared by separate variance t-test. Frequency data were compared using Fisher's exact tests. 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.
3.Student's t-test or Welch's method were adopted for statistical analysis of organ weight, blood biochemistry and hematological studies.

Clinical signs:

no effects observed

Description (incidence and severity):

2.No exposure-related clinical signs were observed during the study.
3.no external signs of toxicity were observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

2.A depression in body weight gain was observed for the test substance-exposed animals during the first week of the study.
3.Body weight in mice exposed for 2 weeks reached the level of non-exposed mice during the 2-week observation period in non-exposed: 41.0 + 3.7 g, exposed: 41.0 4- 2.4 g). but did not do so in mice exposed for 4 weeks.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

3.Hb was significintly decreased in mice exposed for 4 weeks.

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

3.No effects on organ weight was observed.

Gross pathological findings:

no effects observed

Description (incidence and severity):

2.At necropsy, focal/multifocal color changes of the lungs were noted in ninety percent of the animals of the test substance-exposed group.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

2.Histological examination showed nonspecific irritant, inflammatory, and metaplastic changes within the respiratory tracts of test substance-exposed rats.
Laryngeal lesions included squamous metaplasia and foci of minimal granulomatous laryngitis. Other microscopic changes included the presence of cytoplasmic hyalinization (mild to moderate) within the olfactory mucosa, squamous metaplasia (minimal to mild) within the nasal mucosa, cellular hyperplasia within the trachea, alveolar histiocytosis, bronchopneumonia, interstitial pneumonitis and alveolar type II pneumocyte hyperplasia within the lungs.
3.The kidney of exposed mice developed tubulointerstitial nephritis (TIN). Tubular dilatation was prominent, and polymorphonuclear neutrophils (PMNs) were often seen in mice sacrificed 1 day after exposure. In mice sacrificed 2 weeks after exposure PMNs were not prominent, and disappearance of the tubules was observed. Fibrosis was more severe in mice sacrificed 2 weeks after exposure. The kidneys in mice exposed for 4 weeks were affected more widely than those in mice exposed for 2 weeks. Blood biochemical examinations revealed no evidence of renal dysfunction. Submucosal infiltration of polymorphonuclear neutrophils (PMNs) in the nasal mucosa was observed in all mice killed 1 day after exposure. Infiltration was more severe in mice exposed for 4 weeks than in mice exposed for 2 weeks, and had become mild in the 2-week observation period. The other organs examined showed no findings related to test chemical exposure, and there as no hyperplasia due to inhalation.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Dose descriptor:

NOAEC

Effect level:

147 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Remarks on result:

other: No toxic effects were observed

Dose descriptor:

LOAEC

Effect level:

200 000 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical signs

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Remarks on result:

other: Dose related toxic effects were observed

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Conclusions:

The No observed Adverse Effect concentration (NOAEC) for the test chemical using male rats was considered to be 147mg/m3

Executive summary:

Data available for the test chemical was reviewed to determine the toxic nature of test chemical .The studies are as mentioned below:

Study 2

The subacute inhalation toxicity study of test chemical was performed in maleFischer 344 rats . Target concentrations of 0 and 150 mg/m3 were selected for this study. A 2% test substance hydrolysate solution was prepared daily and used to generate the aerosol atmosphere in the inhalation chamber. Chamber concentrations of test substance hydrolysate were determined by gravimetric methods. The nominal concentration was calculated daily by dividing the total amount of material delivered to the chamber by the total airflow rate. 15 rats per group were exposed to test chemical 6 hours per day for a total of 19 exposures over 4 weeks. Clinical observations were performed daily. All animals were weighed prior to study initiation, weekly during the study, and immediately prior to study termination. Ten animals of the control and treated groups were subjected to a complete necropsy and the following tissues were fixed in 10% neutral buffered formalin for histologic evaluation: gross lesions, larynx, lungs, trachea, nasal turbinates and kidneys. For the satellite groups, the larynges of three control animals and five test substance-treated animals were taken and immersion-fixed in 2% glutaraldehyde for possible electronmicroscopic examination. The remaining two rats from the control group were subjected to a complete necropsy and perfusion-fixed with 5% methanol-free EM grade fomaldehyde. The larynges from these two rats were then further immersion-fixed in 2% glutaraldehyde. Other organs (brain, spinal cord, and peripheral nerves) were taken from these control animals and processed for light microscopic evaluation. No exposure-related clinical signs and mortality were observed during the study. A depression in body weight gain was observed for the test substance-exposed animals during the first week of the study. At necropsy, focal/multifocal color changes of the lungs were noted in ninety percent of the animals of the test substance-exposed group. Histological examination showed nonspecific irritant, inflammatory, and metaplastic changes within the respiratory tracts of test substance-exposed rats. Laryngeal lesions included squamous metaplasia and foci of minimal granulomatous laryngitis. Other microscopic changes included the presence of cytoplasmic hyalinization (mild to moderate) within the olfactory mucosa, squamous metaplasia (minimal to mild) within the nasal mucosa, cellular hyperplasia within the trachea, alveolar histiocytosis, bronchopneumonia, interstitial pneumonitis and alveolar type II pneumocyte hyperplasia within the lungs. HenceThe No observed Adverse Effect concentration (NOAEC) for the test chemical using male rats was considered to be 147mg/m3

Study 3

The subacute inhalation toxicity study was performed in maleICR mice. The test chemical was bubbledwith dry air. The test chemical vapor was diluted with filtered room air, and introduced into a 550-1 stainless steel chamber with a dynamic flow rate of 30 l/min. Groups of ten mice in stainless steel wire cages wereexposed to 200 ppm test chemical 6 h/day, 5 days/week, for 2 or 4 weeks in thesubchronic inhalation study.Control mice were exposed to filtered room air using the same type of exposure chamber. The air sample was collected from the center of the chamber, and the test chemical concentration was monitored at approximately20-min intervals in the subchronic inhalation study using a gas chromatograph (163,Hitachi). At the end of daily exposure, the chamber was quickly ventilated by filtrated room air at flow rate of 150 l/min. The behavior and external appearance of mice were checked everyday and body weight was measured before starting test chemical exposure,during the inhalation period and in the morning during the observation period.Animals were sacrificed 2 days after the final exposure. Blood was collected prior to termination of the study for evaluation of some hematology and clinical chemistry parameters including blood urea nitrogen (BUN), serum creatinine (CRNN), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total serum bilirubin (t-BIL), direct serum bilirubin (d-BIL), red blood cell count (RBC), hemoglobin concentration (Hb), packed cell volume (Ht), and total and differential white blood cell counts (WBC). Urine samples were collected using metabolic cages for evaluation of NAG and LDH in urine. Body weights were recorded at initiation and termination of the study, and 3 times/week during the study. Their organs were examined grossly and the cornea, nasal cavity, respiratory tract, lungs, liver, kidneys, spleen, pancreas, thymus, thyroid and bone marrow were examined histopathologically.All mice survived.No external signs of toxicity were observed..Weight gain was suppressed during the exposure period . Body weight in mice exposed for 2 weeks reached the level of non-exposed mice during the 2-week observation period in non-exposed: 41.0 + 3.7 g, exposed: 41.0 4- 2.4 g).but did not do so in mice exposed for 4 weeks.Hb was significintly decreased in mice exposed for 4 weeks. No effects on organ weight was observed.The kidney of exposed mice developed tubulointerstitial nephritis (TIN). Tubular dilatation was prominent, and polymorphonuclear neutrophils (PMNs) were often seen in mice sacrificed 1 day after exposure. In mice sacrificed 2 weeks after exposure PMNs were not prominent, and disappearance of the tubules was observed. Fibrosis was more severe in mice sacrificed 2 weeks after exposure. The kidneys in mice exposed for 4 weeks were affected more widely than those in mice exposed for 2 weeks. Blood biochemical examinations revealed no evidence of renal dysfunction. Submucosal infiltration of polymorphonuclear neutrophils (PMNs) in the nasal mucosa was observed in all mice killed 1 day after exposure. Infiltration was more severe in mice exposed for 4 weeks than in mice exposed for 2 weeks, and had become mild in the 2-week observation period. The other organs examined showed no findings related to test chemical exposure, and there as no hyperplasia due to inhalation. The kidney and nasal mucosa are the target organs for test chemical inhalation. The low observed Adverse Effect concentration (LOAEC) for the test chemical using male mice was considered to be 200000 mg/m3

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

147 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Data is Klimicsh 2 and from authoritative database

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

 Repeated dose toxicity: Oral

Data available for the test chemical was reviewed to determine the toxic nature of test chemical .The studies are as mentioned below:

Study 2

The combined repeated reprductive and developmental toxicity study of test chemical was performed on male and female Sprague-Dawley rats. The test chemical was dissolved in corn oil and administered via oral gavage route throughout the pre-mating period (15 days), during the mating and post-mating periods until final sacrifice for the males (at least 4 weeks in total)and throughout pre-mating (15 days) and mating period, during pregnancy and lactation, until day 4 post-partum inclusive (or until sacrifice for un-mated females) for the females. Three groups of 10 males and 10 females received the test chemical by oral gavage once a day at 10, 50 or 100 mg/kg/day while A group of 10 males and 10 females was given the vehicle (corn oil) under the same experimental conditions and acted as a control group.

Mortality and clinical signs were checked daily in all animals. Body weight and food consumption were recorded at designated intervals. Detailed clinical observations and neurobehavioral tests were conducted at designated intervals to assess behavior, main functions and reflexes (landing
foot splay, rectal temperature and forelimb grip strength) and to measure motor activity over a 1- and 10-minute periods. Blood samples were collected from all males of the principal groups and from all females of the toxicity groups at the end of the treatment period. At final sacrifice, specified organs were weighed and a complete macroscopic post-mortem examination was performed. A microscopic examination was performed on a range of sampled tissue and organs for all males of the principal groups and females of the toxicity group. slightly lower sodium, potassium and glucose levels in males given 100 mg/kg/day, treatment-related degenerative/necrotic nephropathy in 9/10 males,while  slightly lower sodium, potassium and glucose levels in males given 50 mg/kg/day,minimal degenerative/necrotic nephropathy in 4/10 males at 50 mg/kg/day. In female slight not significant transient decrease in body weight, slight degenerative/necrotic nephropathy in 3/10 females, no other substance-induced toxicity.While  no substance-induced toxicity at these two dose-levels i.e. 10, 50 mg/kg/day. Under the conditions of this combined repeat-dose toxicity study with the reproduction/developmental toxicity screening test, the oral administration of test chemical to male and female Sprague-Dawley rats, from the pre-mating period, during mating and until sacrifice (males) or during gestation and lactation until day 4 post-partum (females), induced a transient decrease in body weight gain during lactation at 100 mg/kg/day. Slight transient decrease in body weight gain was also noted in females of the toxicity group at 100 mg/kg/day but no change in body weight was noted in males of the principal group at any dose-level. Tubular nephropathy was observed among some treated males (principal group) at 50 and 100 mg/kg/day and females (toxicity group) at 100 mg/kg/day. This was associated with slightly lower plasma levels of sodium, potassium and glucose. No signs of substance-induced maternal or paternal toxicity occurred at the low dose-level (10 mg/kg/day). Based on these results, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 50 mg/kg/day for the females and 10 mg/kg/day for the males

Study 3

90 Day oral toxicity study in ratswas performed to determine the toxic nature of the test chemical. The test chemical was dissolved inpeanut oil in dose concentration 0, 70, 200, and 600 mg/kg/day to each dose a group of 15 male and 15 female via oral gavage 91 or 92 consecutive days. A concurrent control group received the vehicle on a comparable regimen. The animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity. Clinical observations were performed on all animals prior to and approximately 1-2 hours following dosing. All significant findings were recorded. Detailed physical examinations were conducted weekly during the study period and just prior to the scheduled necropsy. Mean body weight changes were calculated for each week. A final (fasted) body weight was recorded for each animal on the day of scheduled necropsy. Individual food consumption was measured weekly beginning one week prior to randomization (week 1). Food intake was calculated as g/animal/day for the corresponding body weight intervals. Clinical pathology parameters (hematology and serum chemistry) were evaluated on all animals during week 4 and at study termination (week 13). The animals were fasted overnight prior to the collection of blood samples. Blood samples for general clinical pathology evaluations were collected from a lateral tail vein at both time points. Blood samples for assessment of coagulation parameters were collected from the vena cava at the time of necropsy, Urinalysis parameters were urine osmolality, pH, protein, glucose, ketone, bilirubin, blood and urobilinogen. Vaginal smears for determination of the stage of estrus were obtained from all surviving females once daily beginning 21 days prior to the scheduled necropsy. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P]) beginning 21 days prior to the scheduled necropsy. The final vaginal smear for each female was collected on the day of necropsy. A complete necropsy was conducted on all animals. Animals euthanized in extremis or at the scheduled necropsy were euthanized by carbon dioxide asphyxiation followed by exsanguinations. The necropsy included, but was not limited to, examination of the external surface, all orifices and the cranial, abdominal and pelvic cavities and their viscera.

One control group male (day 28), one 200 mg/kg/day group female (day 65) and two 600 mg/kg/day groups males (day 18) were found dead. Macroscopic and/or microscopic findings indicated that none of these deaths was treatment related; these deaths were attributed to dsoing error. Test article-related lethality was limited to the 600 mg/kg/day group. One male and eight females in this group were found dead or euthanized in extremis. Just prior (one or two days) to death, Test chemical -related clinical signs were generally limited to the 600 mg/kg/day group males and females. The predominant clinical sign was rales. Sporadic occurrences of rales for individual animals were noted as early as the first week of dosing, with as many as one-half of the animals having sporadic observations of rales noted by the third week of dosing, and continuing at a similar incidence at the observations prior to and following dosing through the end of the study. Additional treatment-related clinical signs in the 600 mg/kg/day group included wet and/or dried material (red, yellow, brown and/or clear) on various body surfaces and abnormal excreta (feces smaller than normal, soft and/or mucoid feces and decreased defecation). These additional clinical signs also occurred at the lower dose levels; however, the incidence was generally sporadic. No other treatment-related clinical signs were noted. All other findings occurred similarly in the control group, were limited in incidence (typicallysingle occurrences in one or two animals/group) and/or were common findings in laboratory rats. Body weight gains and food consumption were unaffected by test article administration at all dose levels. No oculopathic changes indicative of a test chemical –related effect were observed at any dose level. No test chemical related changes were present in hematology parameters, estrous cycle data or spermatogenic endpoints. No test chemical related changes were present in mean aspartate aminotransferase values for the males at week 13 and in mean alanine aminotransferase values for the males and females at both evaluations during the study (weeks 4 and 13).Test chemical related organ weight changes were seen in the liver of the 600 mg/kg/day males. In this treatment group, although the mean absolute liver weight was not increased, the mean liver-to-brain weight was increased (not statistically significantly), and the mean liver-to-body weight was statistically increased when compared to controls. Gaseous distention in the intestinal tract (primarily in the cecum) was observed macroscopically at necropsy for most males and females, including those that died prior to the scheduled necropsy. Test chemicalrelated microscopic changes were limited to the males, and consisted of increased severity of centrilobular to generalized hepatocellular vacuolation. No other test article-related microscopic changes were observed. Although several of these test article-related findings (clinical signs, gaseous intestinal distension, hepatocellular vacuolation) also occurred at 0, 70 and/or 200 mg/kg/day, all were limited or sporadic in incidence.HenceThe No observed Adverse Effect level (NOAEL) for the test chemical using male and female rats was considered to be 200 mg/Kg/day.

Repeated dose toxicity: inhalation

Data available for the test chemical was reviewed to determine the toxic nature of test chemical .The studies are as mentioned below:

Study 2

The subacute inhalation toxicity study of test chemical was performed in maleFischer 344 rats . Target concentrations of 0 and 150 mg/m3 were selected for this study. A 2% test substance hydrolysate solution was prepared daily and used to generate the aerosol atmosphere in the inhalation chamber. Chamber concentrations of test substance hydrolysate were determined by gravimetric methods. The nominal concentration was calculated daily by dividing the total amount of material delivered to the chamber by the total airflow rate. 15 rats per group were exposed to test chemical 6 hours per day for a total of 19 exposures over 4 weeks. Clinical observations were performed daily. All animals were weighed prior to study initiation, weekly during the study, and immediately prior to study termination. Ten animals of the control and treated groups were subjected to a complete necropsy and the following tissues were fixed in 10% neutral buffered formalin for histologic evaluation: gross lesions, larynx, lungs, trachea, nasal turbinates and kidneys. For the satellite groups, the larynges of three control animals and five test substance-treated animals were taken and immersion-fixed in 2% glutaraldehyde for possible electronmicroscopic examination. The remaining two rats from the control group were subjected to a complete necropsy and perfusion-fixed with 5% methanol-free EM grade fomaldehyde. The larynges from these two rats were then further immersion-fixed in 2% glutaraldehyde. Other organs (brain, spinal cord, and peripheral nerves) were taken from these control animals and processed for light microscopic evaluation. No exposure-related clinical signs and mortality were observed during the study. A depression in body weight gain was observed for the test substance-exposed animals during the first week of the study. At necropsy, focal/multifocal color changes of the lungs were noted in ninety percent of the animals of the test substance-exposed group. Histological examination showed nonspecific irritant, inflammatory, and metaplastic changes within the respiratory tracts of test substance-exposed rats. Laryngeal lesions included squamous metaplasia and foci of minimal granulomatous laryngitis. Other microscopic changes included the presence of cytoplasmic hyalinization (mild to moderate) within the olfactory mucosa, squamous metaplasia (minimal to mild) within the nasal mucosa, cellular hyperplasia within the trachea, alveolar histiocytosis, bronchopneumonia, interstitial pneumonitis and alveolar type II pneumocyte hyperplasia within the lungs. HenceThe No observed Adverse Effect concentration (NOAEC) for the test chemical using male rats was considered to be 147mg/m3

Study 3

The subacute inhalation toxicity study was performed in maleICR mice. The test chemical was bubbledwith dry air. The test chemical vapor was diluted with filtered room air, and introduced into a 550-1 stainless steel chamber with a dynamic flow rate of 30 l/min. Groups of ten mice in stainless steel wire cages wereexposed to 200 ppm test chemical 6 h/day, 5 days/week, for 2 or 4 weeks in thesubchronic inhalation study.Control mice were exposed to filtered room air using the same type of exposure chamber. The air sample was collected from the center of the chamber, and the test chemical concentration was monitored at approximately20-min intervals in the subchronic inhalation study using a gas chromatograph (163,Hitachi). At the end of daily exposure, the chamber was quickly ventilated by filtrated room air at flow rate of 150 l/min. The behavior and external appearance of mice were checked everyday and body weight was measured before starting test chemical exposure,during the inhalation period and in the morning during the observation period.Animals were sacrificed 2 days after the final exposure. Blood was collected prior to termination of the study for evaluation of some hematology and clinical chemistry parameters including blood urea nitrogen (BUN), serum creatinine (CRNN), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total serum bilirubin (t-BIL), direct serum bilirubin (d-BIL), red blood cell count (RBC), hemoglobin concentration (Hb), packed cell volume (Ht), and total and differential white blood cell counts (WBC). Urine samples were collected using metabolic cages for evaluation of NAG and LDH in urine. Body weights were recorded at initiation and termination of the study, and 3 times/week during the study. Their organs were examined grossly and the cornea, nasal cavity, respiratory tract, lungs, liver, kidneys, spleen, pancreas, thymus, thyroid and bone marrow were examined histopathologically.All mice survived.No external signs of toxicity were observed..Weight gain was suppressed during the exposure period . Body weight in mice exposed for 2 weeks reached the level of non-exposed mice during the 2-week observation period in non-exposed: 41.0 + 3.7 g, exposed: 41.0 4- 2.4 g).but did not do so in mice exposed for 4 weeks.Hb was significintly decreased in mice exposed for 4 weeks. No effects on organ weight was observed.The kidney of exposed mice developed tubulointerstitial nephritis (TIN). Tubular dilatation was prominent, and polymorphonuclear neutrophils (PMNs) were often seen in mice sacrificed 1 day after exposure. In mice sacrificed 2 weeks after exposure PMNs were not prominent, and disappearance of the tubules was observed. Fibrosis was more severe in mice sacrificed 2 weeks after exposure. The kidneys in mice exposed for 4 weeks were affected more widely than those in mice exposed for 2 weeks. Blood biochemical examinations revealed no evidence of renal dysfunction. Submucosal infiltration of polymorphonuclear neutrophils (PMNs) in the nasal mucosa was observed in all mice killed 1 day after exposure. Infiltration was more severe in mice exposed for 4 weeks than in mice exposed for 2 weeks, and had become mild in the 2-week observation period. The other organs examined showed no findings related to test chemical exposure, and there as no hyperplasia due to inhalation. The kidney and nasal mucosa are the target organs for test chemical inhalation. The low observed Adverse Effect concentration (LOAEC) for the test chemical using male mice was considered to be 200000 mg/m3

Repeated dose toxicity: dermal

The acute toxicity value for test chemical (as provided in section 7.2.3) is >2000 mg/kg body weight. Also, given the use of the chemical; repeated exposure by the dermal route is unlikely since the use of gloves is common practice in industries. Thus, it is expected that test chemical shall not exhibit 28 day repeated dose toxicity by the dermal route. In addition, there is no data available that suggests that test chemical shall exhibit repeated dose toxicity by the dermal route. Hence this end point was considered for waiver.

Justification for classification or non-classification

Thus based on the above studies for the test chemical does not exhibit toxic nature upon repeated exposure by oral, inhalation and dermal route of exposure and hence is not likely to classify as per the criteria mentioned in CLP regulation.