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Description of key information

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the analogue substance Pigment Orange 36 to rats (according to OECD 422, GLP compliant). The test item was administered in vehicle (highly purified water) at dosages of 0, 100, 300, and 1000 mg/kg body weight/day, animals in control groups received the vehicle only. Test item was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.


All animals survived the scheduled study period.


During the treatment, faeces stained red with dose-dependent intensity of discoloration were noted in all males and females receiving test material. This observation was due to staining properties of the test item.


No effects on food consumption were noted in males at any dose level. Body weight gain was slightly but statistically significantly reduced in males at the dose level of 1000 mg/kg bw/day during the pre-pairing period. No differences in body weight gain were noted at any dose level during the remaining study period. Body weights of males in all dose groups were similar to the respective control values during the entire study period. Because lower body weight gain at the high-dose levels was reversible and did not cause significant changes in body weights, this effect was considered not to be adverse.


Food consumption, body weights and body weight gain of females were not affected by the treatment at any dose level.


No further test item-related observations were noted in males or females at any dose level during the live part of the study.


Terminal examinations revealed changes in motility of sperms in all dose groups. These effects, however could not be confirmed in a separate study designed to verify these effects.


No further effects on male reproductive system were noted during the study. Sperm morphology and sperm count at the high-dose level was similar to the control values. Weights of male reproductive organs, macroscopical and histopathological examination of testes and epididymides gave no indication of any treatment-related effect. Further, no indication of effects on reproduction was noted within this study up to and including the highest dose level. For this reason, changes in motility of sperms were considered not to be adverse in this study.


Based on these results, the NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females and in this study was considered to be 1000 mg/kg bw/day, the highest dose level tested.


 


Based on the observed results in a subacute inhalation study in rats, it is concluded that the No Observed Adverse Effect Concentration (NOAEC) of test item was found to be 0.03 mg/L when exposed for 6 hours/day, 5 days/week, for 4 weeks by flow-past nose-only inhalation route to Sprague Dawley rats. The nominal dose of 0.03 mg/L (6 hrs/day, 5 days/week) is the actual exposure concentration in males and females.


 


As there were no treatment related effects on systemic, reproduction and fertility parameters up to and including the highest dose tested 1000 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Wistar Rats for the test item is determined to be 1000 mg/kg bwt/day under the test conditions and doses employed.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
See Read Across Justification document in chapter 13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Source: Hylasco Biotechnology (India) Pvt. Ltd., 4B MN Park, Turkapally Village, Shameerpet Mandal, Medchal Dist, Telangana 500078
Justification for selection of species: Rat is the standard laboratory rodent species used for toxicity assessment and recommended by various regulatory authorities. The Wistar rat was selected due to the large amount of background data available for this strain.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Source: Hylasco Biotechnology (India) Pvt. Ltd., 4B MN Park, Turkapally Village, Shameerpet Mandal, Medchal Dist, Telangana 500078
Justification for selection of species: Rat is the standard laboratory rodent species used for toxicity assessment and recommended by various regulatory authorities. The Wistar rat was selected due to the large amount of background data available for this strain.
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
The test item was administered by oral gavage in graduated doses to three groups of male and female rats. The males were dosed for 57 days, up to and including the day before scheduled sacrifice (this includes two weeks prior to mating, during mating period and approximately, two weeks post mating period).
Females were dosed throughout the treatment period. This includes two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and up to and including the day before scheduled sacrifice (i.e., up to LD13).
Animals in the recovery groups were kept only for observations of reversibility, persistence or delayed occurrence of systemic toxic effects for 14 days of recovery period and these animals were not mated and consequently were not used for assessment of reproduction/developmental toxicity. The recovery period of the study started from the first scheduled kill of dams.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For homogeneity and test item concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during Week 4 (Day 23) of treatment period and was analysed in-house. For each set, duplicate samples were drawn from the top, middle and bottom layers of each preparation and in case of the control duplicate samples from the middle layer were drawn.
The analysis was done as per the method validated under Eurofins Advinus Study No.: G19468. One set of samples (first set) were analysed for test item concentration analysis and other set (second set) of samples were stored at ambient condition for reanalysis purpose as a backup.
Both set I and set II analysis results of G3 and G4 groups prepared on Day 1(16 June 2020) were out of acceptance limits. Hence, the samples from subsequent preparations were analysed on 17 June 2020. The results of G3 group from the subsequent preparation (17 June 2020) was within the acceptance limits, however G4 group results were out of acceptance limits. Hence the backup samples (formulations prepared on 17 June) of G4 group samples were analysed on 18 June 2020.
Formulations were considered acceptable when the mean results (calculated using all the replicate values) of all the layers and mean of each layer was within ±15.0 % of the claimed concentration and the relative standard deviation (% RSD) was equal to or less than 10.0 %.
Duration of treatment / exposure:
Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily at approximately the same time each day (varying by ± 3 hours) for 57 days which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled sacrifice.
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours) for total of 51-58 days which includes 2 weeks prior to the mating, during mating, pregnancy and up to LD 13.
The dose formulations were administered to the high dose recovery group of rats once daily at approximately the same time each day (varying by ± 3 hours).
The dose volume administered to each rat was at an equivolume of 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.
Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups at an equivolume of 10 mL/kg bwt.
The vehicle and the test item were not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days from the first scheduled kill of dams.
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control
Dose / conc.:
111 mg/kg bw/day (actual dose received)
Dose / conc.:
333 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:
10 males & 10 females (main groups)
5 males & 5 females (recovery)
Control animals:
yes, concurrent vehicle
Details on study design:
Group
No. Group Colour
of
cage card Dose
(mg/kg bwt/day) Concen-tration (mg/mL) Dose volume (mL/ kg bwt/day) No. of rats Sex Rat Numbers
From To
Main Groups
G1 Vehicle Control White 0 0 10 10 M Rx7481 Rx7490
10 F Rx7491 Rx7500
G2 Low dose Yellow 111 11.1 10 10 M Rx7501 Rx7510
10 F Rx7511 Rx7520
G3 Mid dose Green 333 33.3 10 10 M Rx7521 Rx7530
10 F Rx7531 Rx7540
G4 High dose Pink 1000 100 10 10 M Rx7541 Rx7550
10 F Rx7551 Rx7560
Recovery Groups
G1R Vehicle Control recovery White 0 0 10 5 M Rx7561 Rx7565
5 F Rx7566 Rx7570
G4R High dose recovery Pink 1000 100 10 5 M Rx7571 Rx7575
5 F Rx7576 Rx7580
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked:
- ease of removal from home cage
- handling reactivity
- palpebral closure
- eye examination
- piloerection
- lacrimation
- salivation
- skin/fur examination
- perineum wetness
- respiration
- muscle tone and
- extensor thrust response

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
- gait -posture -tremors -mobility score -arousal level -clonic or tonic movements - behaviour -urination -defecation -rearing -abnormal vocalizations
- motoractivity
- sensory reactivity
- landing foot splay
- grip performance
- temperature

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of pre-mating period and end of recovery
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes
- How many animals: 5 per sex per group
- Parameters examined.: RBC,HGB, HCT, MCV, MCH, MCHC, Retic, WBC, DLC, PT, APTT

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of pre-mating period and end of recovery
- Animals fasted: Yes
- How many animals: 5 per sex per group
- Parameters examined.: ALT, Albumen, Albumen/Globuline, ALP, AST, BUN, Bile acids, Ca, Cl, Creatinine, CK, gamma-GT, Glc, PO4, K,Na, Bilirubin, Cholesterol, Protein, Fat

URINALYSIS: Yes
- Time schedule for collection of urine: end of pre-mating period and end of recovery
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined.: spec.gravity, nitrite, pH, protein, Glc, ketone, Ery, Leuco, color, turbitity, Volume, Bilirubin, Urobilinogen

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
All adult animals and pups were subjected for detailed necropsy and findings were recorded. The adult animals sacrificed at term were fasted overnight (water allowed), weighed and exsanguinated under isoflurane anaesthesia. The dams were sacrificed on LD 14.

Tissues & Organ collected:
All gross lesions
Cervix
Epididymides
Ovaries
Oviducts
Uterus
Vagina
Prostate
Seminal vesicles with coagulating glands
Testes
Thyroid with parathyroid
Levator ani bulbocavernosus muscle complex
Cowper’s glands
Glans penis
Brain (Cerebrum, Cerebellum and pons)
Bone marrow smear
Cecum
Colon
Duodenum
Eyes
Femoral Muscle
Femur/bone, femorotibial Joint
Glands, Adrenals
Glands, Mammary
Gut associated lymphoid tissue
Heart
Ileum
Jejunum
Kidneys
Liver
Lungs
Lymph nodes, mandibular
Lymph nodes, mesenteric
Pituitary
Rectum
Sciatic nerve
Spinal cord (cervical, thoracic and lumbar)
Spleen
Sternum with marrow
Stomach
Thymus
Trachea
Urinary bladder
Other examinations:
Data of offspring were also determined (see chapter 7.8.1)
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Endocrine findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Yellow colored intestinal content (colored test material)
Neuropathological findings:
no effects observed
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:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
mortality
ophthalmological examination
organ weights and organ / body weight ratios
serum/plasma biochemistry
sperm measures
urinalysis
Key result
Critical effects observed:
no
Conclusions:
As there were no treatment related effects on systemic, reproduction and fertility parameters up to and including the highest dose tested 1000 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Wistar Rats for the test item is determined to be 1000 mg/kg bwt/day under the test conditions and doses employed.
Executive summary:

To summarize, oral (gavage) administration of the test item to Wistar rats at the dose levels of 111, 333, and 1000 mg/kg bwt/day for 2 weeks prior to mating, during mating, and post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females) had no effects on general health, neurological parameters, body weights, food consumption, pre-coital time, gestation length, mating and fertility parameters. There were no treatmentrelated


effects on the uterine/implantation data and mean litter size. There were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.


Treatment did not induce any test item-related adverse changes with respect to terminal fasting body weights, organ weights/ratios (including reproductive organs), clinical pathology parameters and thyroid hormone profile. Grossly, yellowish gastro-intestinal contents noted in parental rats at 333 mg/kg and 1000 mg/kg was attributed to the physical appearance of the test item.


Histopathology did not reveal any changes in adult animals and pups at all dose levels tested.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This information is submitted based on ECHA decision number : CCH-D-2114479843-34-01/F
A study according to OED TG 422 has been requested by ECHA.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Source: Hylasco Biotechnology (India) Pvt. Ltd., 4B MN Park, Turkapally Village, Shameerpet Mandal, Medchal Dist, Telangana 500078
Justification for selection of species: Rat is the standard laboratory rodent species used for toxicity assessment and recommended by various regulatory authorities. The Wistar rat was selected due to the large amount of background data available for this strain.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Source: Hylasco Biotechnology (India) Pvt. Ltd., 4B MN Park, Turkapally Village, Shameerpet Mandal, Medchal Dist, Telangana 500078
Justification for selection of species: Rat is the standard laboratory rodent species used for toxicity assessment and recommended by various regulatory authorities. The Wistar rat was selected due to the large amount of background data available for this strain.
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
The test item was administered by oral gavage in graduated doses to three groups of male and female rats. The males were dosed for 57 days, up to and including the day before scheduled sacrifice (this includes two weeks prior to mating, during mating period and approximately, two weeks post mating period).
Females were dosed throughout the treatment period. This includes two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and up to and including the day before scheduled sacrifice (i.e., up to LD13).
Animals in the recovery groups were kept only for observations of reversibility, persistence or delayed occurrence of systemic toxic effects for 14 days of recovery period and these animals were not mated and consequently were not used for assessment of reproduction/developmental toxicity. The recovery period of the study started from the first scheduled kill of dams.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For homogeneity and test item concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during Week 4 (Day 23) of treatment period and was analysed in-house. For each set, duplicate samples were drawn from the top, middle and bottom layers of each preparation and in case of the control duplicate samples from the middle layer were drawn.
The analysis was done as per the method validated under Eurofins Advinus Study No.: G19468. One set of samples (first set) were analysed for test item concentration analysis and other set (second set) of samples were stored at ambient condition for reanalysis purpose as a backup.
Both set I and set II analysis results of G3 and G4 groups prepared on Day 1(16 June 2020) were out of acceptance limits. Hence, the samples from subsequent preparations were analysed on 17 June 2020. The results of G3 group from the subsequent preparation (17 June 2020) was within the acceptance limits, however G4 group results were out of acceptance limits. Hence the backup samples (formulations prepared on 17 June) of G4 group samples were analysed on 18 June 2020.
Formulations were considered acceptable when the mean results (calculated using all the replicate values) of all the layers and mean of each layer was within ±15.0 % of the claimed concentration and the relative standard deviation (% RSD) was equal to or less than 10.0 %.
Duration of treatment / exposure:
Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily at approximately the same time each day (varying by ± 3 hours) for 57 days which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled sacrifice.
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours) for total of 51-58 days which includes 2 weeks prior to the mating, during mating, pregnancy and up to LD 13.
The dose formulations were administered to the high dose recovery group of rats once daily at approximately the same time each day (varying by ± 3 hours).
The dose volume administered to each rat was at an equivolume of 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.
Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups at an equivolume of 10 mL/kg bwt.
The vehicle and the test item were not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days from the first scheduled kill of dams.
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control
Dose / conc.:
111 mg/kg bw/day (actual dose received)
Dose / conc.:
333 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:
10 males & 10 females (main groups)
5 males & 5 females (recovery)
Control animals:
yes, concurrent vehicle
Details on study design:
Group
No. Group Colour
of
cage card Dose
(mg/kg bwt/day) Concen-tration (mg/mL) Dose volume (mL/ kg bwt/day) No. of rats Sex Rat Numbers
From To
Main Groups
G1 Vehicle Control White 0 0 10 10 M Rx7481 Rx7490
10 F Rx7491 Rx7500
G2 Low dose Yellow 111 11.1 10 10 M Rx7501 Rx7510
10 F Rx7511 Rx7520
G3 Mid dose Green 333 33.3 10 10 M Rx7521 Rx7530
10 F Rx7531 Rx7540
G4 High dose Pink 1000 100 10 10 M Rx7541 Rx7550
10 F Rx7551 Rx7560
Recovery Groups
G1R Vehicle Control recovery White 0 0 10 5 M Rx7561 Rx7565
5 F Rx7566 Rx7570
G4R High dose recovery Pink 1000 100 10 5 M Rx7571 Rx7575
5 F Rx7576 Rx7580
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked:
- ease of removal from home cage
- handling reactivity
- palpebral closure
- eye examination
- piloerection
- lacrimation
- salivation
- skin/fur examination
- perineum wetness
- respiration
- muscle tone and
- extensor thrust response

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
- gait -posture -tremors -mobility score -arousal level -clonic or tonic movements - behaviour -urination -defecation -rearing -abnormal vocalizations
- motoractivity
- sensory reactivity
- landing foot splay
- grip performance
- temperature

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of pre-mating period and end of recovery
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes
- How many animals: 5 per sex per group
- Parameters examined.: RBC,HGB, HCT, MCV, MCH, MCHC, Retic, WBC, DLC, PT, APTT

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of pre-mating period and end of recovery
- Animals fasted: Yes
- How many animals: 5 per sex per group
- Parameters examined.: ALT, Albumen, Albumen/Globuline, ALP, AST, BUN, Bile acids, Ca, Cl, Creatinine, CK, gamma-GT, Glc, PO4, K,Na, Bilirubin, Cholesterol, Protein, Fat

URINALYSIS: Yes
- Time schedule for collection of urine: end of pre-mating period and end of recovery
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined.: spec.gravity, nitrite, pH, protein, Glc, ketone, Ery, Leuco, color, turbitity, Volume, Bilirubin, Urobilinogen

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
All adult animals and pups were subjected for detailed necropsy and findings were recorded. The adult animals sacrificed at term were fasted overnight (water allowed), weighed and exsanguinated under isoflurane anaesthesia. The dams were sacrificed on LD 14.

Tissues & Organ collected:
All gross lesions
Cervix
Epididymides
Ovaries
Oviducts
Uterus
Vagina
Prostate
Seminal vesicles with coagulating glands
Testes
Thyroid with parathyroid
Levator ani bulbocavernosus muscle complex
Cowper’s glands
Glans penis
Brain (Cerebrum, Cerebellum and pons)
Bone marrow smear
Cecum
Colon
Duodenum
Eyes
Femoral Muscle
Femur/bone, femorotibial Joint
Glands, Adrenals
Glands, Mammary
Gut associated lymphoid tissue
Heart
Ileum
Jejunum
Kidneys
Liver
Lungs
Lymph nodes, mandibular
Lymph nodes, mesenteric
Pituitary
Rectum
Sciatic nerve
Spinal cord (cervical, thoracic and lumbar)
Spleen
Sternum with marrow
Stomach
Thymus
Trachea
Urinary bladder
Other examinations:
Data of offspring were also determined (see chapter 7.8.1)
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Endocrine findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Yellow colored intestinal content (colored test material)
Neuropathological findings:
no effects observed
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:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
mortality
ophthalmological examination
organ weights and organ / body weight ratios
serum/plasma biochemistry
sperm measures
urinalysis
Key result
Critical effects observed:
no
Conclusions:
As there were no treatment related effects on systemic, reproduction and fertility parameters up to and including the highest dose tested 1000 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Wistar Rats for the test item Hostaperm-Gelb H6G (C. I. Pigment Yellow 175) is determined to be 1000 mg/kg bwt/day under the test conditions and doses employed.
Executive summary:

To summarize, oral (gavage) administration of the test item “Hostaperm-Gelb H6G (C. I. Pigment Yellow 175)” to Wistar rats at the dose levels of 111, 333, and 1000 mg/kg bwt/day for 2 weeks prior to mating, during mating, and post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females) had no effects on general health, neurological parameters, body weights, food consumption, pre-coital time, gestation length, mating and fertility parameters. There were no treatmentrelated

effects on the uterine/implantation data and mean litter size. There were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

Treatment did not induce any test item-related adverse changes with respect to terminal fasting body weights, organ weights/ratios (including reproductive organs), clinical pathology parameters and thyroid hormone profile. Grossly, yellowish gastro-intestinal contents noted in parental rats at 333 mg/kg and 1000 mg/kg was attributed to the physical appearance of the test item.

Histopathology did not reveal any changes in adult animals and pups at all dose levels tested.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 4NOV 1970 to 4 DEC 1970
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Does not meet important criteria of today standard methods (e.g. no clinical biochemistry, no detailed clinical observations, no functional observations, no reporting of individual data, especially on food consumption and body weight development)
Qualifier:
according to guideline
Guideline:
other: company guideline
Deviations:
no
Principles of method if other than guideline:
Testing of toxicity after repeated oral application
GLP compliance:
no
Remarks:
study was performed previous to GLP implementation
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: in house breeding; SPF
- Weight at study initiation: males: mean 111 g (96 g - 128 g); females: mean 106 g (92 g - 114 g)
- Fasting period before study: no
- Housing: 5 animals/sex/cage
- Diet: Standard diet Altromin R (Altromin GmbH, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-25
- Humidity (%): 35-60


Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): once at the beginning of the study
- Mixing appropriate amounts with standard diet using a Lödige precision mixer, model M 20 E
- Preparation of pellets using a Templewood mixed feed press
- Storage temperature of food: no data
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
30 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
5.0%, 1.0%, 0.2%, 0%
Basis:
nominal in diet
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: no lethality was observed after single oral application of 15000 mg/kg bw; pretest (10 days feeding study) with rats receiving feed with 5%, 1%, 0.2% and 0% of the test item
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined: YES
- 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: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before the beginning and at the end of the experiment
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: each animal of the study
- Parameters examined: haemoglobin concentration, erythrocyte count, leucocyte count, differential blood count, appearance of Heinz bodies

CLINICAL CHEMISTRY: No

URINALYSIS: Yes
- Time schedule for collection of urine: before the beginning and at the end of the experiment
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: appearance, colour, protein, glucose, bilirubin, specific weight (urine collected from 5 animals), sediment


NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (heart, lung, liver, spleen, adrenals, kidneys)

Other examinations:
ORGAN WEIGHTS:
- heart, lung, liver, spleen, adrenals, kidneys
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slightgly reduced body weight gain in the mid-dose and high-dose females, i.e. 6.5% and 8.2% loss, respectively as compared to controls. Weight gains of the males and the remaining females were normal and corresponded to those of the controls.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
not examined
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
- no effects

BODY WEIGHT AND WEIGHT GAIN
- Slightly reduced body weight gain in the mid-dose and high-dose females, i.e. 6.5% and 8.2% loss, respectively as compared to controls. Weight gains of the males and the remaining females were normal and corresponded to those of the controls.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
-no effects

HAEMATOLOGY
- no pathological findings

URINALYSIS
- no test item related effects
- at the end of the study male rats excreted protein in the urine which was judged to be normal in adult male rats

ORGAN WEIGHTS
- no effects

GROSS PATHOLOGY
- no effects

HISTOPATHOLOGY: NON-NEOPLASTIC
- no effects
Dose descriptor:
NOAEL
Effect level:
50 000 mg/kg diet
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no toxic effects were observed (50000 mg/kg diet correspond to 2000 mg/kg bw in male rats, calculated according to Guidance on information requirements R.8)
Dose descriptor:
NOAEL
Effect level:
500 000 mg/kg diet
Based on:
test mat.
Sex:
female
Basis for effect level:
other: minimal impairment of body weight development in high-dose and mid-dose group was not accounted as adverse toxic effect (50000 mg/kg diet correspond to 2500 mg/kg bw in female rats, calculated according to Guidance on information requirements R.8)
Critical effects observed:
not specified

 Dose sex   body weight  standard deviation
 [%]   [g]  [g] 
 5     males 211.4 28.32
 females 152.8 15.18
 1     males 210.4 13.88
 females 155.4 6.87
 0.2     males 221.0 16.25
 females 162.6 11.74
 0   (control)  males 223.4 26.15
 females 166.4 9.97
Conclusions:
The test item did not reveal substance related adverse effects under the conditions tested. Minimal effects on body weight development in the mid-and high dose group were not accounted for as adverse. Therefore the NOAEL in this subacute oral feeding study was 5% in the diet (corresponding to 2500 mg/kg bw in females and 2000 mg/kg bw in males; high dose group). Although the study design and reporting does not fulfil todays standard requirements (e.g. no clinical biochemistry, no detailed clinical observations), these data indicated that the test substance is not toxic after repeated oral exposure.
Executive summary:

Male and female Wistar rats were exposed to 5%, 1%, 0.2% or 0% test item in the diet for 30 days. Cage side observations, food consumption, body weight development, haematology, urine analysis, macroscopic investigations and histopathology of selected organs did not reveal any substance related toxic effects, except for slightly reduced body weights in high-dose and mid-dose female animals (i.e. 8.2% and 6.5% respectively compared to controls). These effects were minimal and therefore not accounted for as adverse. The NOAEL in this study was 5% in the diet (corresponding to 2500 mg/kg bw in females and 2000 mg/kg bw in males).

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 422, GLP-compliant)
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Tocixity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Source: Harlan Laboratories, Inc., Maasheseweg 87c, 5800 AN Vernay / Netherlands
- Number of Animals: 44 males (11 per group) and 44 females (11 per group)
- Age (at Start of Treatment): 11 weeks
- Body Weight Range (at Start of Treatment): 301 to 362 g (males), 216 to 247 g (females)
- Identification: Parent animals had cage card and individual animal number (ear tattoo), pups were individually tattooed with Indian ink on day 1 post partum
- Randomization: Performed after at least three days of acclimatization using a computer-generated random algorithm. Body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Accommodation: In groups of five in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V., Netherlands), batch/lot nos. 02105111001, 02105111201, 02105120301 and 6960C.CS-100099). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 80/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
Standard laboratory conditions, continuously monitored.
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 to 70
- Air changes (per hr): . Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12 (with at least eight hours music during the light period)
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
DOSE FORMULATIONS
The dose formulations were prepared weekly using the test item as supplied by the Sponsor.
The test item was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using a magnetic stirrer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATIONS
Dose formulations were stored at room temperature (20 +/- 5 °C) in glass beakers.
Based upon the results of stability analyses performed within the non-GLP Harlan Laboratories study (Dose Range-Finding Study for a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test in the Han Wistar Rat), dose formulations were stable for at least 8 days if stored at room temperature.

TREATMENT
- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of studies.
- Frequency of Administration: Once daily
- Target Dose Levels: 0 mg/kg/day (control group), 100 mg/kg/day (group 2), 300 mg/kg/day (group 3) and 1000 mg/kg/day (group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous non-GLP dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories Study D33711, using dose levels of 0, 100, 300 and 1000 mg/kg/ day, where no adverse effects were observed up to and including the highest dose level.
- Dose Volume: 10 mL/kg body weight
- Dose Concentrations: 0 mg/mL/day (control group), 10 mg/mL/day (group 2), 30 mg/mL/day (group 3) and 100 mg/mL/day (group 4).
- Duration of Acclimatization Period: 7 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
METHOD
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 0.5 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 0.5 g of each concentration were taken from the middle only to confirm stability (8 days). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 +/- 5 °C) and delivered on dry ice to the responsible for formulation analysis (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 +/- 5 °C until analysis.
The samples were analyzed by UV-VIS spectroscopy following an analytical procedure developed at Harlan Laboratories. The test item was used as the analytical standard.

RESULTS
Blank samples showed no significant absorbance and, therefore, it was confirmed that only highly purified water was applied within the control experiment.
The application formulations investigated during the study were found to comprise test material in the range of 93.1% to 105.6% and, thus, the required content limit of +/-20% with reference to the nominal content was met. The homogeneous distribution of test item in the preparations was approved because single results found did not deviate more than 5.5% (<15%) from the corresponding mean.
The test item was found to be stable in application formulations when kept eight days at 20 +/- 5 °C due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item and highly purified water as vehicle during this study. Application formulations were found to be homogeneously prepared and stable over a storage period of eight days (20 +/- 5 °C).
Duration of treatment / exposure:
MALES: 40 days
FEMALES: Approximately 7 weeks
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
11

Control animals:
yes, concurrent vehicle
Details on study design:
MALES
- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Treatment Ends: On day before sacrifice
- Blood Sampling: After 28 days of Treatment
- Necropsy: After treatment for 39 days, when no longer needed for assessment of reproductive effects

FEMALES
- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Gestation: Approximately 21 days
- Treatment Ends: On day 4 post partum
- Blood Sampling: Day 5 post partum
- Necropsy: On day 5 post partum (pups on day 4 post partum)
Positive control:
Not required
Observations and examinations performed and frequency:
VIABILITY/MORTALITY: Twice daily

CLINICAL SIGNS
Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION
Males: on days 1 - 4, 4 - 8, 8 - 11 and 11 - 14 during pre-pairing period and weekly during after pairing period.
Females: on days 1 - 4, 4 - 8, 8 - 11 and 11 - 14 during pre-pairing period; on days 0 - 7, 7 14 and 14 - 21 during gestation period and on days 1 - 4 of during lactation period.
No food consumption was recorded during the pairing period.

BODY WEIGHTS: Recorded daily from treatment start to day of necropsy.

DETAILED CLINICAL OBSERVATIONS
Detailed clinical observations were performed outside the home cage in all animals. In males, it was performed once prior to the first administration of the test item and weekly thereafter. In females, it was performed once prior to the first administration of the test item, weekly during the pre-pairing and pairing periods and on days 0, 6, 13 and 20 of the gestation period.
Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

FUNCTIONAL OBSERVATIONAL BATTERY
At one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 post partum) relevant parameters were performed with five P generation males and five P generation females from each group. This FOB assessment was conducted following the daily dose administration. Animals were observed for the following:
- Cage-side observations: faeces-balls, urine and posture as well as resistance to removal.
- Hand-held observations: muscle tone, constituation, skin, pupile size, palpebral closure, lacrimation, salivation, reaction to handling and general abnormalities.
- Open field observations: level of ambulatory activity including rearing (one minute evaluation), unusual body movements (e.g. spasms, convulsions), gait evaluation, behavior, hair coat, respiration, quantity of faeces-balls and urine.
- Reflexes: blinking, palpebral closure, pinna reflex, extensor thrust response, paw pinch, responsiveness to sharp noise, righting reflex and hearing ability (Preyer’s reflex).
- Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.

Any abnormal findings were recorded and, where appropriate, graded in severity.

Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.

CLINICAL LABORATORY INVESTIGATIONS
Blood samples were obtained on the day of the scheduled necropsy from 5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum. Blood samples were drawn sublingually from all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

The following hematology parameters were determined:
- Erythrocyte count
- Hemoglobin
- Hematocrit
- Mean corpuscular volume
- Red cell volume distribution width
- Mean corpuscular hemoglobin
- Mean corpuscular hemoglobin concentration
- Hemoglobin concentration distribution width
- Leukocyte count, total
- Differential leukocyte count
- Platelet count
- Prothrombin time (= Thromboplastin time)
- Activated partial Thromboplastin time

The following clinical biochemistry parameters were determined:
- Glucose
- Urea
- Creatinine
- Bilirubin, total
- Cholesterol, total
- Triglycerides
- Aspartate aminotransferase
- Alanine aminotransferase
- Alkaline phosphatase
- Gamma-glutamyl-transferase
- Bile acids
- Sodium
- Potassium
- Chloride
- Calcium
- Phosphorus
- Protein, total
- Albumin
- Globulin
- Albumin/Globulin ratio

URINALYSIS
The following urinalysis parameters were determined in five males of each group, which are allocated to the blood analysis, during the last week of the study using timed urine volume collection:
- Volume (18 hours)
- Specific gravity (relative density)
- Color
- Appearance
- pH
- Nitrite
- Osmolality
- Protein
- Glucose
- Ketones
- Urobilinogen
- Bilirubin
- Blood/Blood cells
Sacrifice and pathology:
TERMINATION AND NECROPSY

Males were sacrificed after treatment for 39 days, when no longer needed for the assessment of reproductive effects. Dams were sacrificed on day 5 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

All animals sacrificed were subjected to a detailed macroscopic examination. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. At the scheduled sacrifice, all animals were sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated. All parent animals were examined macroscopically for any structural changes. For the parent animals, special attention was directed at the organs of the reproductive system.The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

SEMINOLOGY AND SPERMATID COUNT
Sperm analysis was performed on the first 5 males per group.

Motility:
At necropsy of adult males an epididymal sperm sample was obtained from the left cauda epididymidis of each male. The sample was diluted with a pre-warmed (about 35 °C) physiological medium, and shortly after being obtained, one hundred sperm were counted microscopically for determination of percentage of not motile, stationary motile and progressively motile sperm.

Morphology:
A second sperm sample from the left cauda epididymidis was used for morphological assessment after fixation and Eosin staining. 500 sperm per sample were evaluated microscopically and classified into the following categories:
A: Normal, complete sperm
B: Normal head only (tail detached)
C: Complete sperm, misshapen hook
D: Complete sperm, abnormally curved hook
E: Complete sperm, reversed head
F: Abnormal head only (tail detached)

Morphological sperm evaluation was performed only for group 1 and 4 males. In the absence of a treatment-related effect the slides for the group 2 and 3 males were not evaluated.

Sperm, Spermatid Count:
The left caudal epididymis and left testis were taken for determination of homogenization-resistant spermatids and caudal epididymal sperm reserve. These tissues were frozen at -20 +/- 5 °C pending evaluation. For evaluation the weighed tissues were placed in Triton-X-100 solution and homogenized with a blender (Ultra Turrax) and an ultrasonic water bath. Sperm or spermatid heads were counted microscopically using a modified Neubauer chamber. These evaluations were performed in the first instance only for group 1 and 4 males. In the absence of a treatment-related effect the remaining frozen tissues were not evaluated.

ORGAN WEIGHTS
At the scheduled sacrifice, testes and epididymides from all parental males were weighed separately. In addition, from 5 males and 5 females sacrificed at the end of the study which were selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken.
- Adrenal glands (weighed as pairs)
- Brain
- Heart
- Kidneys (weighed as pairs)
- Liver
- Thymus
- Spleen

TISSUE PRESERVATION
The following tissues from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Prostate
- Seminal vesicles with coagulating gland
- Testes (in Bouin’s fixative)*
- Epididymides (in Bouin’s fixative)*
*From the first five males in each group which were used for sperm analysis, only the right testis and right epididymis were preserved for histopathological examination.

The following tissues from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Ovaries

In addition, from 5 males and 5 females per group selected for organ weights, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Gross lesions
- Brain (representative regions including cerebrum, cerebellum and pons)
- Spinal chord
- Small and large intestines (incl. Peyer’s patches)
- Stomach
- Liver
- Kidneys
- Adrenals
- Spleen
- Heart
- Thymus
- Thyroids, and parathyroids if possible
- Trachea and lungs (preserved by inflation with fixative and then immersion)
- Uterus (with vagina)
- Urinary bladder
- Lymph nodes (mesenterial, mandibular)
- Peripheral nerve (sciatic)
- Bone marrow

HISTOTECHNIQUE
All organ and tissue samples to be examined by the study pathologist were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin. Special stains were used at the discretion of the study pathologist.

HISTOPATHOLOGY
Slides of all organs and tissues listed collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the study pathologist. The same applied to all occurring gross lesions.
Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, where necessary.
A histopathology peer review was performed. A histopathology phase report was provided by the principal investigator which was included in the report.
Other examinations:
MATING, GESTATION, LACTATION
During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed. The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum. For a female which did not mate during the 14-day pairing period, a second pairing of this female with a male in the same group, which had already mated successfully, was performed. All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

REPRODUCTIVE AND OFFSPRING VIABILITY INDICES
From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, post-implantation losses, mean litter size, pup sex ratios and viability indices.

LITTER OBSERVATIONS
The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.

POSTMORTEM EXAMINATION OF OFFSPRING
Pups were sacrificed on day 4 post partum. All animals were sacrificed by by an injection of sodium pentobarbital and subjected to a detailed macroscopic examination. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. Pups found dead during the study, except those excessively cannibalized, were examined macroscopically.

Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied when the variables could be dichotomized without loss of information.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Red stained faeces were noted in all males and females in dose groups; this was due to the staining properties of the test item
Mortality:
mortality observed, treatment-related
Description (incidence):
Red stained faeces were noted in all males and females in dose groups; this was due to the staining properties of the test item
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In males at the dose level of 1000 mg/kg bw/day, lower body weight gain during the pre-pairing period
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:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Locomotor activity was not affected and functional observational battery gave no indication of a test item-related effect.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
1. IN-LIFE DATA OF PARENTAL ANIMALS
VIABILITY / MORTALITY
All animals survived scheduled study period.

DAILY CLINICAL SIGNS OR OBSERVATIONS
Red stained feces was noted in all males and females in all dose groups starting from day 2 of the treatment until completion of the study with dose-related intensity of discoloration. This observation was due to staining properties of the test item.
No further test item-related clinical signs or observations were noted in males or females at any dose level.
Incidentally, in one male (no. 16) at the dose level of 100 mg/kg bw/day chromodacryorrhea was noted during the study (starting on day 1 of the pre-pairing period) and eye reduced in size was noted in the same animal from day 13 of the pre-pairing period.
No further test item-related findings were noted at any dose level.

FINDINGS AT DETAILED WEEKLY CLINICAL OBSERVATIONS
No test item-related findings were noted during detailed weekly clinical observations.
The only findings noted were chromodacryorrhea and eye reduced in size in male no. 16 at the dose level of 100 mg/kg bw/day recorded already during the daily clinical observations.

FUNCTIONAL OBSERVATIONAL BATTERY
No test item-related findings were noted during the functional observational battery tests in males or females at any dose level.
Statistically significantly lower body temperature was noted in both sexes. In males mean body temperature was 37.9 °C and 37.8 °C at the high- and mid-dose levels, respectively, compared to 38.4 °C in the control group. In females, 38.5 °C was noted at the high-dose level, compared to 38.9 °C in the control group. The differences noted in males and females were only minor, not clearly dose dependent and all values remained in the historical control range. For these reasons, changes in body temperature were considered not to be test item-related.
No further findings were noted during functional observational battery in males or females at any dose level except for the eye findings in male no. 16 at 100 mg/kg bw/day.

LOCOMOTOR ACTIVITY
No effects on locomotor activity were noted in males or females at any dose level.
Mean beam counts during the 30 minutes of measurement at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: 1255, 1137, 1219 and 1209 in males and 924, 882, 1002 and 1050 in females.

FOOD CONSUMPTION OF MALES: No effects on food consumption were noted in males at any dose level.
Mean differences in food consumption at the dose levels 100, 300 and 1000 mg/kg bw/day were respectively: +1.1%, -3.4% and -3.4% during the pre-pairing period and -1.5%, -3.1% and -3.8% during the after pairing period ( percentages refer to the respective values in the control group).

FOOD CONSUMPTION OF FEMALES: No test item-related effects on food consumption were noted in females at any dose level.
Incidentally, statistically significantly higher food consumption was noted at the dose level of 100 mg/kg bw/day during lactation period. In the absence of an effect in females at the dose levels of 300 and 1000 mg/kg bw/day, this difference was considered not to be related to the treatment.
Mean differences in food consumption at the dose levels 100, 300 and 1000 mg/kg bw/day were respectively: +5.9%, +2.7%, and +1.1% during the pre-pairing period, +7.0%, +2.5% and +2.9% during the gestation period and +23.2%, -5.9% and +11.0% during the lactation period (percentages refer to the respective values in the control group).

BODY WEIGHTS OF MALES
At the dose level of 1000 mg/kg bw/day, a slightly lower body weight gain if compared to the controls was noted during the pre-pairing period. Mean body weight gain within this period was +10%, compared to +13% in the control group. The difference in body weight gain was statistically significant during the most days starting from day 3 until the end of the pre-pairing period. This effect was considered to be test item-related. During the pairing and after pairing periods, body weight gain was similar at all dose levels.
No significant changes in body weights were noted in males at any time during the study.
Because the lower body weight gain at the high-dose level was reversible despite treatment continued and did not result in any significant changes in body weights, this finding was considered not to be adverse.
No significant changes in body weight gain or body weights were noted in males at the dose levels of 100 and 300 mg/kg bw/day.
Mean differences in body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: +13%, +13%, +11% and +10% during the pre-pairing period, +10%, +10%, +9% and +9% during the pairing period and +7%, +6%, +7% and +6% during the after pairing period (percentages refer to the body weight change within the respective period).

BODY WEIGHTS OF FEMALES
Body weights and body weight gain of females were not affected by the treatment with the test item at any dose level.
On individual days some statistically significantly changed values of body weight gain were noted at the low-, mid- and high-dose levels. The changes did not follow a dose dependency and were therefore not related to the treatment.
Mean differences in body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: 5%, 7%, 6% and 7% during the pre-pairing period, 48%, 56%, 47% and 54% during the gestation period and 3%, 8%, 4% and 5% during the lactation period (percentages refer to the body weight change within the respective period).

2. CLINICAL LABORATORY INVESTIGATIONS
HEMATOLOGY
No test item-related effects on hematology parameters were noted in males or females at any dose level.
In males, statistically significant changes of several parameters: higher distribution width of red cell volume (RDW) at the low-dose level and higher distribution width of hemoglobin concentration (HDW) at the low- and mid-dose levels occurred in the absence of an effect at the high dose and therefore were considered not to be test item-related.
In females, at the low-dose level, statistically significantly higher platelets count was noted in the absence of any increase of this value at the mid- and high-dose levels and therefore it was not test item-related.
No further changes of hematology parameters were noted in males or females at any dose level.

CLINICAL BIOCHEMISTRY
No test item-related effects on biochemistry parameters were noted in males or females at any dose level.
In males, at the mid-dose level, statistically significantly lower concentration of triglycerides was noted. In the absence of dose dependency, this finding was not test item-related.
In females at the low dose level, following statistically significant changes were noted: higher concentration of cholesterol, higher concentration of globulin, and lower globulin to albumin ratio. These changes were not dose-dependent and therefore they were considered not to be test item-related.
No further changes of biochemistry parameters were noted in either males or females at any dose level.

URINALYSIS
No changes in urine parameters were noted in males at any dose level.

3. TERMINAL FINDINGS - PARENTAL ANIMALS
SEMINOLOGY AND SPERMATID COUNT
In all dose groups, statistically significant changes in motility of sperms were noted. Following values were assessed in sperm samples at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day respectively: 81.1%, 64.6%, 57.3% and 47.5% of progressive sperms (changes were statistically significant in all dose groups), 3.7%, 11.3%, 6.0% and 11.9% of stationary sperms (changes were statistically significant at the dose levels of 1000 and 100 mg/kg bw/day) and 15.2%, 24.1, 36.7 and 40.6% of not motile sperms (changes were statistically significant at the dose levels of 1000 and 300 mg/kg bw/day). These changes might be test item-related. However no significant dose dependent trend indicated by probability values of <0.05 was determined for any of these changes when performing a linear regression analysis (least squares).

No further changes were noted during sperm analysis. At the high-dose level, all morphological categories of sperms were represented with similar frequency to that in the control group whereas sperm count was similar to the respective control values in samples from both testis and epididymidis.

ORGAN WEIGHTS
No changes in absolute organ weights or organ weights to body weights and to brain weights ratios were noted in males or females at any dose level.

MACROSCOPICAL FINDINGS
Type and distribution of findings noted during macroscopical examination of males or females did not indicate any test item-related effect.

HISTOPATHOLOGY FINDINGS
Under the conditions of this experiment, treatment with test item did not cause pathological findings. All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.


Dose descriptor:
NOAEL
Remarks:
P (for general toxicity)
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 1000 mg/kg bw/day, the highest dose level tested.
Dose descriptor:
NOAEL
Remarks:
P (for reproduction)
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 1000 mg/kg bw/day, the highest dose level tested.
Dose descriptor:
NOAEL
Remarks:
F1 (for development)
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 1000 mg/kg bw/day, the highest dose level tested.
Critical effects observed:
not specified

1. REPRODUCTION, BREEDING AND PUP DATA

SUMMARY OF PERFORMANCE

P Animals Breeding for F1 Litters

Group
(mg/kg/day)

1
(0)

2
(100)

3
(300)

4
(100)

Female numbers

45-55

56-66

67-77

78-88

Number of females paired

11

11

11

11

Number of females mated

11

11

11

11

Number of non pregnant females (A)

3

1

3

0

Numbers of pregnant females,
which did not deliver any pups (B)

0

0

0

1

Number of females which reared their pups until day 4 post partum

8

10

8

10

(A)  Female Nos. 45, 46, 55, 62, 74, 75 and 77.

(B)  Female No. 85 had implantations only.

 

MATING PERFORMANCE AND FERTILITY

 Mating performance and fertility were not affected by the treatment at any dose level.

All females in groups 2, 3 and 4 mated within the first pairing period. In group 1, one female (no. 54) was mated during the second pairing period.

 Mean (median) precoital times were 4.5 (3), 2.5 (3), 4.0 (2) and 2.6 (3) days at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

 Seven females were not pregnant: three in the control group and in the mid-dose level and one in the low-dose level. Consequently, fertility indexes (number of females pregnant as percentages of females paired) and conception rate (number of females pregnant as percentages of females mated) were 72.7%, 90.9%, 72.7% and 100.0% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

 

One female at the high dose level had one implantation site but delivered no pups. Consequently, gestation index (number of females with living pups as percentages of females pregnant) was 100% in the control group and at low- and mid-dose levels and 90.9% at the high-dose level.

 

CORPORA LUTEA COUNT

 No test item-related effects on corpora lutea count were observed at any dose level.

Mean number of corpora lutea per dam was 16.0, 17.2, 16.3 and 18.4 in order of ascending dose levels.

 

DURATION OF GESTATION

 No effects on duration of gestation were observed at any dose level.

 Mean duration of gestation was 21.6, 21.6, 21.5 and 21.7 days, in order of ascending dose level.

 

IMPLANTATION RATE AND POST-IMPLANTATION LOSS

 No effects on implantation rate and post-implantation loss were observed at any dose level.

 In order of ascending dose levels, mean number of implantations per dam was 12.6, 14.9, 12.6 and 14.0 whereas mean incidence of post-implantation loss per dam was 1.5, 0.8, 0.6 and 0.5 per dam.

 

LITTER SIZE AT FIRST LITTER CHECK

 No effects on litter size were noted at any dose level.

 During the first litter check, one dead pup was found in a litter at the dose level of 1000 mg/kg bw/day. Because of isolated occurrence, this finding was considered to be incidental.

 Mean number of living pups per dam at first litter check was 11.1, 14.3, 12.0 and 13.5 in order of ascending dose levels.

 Birth index (number of pups born alive as a percentage of implantations) was 88.1%, 94.8%, 95.0% and 96.4% at the dose level of 0, 100, 300 and 1000 mg/kg bw/day.

 Birth index at the dose level of 1000 mg kg bw/day was statistically significantly higher than the respective control value. This was considered to be a result of biological variability.

 

POSTNATAL LOSS DAYS 0 - 4 POST PARTUM

 No test item-related effects on postnatal loss were noted at any dose level.

 In the control group one pup was missing on day 4, at the low-dose level one pup was missing on day 2, at the mid dose level three pups (from two litters) were missing on day 2 and at the high dose level no postnatal loss was noted in any litter.

 Mean postnatal loss per dam during four days of lactation was 0.1%, 0.1%, 0.4% and 0.0% at the dose level of 0, 100, 300 and 1000 mg/kg bw/day, respectively. Consequently, viability index (number of pups alive at termination on day 4 p.p. as a percentage of pups born alive) was 98.9%, 99.3%, 96.9% and 100% in order of ascending dose levels.

 

EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION

 No test item-related findings were noted in pups during first litter check and during lactation at any dose level.

 Incidentally, one pup in the control group was found with a wound and missing tail tip, two further pups, each one at the low- and mid-dose levels, had a wound at first litter check. These findings were also noted during the remaining lactation period.

 

SEX RATIOS

 Pups sex ratio was not affected by exposure to the test item at any dose level.

 At first litter check, percentages of male pups were 56%, 48%, 51% and 56% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day.

 

 BODY WEIGHTS TO DAY 4 POST PARTUM

 Body weights and body weight gain of pups were not affected by the treatment with the test item at any dose level.

Mean body weights of pups on day 1 post partum were: 6.4 g, 6.1 g, 6.4 and 6.2 g and mean differences in body weights during lactation were +49.9%, +43.6%, +47.8% and +42.6%, at the dose levels of 0, 100, 300 and 1000 mg/kg/day, respectively.

 

At the dose levels of 100 and 1000 mg/kg bw/day, slightly not statistically significantly lower body weight gain of pups was noted. This effect was considered to be due to a higher number of pups at these dose levels which was supported by observation that reduction of body weight gain was more pronounced in litters of higher size. Therefore this effect was considered not to be test item-related.

 

MACROSCOPICAL FINDINGS

 No test item-related findings were noted at macroscopic examination of pups at any dose level.

 Incidentally, in the control group one pup had a sore in the thoraco-dorsal region, one further pup in this group had a missing tail tip. These findings were already recorded during the in life phase. At the high-dose level, one pup had a watery cyst in the left kidney.

 No further findings were noted in pups at any dose level.

 

Conclusions:
This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item to rats (according to OECD 422, GLP compliant). The test item was administered in vehicle (highly purified water) at dosages of 0, 100, 300, and 1000 mg/kg body weight/day, animals in control groups received the vehicle only. Test item was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.
Under the conditions of this study, no adverse effects were found in males or females up to the highest dose level of 1000 mg/kg bw/day.

All animals survived the scheduled study period.
During the treatment, faeces stained red with dose-dependent intensity of discoloration were noted in all males and females receiving test material. This observation was due to staining properties of the test item.

No effects on food consumption were noted in males at any dose level. Body weight gain was slightly but statistically significantly reduced in males at the dose level of 1000 mg/kg bw/day during the pre-pairing period. No differences in body weight gain were noted at any dose level during the remaining study period. Body weights of males in all dose groups were similar to the respective control values during the entire study period. Because lower body weight gain at the high-dose levels was reversible and did not cause significant changes in body weights, this effect was considered not to be adverse.

Food consumption, body weights and body weight gain of females were not affected by the treatment at any dose level.

No further test item-related observations were noted in males or females at any dose level during the live part of the study.

Terminal examinations revealed changes in motility of sperms in all dose groups. Statistically significant decrease in mean count of progressive sperms was noted at the dose levels of 100, 300 and 1000 mg/kg bw/day, statistically significant increase in mean count of stationary sperms was noted at the dose levels of 100 and 1000 mg/kg bw/day and statistically significant increase in mean count in not motile sperms was noted at the dose levels of 300 and 1000 mg/kg bw/day. However a significant dose dependent trend indicated by probability values of <0.05 was not established for any of these changes when performing a linear regression analysis (least squares).
No further effects on male reproductive system were noted during the study. Sperm morphology and sperm count at the high-dose level was similar to the control values. Weights of male reproductive organs, macroscopical and histopathological examination of testes and epididymides gave no indication of any treatment-related effect. Further, no indication of effects on reproduction was noted within this study up to and including the highest dose level. For this reason, changes in motility of sperms were considered not to be adverse in this study.


Reproduction and development were not affected by the treatment. Mating performance, fertility, duration of gestation, corpora lutea count, implantation rate, post implantation and postnatal loss or litter size were similar in the control and all dose groups. There were no test item-related findings in pups noted during the first litter check, the first 4 days post partum or during the necropsy, pups body weights and body weight gain were not affected by the treatment at any dose level.

Based on these results, the NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females and for reproduction/developmental toxicity in this study was considered to be 1000 mg/kg bw/day, the highest dose level tested.
Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of the test itemon the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provides information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

 

The test item was administered to male rats for 39 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

 

The following dose levels were applied:

Group 1:                        0 mg/kg body weight/day (control group)

Group 2:                    100 mg/kg body weight/day

Group 3:                    300 mg/kg body weight/day

Group 4:                   1000 mg/kg body weight/day

 

A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (highly purified water).

 

The following results were obtained:

 MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

 All animals survived the scheduled study period. Feces stained red with dose-dependent intensity of discoloration were noted in all males and females in all dose groups starting from day 2 of the treatment until completion of the study. This observation was due to staining properties of the test item.

 No further test item-related clinical signs or observations were noted in males or females at any dose level.

 

FUNCTIONAL OBSERVATIONAL BATTERY IN PARENTAL ANIMALS

 No test item-related findings were noted during the functional observational battery tests in males or females at any dose level.

 

FOOD CONSUMPTION OF PARENTAL ANIMALS

 No effects on food consumption were noted in males or females at any dose level.

 

BODY WEIGHTS OF PARENTAL ANIMALS

 In males at the dose level of 1000 mg/kg bw/day, a slight but statistically significant lower body weight gain was noted during the pre-pairing period. No differences in body weight gain were noted at any dose level during the remaining study period. Body weights of males in all dose groups were similar to the respective control values during the entire study period. Because reduction in body weight gain at the high-dose levels was reversible and did not cause significant changes in body weights, this effect was considered not to be adverse.

 Body weights and body weight gain of females were not affected by the treatment at any dose level.

 

CLINICAL LABORATORY INVESTIGATIONS IN PARENTAL ANIMALS

 No test item-related effects on hematology and clinical biochemistry parameters were noted in males or females at any dose level.

 No changes in urine parameters were noted in males at any dose level.

 

REPRODUCTION AND BREEDING DATA

 Mating performance, fertility, corpora lutea count, duration of gestation, implantation rate and post-implantation loss, litter size or postnatal loss were not affected by the treatment with the test item.

 

SEMINOLOGY AND SPERMATID COUNT IN PARENTAL MALES

Effects on sperm motility which might be test item-related were noted in all dose groups. Mean count of progressive sperms was statistically significantly reduced at the dose levels of 1000, 300 and 100 mg/kg bw/day, mean count of stationary sperms was statistically significantly increased at the dose levels of 1000 and 100 mg/kg bw/day and mean count of not motile sperms was statistically significantly increased at the dose level of 1000 and 300 mg/kg bw/day. But a significant dose dependent trend couldn’t be established.

In the absence of any findings during necropsy or histopathological examination of male reproductive organs as well as in the absence of any effects on reproduction, the differences in sperm motility were considered not to be adverse.

ORGAN WEIGHTS OF PARENTAL ANIMALS

 No changes in absolute organ weights or organ weights to body weights and to brain weights ratios were noted in males or females at any dose level.

 

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATIONS OF PARENTAL ANIMALS

 Type and distribution of findings noted during macroscopical examination did not indicate any test item-related effect.

 Treatment with the test item did not cause pathological findings. All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

 

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

 No test item-related findings were noted in pups during first litter check and during lactation at any dose level.

 Pups sex ratio was not affected by the exposure to the test item at any dose level.

 

PUP WEIGHTS TO DAY 4 POST PARTUM

 Body Weights and body weight gain of pups were not affected by the treatment with the test item at any dose level.

 

MACROSCOPICAL FINDINGS OF PUPS

 No test item-related findings were noted at macroscopic examination of pups at any dose level.

 

CONCLUSION

Based on these results, the NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females and for reproduction/developmental toxicity in this study was considered to be 1000 mg/kg bw/day, the highest dose level tested.

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 422, GLP-compliant)
Justification for type of information:
See read across justification document in chapter 13
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Tocixity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Source: Harlan Laboratories, Inc., Maasheseweg 87c, 5800 AN Vernay / Netherlands
- Number of Animals: 44 males (11 per group) and 44 females (11 per group)
- Age (at Start of Treatment): 11 weeks
- Body Weight Range (at Start of Treatment): 301 to 362 g (males), 216 to 247 g (females)
- Identification: Parent animals had cage card and individual animal number (ear tattoo), pups were individually tattooed with Indian ink on day 1 post partum
- Randomization: Performed after at least three days of acclimatization using a computer-generated random algorithm. Body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Accommodation: In groups of five in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V., Netherlands), batch/lot nos. 02105111001, 02105111201, 02105120301 and 6960C.CS-100099). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 80/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
Standard laboratory conditions, continuously monitored.
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 to 70
- Air changes (per hr): . Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12 (with at least eight hours music during the light period)
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
DOSE FORMULATIONS
The dose formulations were prepared weekly using the test item as supplied by the Sponsor.
The test item was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using a magnetic stirrer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATIONS
Dose formulations were stored at room temperature (20 +/- 5 °C) in glass beakers.
Based upon the results of stability analyses performed within the non-GLP Harlan Laboratories study (Dose Range-Finding Study for a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test in the Han Wistar Rat), dose formulations were stable for at least 8 days if stored at room temperature.

TREATMENT
- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of studies.
- Frequency of Administration: Once daily
- Target Dose Levels: 0 mg/kg/day (control group), 100 mg/kg/day (group 2), 300 mg/kg/day (group 3) and 1000 mg/kg/day (group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous non-GLP dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories Study D33711, using dose levels of 0, 100, 300 and 1000 mg/kg/ day, where no adverse effects were observed up to and including the highest dose level.
- Dose Volume: 10 mL/kg body weight
- Dose Concentrations: 0 mg/mL/day (control group), 10 mg/mL/day (group 2), 30 mg/mL/day (group 3) and 100 mg/mL/day (group 4).
- Duration of Acclimatization Period: 7 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
METHOD
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 0.5 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 0.5 g of each concentration were taken from the middle only to confirm stability (8 days). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 +/- 5 °C) and delivered on dry ice to the responsible for formulation analysis (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 +/- 5 °C until analysis.
The samples were analyzed by UV-VIS spectroscopy following an analytical procedure developed at Harlan Laboratories. The test item was used as the analytical standard.

RESULTS
Blank samples showed no significant absorbance and, therefore, it was confirmed that only highly purified water was applied within the control experiment.
The application formulations investigated during the study were found to comprise test material in the range of 93.1% to 105.6% and, thus, the required content limit of +/-20% with reference to the nominal content was met. The homogeneous distribution of test item in the preparations was approved because single results found did not deviate more than 5.5% (<15%) from the corresponding mean.
The test item was found to be stable in application formulations when kept eight days at 20 +/- 5 °C due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item and highly purified water as vehicle during this study. Application formulations were found to be homogeneously prepared and stable over a storage period of eight days (20 +/- 5 °C).
Duration of treatment / exposure:
MALES: 40 days
FEMALES: Approximately 7 weeks
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
11

Control animals:
yes, concurrent vehicle
Details on study design:
MALES
- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Treatment Ends: On day before sacrifice
- Blood Sampling: After 28 days of Treatment
- Necropsy: After treatment for 39 days, when no longer needed for assessment of reproductive effects

FEMALES
- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Gestation: Approximately 21 days
- Treatment Ends: On day 4 post partum
- Blood Sampling: Day 5 post partum
- Necropsy: On day 5 post partum (pups on day 4 post partum)
Positive control:
Not required
Observations and examinations performed and frequency:
VIABILITY/MORTALITY: Twice daily

CLINICAL SIGNS
Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION
Males: on days 1 - 4, 4 - 8, 8 - 11 and 11 - 14 during pre-pairing period and weekly during after pairing period.
Females: on days 1 - 4, 4 - 8, 8 - 11 and 11 - 14 during pre-pairing period; on days 0 - 7, 7 14 and 14 - 21 during gestation period and on days 1 - 4 of during lactation period.
No food consumption was recorded during the pairing period.

BODY WEIGHTS: Recorded daily from treatment start to day of necropsy.

DETAILED CLINICAL OBSERVATIONS
Detailed clinical observations were performed outside the home cage in all animals. In males, it was performed once prior to the first administration of the test item and weekly thereafter. In females, it was performed once prior to the first administration of the test item, weekly during the pre-pairing and pairing periods and on days 0, 6, 13 and 20 of the gestation period.
Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

FUNCTIONAL OBSERVATIONAL BATTERY
At one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 post partum) relevant parameters were performed with five P generation males and five P generation females from each group. This FOB assessment was conducted following the daily dose administration. Animals were observed for the following:
- Cage-side observations: faeces-balls, urine and posture as well as resistance to removal.
- Hand-held observations: muscle tone, constituation, skin, pupile size, palpebral closure, lacrimation, salivation, reaction to handling and general abnormalities.
- Open field observations: level of ambulatory activity including rearing (one minute evaluation), unusual body movements (e.g. spasms, convulsions), gait evaluation, behavior, hair coat, respiration, quantity of faeces-balls and urine.
- Reflexes: blinking, palpebral closure, pinna reflex, extensor thrust response, paw pinch, responsiveness to sharp noise, righting reflex and hearing ability (Preyer’s reflex).
- Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.

Any abnormal findings were recorded and, where appropriate, graded in severity.

Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.

CLINICAL LABORATORY INVESTIGATIONS
Blood samples were obtained on the day of the scheduled necropsy from 5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum. Blood samples were drawn sublingually from all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

The following hematology parameters were determined:
- Erythrocyte count
- Hemoglobin
- Hematocrit
- Mean corpuscular volume
- Red cell volume distribution width
- Mean corpuscular hemoglobin
- Mean corpuscular hemoglobin concentration
- Hemoglobin concentration distribution width
- Leukocyte count, total
- Differential leukocyte count
- Platelet count
- Prothrombin time (= Thromboplastin time)
- Activated partial Thromboplastin time

The following clinical biochemistry parameters were determined:
- Glucose
- Urea
- Creatinine
- Bilirubin, total
- Cholesterol, total
- Triglycerides
- Aspartate aminotransferase
- Alanine aminotransferase
- Alkaline phosphatase
- Gamma-glutamyl-transferase
- Bile acids
- Sodium
- Potassium
- Chloride
- Calcium
- Phosphorus
- Protein, total
- Albumin
- Globulin
- Albumin/Globulin ratio

URINALYSIS
The following urinalysis parameters were determined in five males of each group, which are allocated to the blood analysis, during the last week of the study using timed urine volume collection:
- Volume (18 hours)
- Specific gravity (relative density)
- Color
- Appearance
- pH
- Nitrite
- Osmolality
- Protein
- Glucose
- Ketones
- Urobilinogen
- Bilirubin
- Blood/Blood cells
Sacrifice and pathology:
TERMINATION AND NECROPSY

Males were sacrificed after treatment for 39 days, when no longer needed for the assessment of reproductive effects. Dams were sacrificed on day 5 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

All animals sacrificed were subjected to a detailed macroscopic examination. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. At the scheduled sacrifice, all animals were sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated. All parent animals were examined macroscopically for any structural changes. For the parent animals, special attention was directed at the organs of the reproductive system.The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

SEMINOLOGY AND SPERMATID COUNT
Sperm analysis was performed on the first 5 males per group.

Motility:
At necropsy of adult males an epididymal sperm sample was obtained from the left cauda epididymidis of each male. The sample was diluted with a pre-warmed (about 35 °C) physiological medium, and shortly after being obtained, one hundred sperm were counted microscopically for determination of percentage of not motile, stationary motile and progressively motile sperm.

Morphology:
A second sperm sample from the left cauda epididymidis was used for morphological assessment after fixation and Eosin staining. 500 sperm per sample were evaluated microscopically and classified into the following categories:
A: Normal, complete sperm
B: Normal head only (tail detached)
C: Complete sperm, misshapen hook
D: Complete sperm, abnormally curved hook
E: Complete sperm, reversed head
F: Abnormal head only (tail detached)

Morphological sperm evaluation was performed only for group 1 and 4 males. In the absence of a treatment-related effect the slides for the group 2 and 3 males were not evaluated.

Sperm, Spermatid Count:
The left caudal epididymis and left testis were taken for determination of homogenization-resistant spermatids and caudal epididymal sperm reserve. These tissues were frozen at -20 +/- 5 °C pending evaluation. For evaluation the weighed tissues were placed in Triton-X-100 solution and homogenized with a blender (Ultra Turrax) and an ultrasonic water bath. Sperm or spermatid heads were counted microscopically using a modified Neubauer chamber. These evaluations were performed in the first instance only for group 1 and 4 males. In the absence of a treatment-related effect the remaining frozen tissues were not evaluated.

ORGAN WEIGHTS
At the scheduled sacrifice, testes and epididymides from all parental males were weighed separately. In addition, from 5 males and 5 females sacrificed at the end of the study which were selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken.
- Adrenal glands (weighed as pairs)
- Brain
- Heart
- Kidneys (weighed as pairs)
- Liver
- Thymus
- Spleen

TISSUE PRESERVATION
The following tissues from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Prostate
- Seminal vesicles with coagulating gland
- Testes (in Bouin’s fixative)*
- Epididymides (in Bouin’s fixative)*
*From the first five males in each group which were used for sperm analysis, only the right testis and right epididymis were preserved for histopathological examination.

The following tissues from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Ovaries

In addition, from 5 males and 5 females per group selected for organ weights, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Gross lesions
- Brain (representative regions including cerebrum, cerebellum and pons)
- Spinal chord
- Small and large intestines (incl. Peyer’s patches)
- Stomach
- Liver
- Kidneys
- Adrenals
- Spleen
- Heart
- Thymus
- Thyroids, and parathyroids if possible
- Trachea and lungs (preserved by inflation with fixative and then immersion)
- Uterus (with vagina)
- Urinary bladder
- Lymph nodes (mesenterial, mandibular)
- Peripheral nerve (sciatic)
- Bone marrow

HISTOTECHNIQUE
All organ and tissue samples to be examined by the study pathologist were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin. Special stains were used at the discretion of the study pathologist.

HISTOPATHOLOGY
Slides of all organs and tissues listed collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the study pathologist. The same applied to all occurring gross lesions.
Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, where necessary.
A histopathology peer review was performed. A histopathology phase report was provided by the principal investigator which was included in the report.
Other examinations:
MATING, GESTATION, LACTATION
During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed. The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum. For a female which did not mate during the 14-day pairing period, a second pairing of this female with a male in the same group, which had already mated successfully, was performed. All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

REPRODUCTIVE AND OFFSPRING VIABILITY INDICES
From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, post-implantation losses, mean litter size, pup sex ratios and viability indices.

LITTER OBSERVATIONS
The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.

POSTMORTEM EXAMINATION OF OFFSPRING
Pups were sacrificed on day 4 post partum. All animals were sacrificed by by an injection of sodium pentobarbital and subjected to a detailed macroscopic examination. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. Pups found dead during the study, except those excessively cannibalized, were examined macroscopically.

Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied when the variables could be dichotomized without loss of information.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Red stained faeces were noted in all males and females in dose groups; this was due to the staining properties of the test item
Mortality:
mortality observed, treatment-related
Description (incidence):
Red stained faeces were noted in all males and females in dose groups; this was due to the staining properties of the test item
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In males at the dose level of 1000 mg/kg bw/day, lower body weight gain during the pre-pairing period
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:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Locomotor activity was not affected and functional observational battery gave no indication of a test item-related effect.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
1. IN-LIFE DATA OF PARENTAL ANIMALS
VIABILITY / MORTALITY
All animals survived scheduled study period.

DAILY CLINICAL SIGNS OR OBSERVATIONS
Red stained feces was noted in all males and females in all dose groups starting from day 2 of the treatment until completion of the study with dose-related intensity of discoloration. This observation was due to staining properties of the test item.
No further test item-related clinical signs or observations were noted in males or females at any dose level.
Incidentally, in one male (no. 16) at the dose level of 100 mg/kg bw/day chromodacryorrhea was noted during the study (starting on day 1 of the pre-pairing period) and eye reduced in size was noted in the same animal from day 13 of the pre-pairing period.
No further test item-related findings were noted at any dose level.

FINDINGS AT DETAILED WEEKLY CLINICAL OBSERVATIONS
No test item-related findings were noted during detailed weekly clinical observations.
The only findings noted were chromodacryorrhea and eye reduced in size in male no. 16 at the dose level of 100 mg/kg bw/day recorded already during the daily clinical observations.

FUNCTIONAL OBSERVATIONAL BATTERY
No test item-related findings were noted during the functional observational battery tests in males or females at any dose level.
Statistically significantly lower body temperature was noted in both sexes. In males mean body temperature was 37.9 °C and 37.8 °C at the high- and mid-dose levels, respectively, compared to 38.4 °C in the control group. In females, 38.5 °C was noted at the high-dose level, compared to 38.9 °C in the control group. The differences noted in males and females were only minor, not clearly dose dependent and all values remained in the historical control range. For these reasons, changes in body temperature were considered not to be test item-related.
No further findings were noted during functional observational battery in males or females at any dose level except for the eye findings in male no. 16 at 100 mg/kg bw/day.

LOCOMOTOR ACTIVITY
No effects on locomotor activity were noted in males or females at any dose level.
Mean beam counts during the 30 minutes of measurement at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: 1255, 1137, 1219 and 1209 in males and 924, 882, 1002 and 1050 in females.

FOOD CONSUMPTION OF MALES: No effects on food consumption were noted in males at any dose level.
Mean differences in food consumption at the dose levels 100, 300 and 1000 mg/kg bw/day were respectively: +1.1%, -3.4% and -3.4% during the pre-pairing period and -1.5%, -3.1% and -3.8% during the after pairing period ( percentages refer to the respective values in the control group).

FOOD CONSUMPTION OF FEMALES: No test item-related effects on food consumption were noted in females at any dose level.
Incidentally, statistically significantly higher food consumption was noted at the dose level of 100 mg/kg bw/day during lactation period. In the absence of an effect in females at the dose levels of 300 and 1000 mg/kg bw/day, this difference was considered not to be related to the treatment.
Mean differences in food consumption at the dose levels 100, 300 and 1000 mg/kg bw/day were respectively: +5.9%, +2.7%, and +1.1% during the pre-pairing period, +7.0%, +2.5% and +2.9% during the gestation period and +23.2%, -5.9% and +11.0% during the lactation period (percentages refer to the respective values in the control group).

BODY WEIGHTS OF MALES
At the dose level of 1000 mg/kg bw/day, a slightly lower body weight gain if compared to the controls was noted during the pre-pairing period. Mean body weight gain within this period was +10%, compared to +13% in the control group. The difference in body weight gain was statistically significant during the most days starting from day 3 until the end of the pre-pairing period. This effect was considered to be test item-related. During the pairing and after pairing periods, body weight gain was similar at all dose levels.
No significant changes in body weights were noted in males at any time during the study.
Because the lower body weight gain at the high-dose level was reversible despite treatment continued and did not result in any significant changes in body weights, this finding was considered not to be adverse.
No significant changes in body weight gain or body weights were noted in males at the dose levels of 100 and 300 mg/kg bw/day.
Mean differences in body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: +13%, +13%, +11% and +10% during the pre-pairing period, +10%, +10%, +9% and +9% during the pairing period and +7%, +6%, +7% and +6% during the after pairing period (percentages refer to the body weight change within the respective period).

BODY WEIGHTS OF FEMALES
Body weights and body weight gain of females were not affected by the treatment with the test item at any dose level.
On individual days some statistically significantly changed values of body weight gain were noted at the low-, mid- and high-dose levels. The changes did not follow a dose dependency and were therefore not related to the treatment.
Mean differences in body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: 5%, 7%, 6% and 7% during the pre-pairing period, 48%, 56%, 47% and 54% during the gestation period and 3%, 8%, 4% and 5% during the lactation period (percentages refer to the body weight change within the respective period).

2. CLINICAL LABORATORY INVESTIGATIONS
HEMATOLOGY
No test item-related effects on hematology parameters were noted in males or females at any dose level.
In males, statistically significant changes of several parameters: higher distribution width of red cell volume (RDW) at the low-dose level and higher distribution width of hemoglobin concentration (HDW) at the low- and mid-dose levels occurred in the absence of an effect at the high dose and therefore were considered not to be test item-related.
In females, at the low-dose level, statistically significantly higher platelets count was noted in the absence of any increase of this value at the mid- and high-dose levels and therefore it was not test item-related.
No further changes of hematology parameters were noted in males or females at any dose level.

CLINICAL BIOCHEMISTRY
No test item-related effects on biochemistry parameters were noted in males or females at any dose level.
In males, at the mid-dose level, statistically significantly lower concentration of triglycerides was noted. In the absence of dose dependency, this finding was not test item-related.
In females at the low dose level, following statistically significant changes were noted: higher concentration of cholesterol, higher concentration of globulin, and lower globulin to albumin ratio. These changes were not dose-dependent and therefore they were considered not to be test item-related.
No further changes of biochemistry parameters were noted in either males or females at any dose level.

URINALYSIS
No changes in urine parameters were noted in males at any dose level.

3. TERMINAL FINDINGS - PARENTAL ANIMALS
SEMINOLOGY AND SPERMATID COUNT
In all dose groups, statistically significant changes in motility of sperms were noted. Following values were assessed in sperm samples at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day respectively: 81.1%, 64.6%, 57.3% and 47.5% of progressive sperms (changes were statistically significant in all dose groups), 3.7%, 11.3%, 6.0% and 11.9% of stationary sperms (changes were statistically significant at the dose levels of 1000 and 100 mg/kg bw/day) and 15.2%, 24.1, 36.7 and 40.6% of not motile sperms (changes were statistically significant at the dose levels of 1000 and 300 mg/kg bw/day). These changes might be test item-related. However no significant dose dependent trend indicated by probability values of <0.05 was determined for any of these changes when performing a linear regression analysis (least squares).

No further changes were noted during sperm analysis. At the high-dose level, all morphological categories of sperms were represented with similar frequency to that in the control group whereas sperm count was similar to the respective control values in samples from both testis and epididymidis.

ORGAN WEIGHTS
No changes in absolute organ weights or organ weights to body weights and to brain weights ratios were noted in males or females at any dose level.

MACROSCOPICAL FINDINGS
Type and distribution of findings noted during macroscopical examination of males or females did not indicate any test item-related effect.

HISTOPATHOLOGY FINDINGS
Under the conditions of this experiment, treatment with test item did not cause pathological findings. All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.


Dose descriptor:
NOAEL
Remarks:
P (for general toxicity)
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 1000 mg/kg bw/day, the highest dose level tested.
Dose descriptor:
NOAEL
Remarks:
P (for reproduction)
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 1000 mg/kg bw/day, the highest dose level tested.
Dose descriptor:
NOAEL
Remarks:
F1 (for development)
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 1000 mg/kg bw/day, the highest dose level tested.
Critical effects observed:
not specified

1. REPRODUCTION, BREEDING AND PUP DATA

SUMMARY OF PERFORMANCE

P Animals Breeding for F1 Litters

Group
(mg/kg/day)

1
(0)

2
(100)

3
(300)

4
(100)

Female numbers

45-55

56-66

67-77

78-88

Number of females paired

11

11

11

11

Number of females mated

11

11

11

11

Number of non pregnant females (A)

3

1

3

0

Numbers of pregnant females,
which did not deliver any pups (B)

0

0

0

1

Number of females which reared their pups until day 4 post partum

8

10

8

10

(A)  Female Nos. 45, 46, 55, 62, 74, 75 and 77.

(B)  Female No. 85 had implantations only.

 

MATING PERFORMANCE AND FERTILITY

 Mating performance and fertility were not affected by the treatment at any dose level.

All females in groups 2, 3 and 4 mated within the first pairing period. In group 1, one female (no. 54) was mated during the second pairing period.

 Mean (median) precoital times were 4.5 (3), 2.5 (3), 4.0 (2) and 2.6 (3) days at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

 Seven females were not pregnant: three in the control group and in the mid-dose level and one in the low-dose level. Consequently, fertility indexes (number of females pregnant as percentages of females paired) and conception rate (number of females pregnant as percentages of females mated) were 72.7%, 90.9%, 72.7% and 100.0% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

 

One female at the high dose level had one implantation site but delivered no pups. Consequently, gestation index (number of females with living pups as percentages of females pregnant) was 100% in the control group and at low- and mid-dose levels and 90.9% at the high-dose level.

 

CORPORA LUTEA COUNT

 No test item-related effects on corpora lutea count were observed at any dose level.

Mean number of corpora lutea per dam was 16.0, 17.2, 16.3 and 18.4 in order of ascending dose levels.

 

DURATION OF GESTATION

 No effects on duration of gestation were observed at any dose level.

 Mean duration of gestation was 21.6, 21.6, 21.5 and 21.7 days, in order of ascending dose level.

 

IMPLANTATION RATE AND POST-IMPLANTATION LOSS

 No effects on implantation rate and post-implantation loss were observed at any dose level.

 In order of ascending dose levels, mean number of implantations per dam was 12.6, 14.9, 12.6 and 14.0 whereas mean incidence of post-implantation loss per dam was 1.5, 0.8, 0.6 and 0.5 per dam.

 

LITTER SIZE AT FIRST LITTER CHECK

 No effects on litter size were noted at any dose level.

 During the first litter check, one dead pup was found in a litter at the dose level of 1000 mg/kg bw/day. Because of isolated occurrence, this finding was considered to be incidental.

 Mean number of living pups per dam at first litter check was 11.1, 14.3, 12.0 and 13.5 in order of ascending dose levels.

 Birth index (number of pups born alive as a percentage of implantations) was 88.1%, 94.8%, 95.0% and 96.4% at the dose level of 0, 100, 300 and 1000 mg/kg bw/day.

 Birth index at the dose level of 1000 mg kg bw/day was statistically significantly higher than the respective control value. This was considered to be a result of biological variability.

 

POSTNATAL LOSS DAYS 0 - 4 POST PARTUM

 No test item-related effects on postnatal loss were noted at any dose level.

 In the control group one pup was missing on day 4, at the low-dose level one pup was missing on day 2, at the mid dose level three pups (from two litters) were missing on day 2 and at the high dose level no postnatal loss was noted in any litter.

 Mean postnatal loss per dam during four days of lactation was 0.1%, 0.1%, 0.4% and 0.0% at the dose level of 0, 100, 300 and 1000 mg/kg bw/day, respectively. Consequently, viability index (number of pups alive at termination on day 4 p.p. as a percentage of pups born alive) was 98.9%, 99.3%, 96.9% and 100% in order of ascending dose levels.

 

EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION

 No test item-related findings were noted in pups during first litter check and during lactation at any dose level.

 Incidentally, one pup in the control group was found with a wound and missing tail tip, two further pups, each one at the low- and mid-dose levels, had a wound at first litter check. These findings were also noted during the remaining lactation period.

 

SEX RATIOS

 Pups sex ratio was not affected by exposure to the test item at any dose level.

 At first litter check, percentages of male pups were 56%, 48%, 51% and 56% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day.

 

 BODY WEIGHTS TO DAY 4 POST PARTUM

 Body weights and body weight gain of pups were not affected by the treatment with the test item at any dose level.

Mean body weights of pups on day 1 post partum were: 6.4 g, 6.1 g, 6.4 and 6.2 g and mean differences in body weights during lactation were +49.9%, +43.6%, +47.8% and +42.6%, at the dose levels of 0, 100, 300 and 1000 mg/kg/day, respectively.

 

At the dose levels of 100 and 1000 mg/kg bw/day, slightly not statistically significantly lower body weight gain of pups was noted. This effect was considered to be due to a higher number of pups at these dose levels which was supported by observation that reduction of body weight gain was more pronounced in litters of higher size. Therefore this effect was considered not to be test item-related.

 

MACROSCOPICAL FINDINGS

 No test item-related findings were noted at macroscopic examination of pups at any dose level.

 Incidentally, in the control group one pup had a sore in the thoraco-dorsal region, one further pup in this group had a missing tail tip. These findings were already recorded during the in life phase. At the high-dose level, one pup had a watery cyst in the left kidney.

 No further findings were noted in pups at any dose level.

 

Conclusions:
This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item to rats (according to OECD 422, GLP compliant). The test item was administered in vehicle (highly purified water) at dosages of 0, 100, 300, and 1000 mg/kg body weight/day, animals in control groups received the vehicle only. Test item was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.
Under the conditions of this study, no adverse effects were found in males or females up to the highest dose level of 1000 mg/kg bw/day.

All animals survived the scheduled study period.
During the treatment, faeces stained red with dose-dependent intensity of discoloration were noted in all males and females receiving test material. This observation was due to staining properties of the test item.

No effects on food consumption were noted in males at any dose level. Body weight gain was slightly but statistically significantly reduced in males at the dose level of 1000 mg/kg bw/day during the pre-pairing period. No differences in body weight gain were noted at any dose level during the remaining study period. Body weights of males in all dose groups were similar to the respective control values during the entire study period. Because lower body weight gain at the high-dose levels was reversible and did not cause significant changes in body weights, this effect was considered not to be adverse.

Food consumption, body weights and body weight gain of females were not affected by the treatment at any dose level.

No further test item-related observations were noted in males or females at any dose level during the live part of the study.

Terminal examinations revealed changes in motility of sperms in all dose groups. Statistically significant decrease in mean count of progressive sperms was noted at the dose levels of 100, 300 and 1000 mg/kg bw/day, statistically significant increase in mean count of stationary sperms was noted at the dose levels of 100 and 1000 mg/kg bw/day and statistically significant increase in mean count in not motile sperms was noted at the dose levels of 300 and 1000 mg/kg bw/day. However a significant dose dependent trend indicated by probability values of <0.05 was not established for any of these changes when performing a linear regression analysis (least squares).
No further effects on male reproductive system were noted during the study. Sperm morphology and sperm count at the high-dose level was similar to the control values. Weights of male reproductive organs, macroscopical and histopathological examination of testes and epididymides gave no indication of any treatment-related effect. Further, no indication of effects on reproduction was noted within this study up to and including the highest dose level. For this reason, changes in motility of sperms were considered not to be adverse in this study.


Reproduction and development were not affected by the treatment. Mating performance, fertility, duration of gestation, corpora lutea count, implantation rate, post implantation and postnatal loss or litter size were similar in the control and all dose groups. There were no test item-related findings in pups noted during the first litter check, the first 4 days post partum or during the necropsy, pups body weights and body weight gain were not affected by the treatment at any dose level.

Based on these results, the NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females and for reproduction/developmental toxicity in this study was considered to be 1000 mg/kg bw/day, the highest dose level tested.
Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of the test itemon the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provides information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

 

The test item was administered to male rats for 39 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

 

The following dose levels were applied:

Group 1:                        0 mg/kg body weight/day (control group)

Group 2:                    100 mg/kg body weight/day

Group 3:                    300 mg/kg body weight/day

Group 4:                   1000 mg/kg body weight/day

 

A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (highly purified water).

 

The following results were obtained:

 MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

 All animals survived the scheduled study period. Feces stained red with dose-dependent intensity of discoloration were noted in all males and females in all dose groups starting from day 2 of the treatment until completion of the study. This observation was due to staining properties of the test item.

 No further test item-related clinical signs or observations were noted in males or females at any dose level.

 

FUNCTIONAL OBSERVATIONAL BATTERY IN PARENTAL ANIMALS

 No test item-related findings were noted during the functional observational battery tests in males or females at any dose level.

 

FOOD CONSUMPTION OF PARENTAL ANIMALS

 No effects on food consumption were noted in males or females at any dose level.

 

BODY WEIGHTS OF PARENTAL ANIMALS

 In males at the dose level of 1000 mg/kg bw/day, a slight but statistically significant lower body weight gain was noted during the pre-pairing period. No differences in body weight gain were noted at any dose level during the remaining study period. Body weights of males in all dose groups were similar to the respective control values during the entire study period. Because reduction in body weight gain at the high-dose levels was reversible and did not cause significant changes in body weights, this effect was considered not to be adverse.

 Body weights and body weight gain of females were not affected by the treatment at any dose level.

 

CLINICAL LABORATORY INVESTIGATIONS IN PARENTAL ANIMALS

 No test item-related effects on hematology and clinical biochemistry parameters were noted in males or females at any dose level.

 No changes in urine parameters were noted in males at any dose level.

 

REPRODUCTION AND BREEDING DATA

 Mating performance, fertility, corpora lutea count, duration of gestation, implantation rate and post-implantation loss, litter size or postnatal loss were not affected by the treatment with the test item.

 

SEMINOLOGY AND SPERMATID COUNT IN PARENTAL MALES

Effects on sperm motility which might be test item-related were noted in all dose groups. Mean count of progressive sperms was statistically significantly reduced at the dose levels of 1000, 300 and 100 mg/kg bw/day, mean count of stationary sperms was statistically significantly increased at the dose levels of 1000 and 100 mg/kg bw/day and mean count of not motile sperms was statistically significantly increased at the dose level of 1000 and 300 mg/kg bw/day. But a significant dose dependent trend couldn’t be established.

In the absence of any findings during necropsy or histopathological examination of male reproductive organs as well as in the absence of any effects on reproduction, the differences in sperm motility were considered not to be adverse.

ORGAN WEIGHTS OF PARENTAL ANIMALS

 No changes in absolute organ weights or organ weights to body weights and to brain weights ratios were noted in males or females at any dose level.

 

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATIONS OF PARENTAL ANIMALS

 Type and distribution of findings noted during macroscopical examination did not indicate any test item-related effect.

 Treatment with the test item did not cause pathological findings. All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

 

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

 No test item-related findings were noted in pups during first litter check and during lactation at any dose level.

 Pups sex ratio was not affected by the exposure to the test item at any dose level.

 

PUP WEIGHTS TO DAY 4 POST PARTUM

 Body Weights and body weight gain of pups were not affected by the treatment with the test item at any dose level.

 

MACROSCOPICAL FINDINGS OF PUPS

 No test item-related findings were noted at macroscopic examination of pups at any dose level.

 

CONCLUSION

Based on these results, the NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females and for reproduction/developmental toxicity in this study was considered to be 1000 mg/kg bw/day, the highest dose level tested.

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 with restrictions: read across

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
repeated dose toxicity: inhalation, other
Remarks:
28-Day Inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 March 2021 to 12 October 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
adopted on 25 June 2018
GLP compliance:
yes (incl. QA statement)
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Rat is the preferred laboratory rodent species for inhalation toxicity assessment and is also recommended by various regulatory guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 7 weeks
- Weight at study initiation: Males: 140.06 g to 159.97 g; Females: 120.63 g to 139.41 g
- Fasting period before study: No
- Housing: Maximum of three animals per cage were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized corn cob was provided as bedding material.
- Diet (e.g. ad libitum): Altromin Maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG).
- Water (e.g. ad libitum): Deep bore-well water passed through a reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: 7 Days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6°C to 22.9°C
- Humidity (%): 46% to 65%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 15 March 2021 To: 10 August 2021
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 2 - ca. 2 µm
Geometric standard deviation (GSD):
3
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow-past, nose-only dynamic inhalation exposure system supplied by CH Technologies, USA.
- Method of holding animals in test chamber: The animals were confined separately in restraint tubes which were positioned radially around the flow-past, nose-only dynamic inhalation exposure chamber.
- Source and rate of air: Air flow from the generation system into the chamber was controlled and monitored through control panel (supplied by CH Technologies, USA) using calibrated flow-meters (rotameters) and outlet of the chamber air flow was controlled by rotameter. The actual flow rate of chamber inlet was 20 L/min with 60 psi pressure and outlet air from chamber was 15 L/min maintained throughout the exposure period. The air inlet flow rate was recorded hourly once during exposure (i.e. ± 10 minutes) after equilibration period (T95) 0.11min.
- Method of conditioning air: Compressed air
- System of generating particulates/aerosols: The Rotating Brush Generator (Palas RBG 1000 - supplied by Palas GmbH) was used to generate the dust particles (aerosols) into an airborne state.
- Temperature, humidity, pressure in air chamber: 22 ± 3°C, 30 to 70%, 60psi
- Air flow rate: 20L/min
- Air change rate: 12 to 15 air changes per cycle
- Method of particle size determination: Cascade impactor
- Treatment of exhaust air: NaOH

TEST ATMOSPHERE
- Brief description of analytical method used: No
- Samples taken from breathing zone: yes
Duration of treatment / exposure:
Animals were exposed to aerosolized test item continuously for 6 hours per day, 5 days per week, for a total of 20 actual exposures, after equilibration period of the chamber concentration.
Following the 28 days exposure period, the animals in the recovery groups (G1R and G4R) were not given any treatment and was maintained for 56 days post treatment period and observed for reversibility or persistence of toxic effects.
Frequency of treatment:
6 hours per day, 5 days per week
Dose / conc.:
0 mg/L air (nominal)
Remarks:
Air Only
Dose / conc.:
0.001 mg/L air (nominal)
Remarks:
Low Dose
Dose / conc.:
0.003 mg/L air (nominal)
Remarks:
Mid Dose
Dose / conc.:
0.03 mg/L air (nominal)
Remarks:
High Dose
No. of animals per sex per dose:
5 Males + 5 Females
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale:
- Rationale for selecting satellite groups: Following the 28 days exposure period, the animals in the recovery groups (G1R and G4R) were not given any treatment and was maintained for 56 days post treatment period and observed for reversibility or persistence of toxic effects.
- Post-exposure recovery period in satellite groups: 56 Days
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All the animals were observed for clinical signs and pre-terminal deaths at 1.5 hrs (±10 mins), 3 hrs (±10 mins), 4.5 hrs (±10 mins) and 6 hrs (±10 mins) during exposure period. Post-exposure clinical signs and 30 to 40 minutes and 1 hour (±10 mins) following exposure to the test chemical, thereafter once a day for clinical signs and twice daily for mortality.
- Cage side observations checked in table No.1

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once in a week

BODY WEIGHT: Yes
- Time schedule for examinations: Individual animal body weights were recorded at receipt, prior to exposure (Day 1) and twice weekly thereafter for first two weeks. Since there was no significant body weight effects in the first two weeks, body weights were recorded weekly thereafter (post exposure period) for remaining period of the study. Fasting body weight of all the animals of all the groups were recorded at their scheduled terminal sacrifice.

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: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examination was carried out once during acclimatization (Pre-treatment) and during week 4 in control and high dose group animals. No, treatment related ocular changes are noted in high dose during week 4, the examination was not extended to lower dose groups (G2, G3) of main study animals and during week 12 for recovery group animals (G1R and G4R).
- Dose groups that were examined: G1/G1R and G4/G4R

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected from all the surviving rats of each group on day 27 (within 24 hours after the last given dose) and day 85 of recovery groups respectively.
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: 60
- Parameters checked in table [No.10] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected from all the surviving rats of each group on day 27 (within 24 hours after the last given dose) and day 85 of recovery groups respectively.
- Animals fasted: Yes
- How many animals: 60
- Parameters checked in table [No.12] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected and analyzed from all rats on day 27 (within 24 hours after the last given dose). The animals were kept in urine collection cages overnight for urine collection. Animal was not given access to feed but water was provided ad libitum during their stay in urine collection cages.
The recovery group animal’s urine was collected on day 85.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.13] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Neurological/Functional examination was carried out during Week 4 and 12 for vehicle control and high dose animals. Since no apparent treatment related effects persisted in high dose animals, the examination was not carried out for low and mid dose groups (G2 and G3) during Week 4 of treatment period.
- Dose groups that were examined: G1/G1R and G4/G4R
- Battery of functions tested: sensory activity / grip strength / motor activity.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table No. 19)
HISTOPATHOLOGY: Yes (see table No. 20)
Statistics:
The raw data were subjected to statistical analysis. The computer printout of the data (in the form of the appendix) was verified with the original raw data. After verification, the data were subjected to statistical analysis using SPSS Software version 22. Body weight, percent change in body weight with respect to Day 1, feed consumption, FOB, organ weight ratios, hematology, and clinical chemistry estimations, urine volume, pH, and specific gravity were subjected to statistical analysis. One-way ANOVA followed by Dunnett’s post-test was done for different treatment groups comparing with the control group data. All analyses and comparisons were evaluated at the 95% level of confidence (P<0.05). Statistically significant changes obtained from the aforementioned tests were designated by the superscripts in the summary table throughout the study report, as stated below:
*: Statistically significant (p<0.05).
Clinical signs:
no effects observed
Description (incidence and severity):
All animals survived to scheduled sacrifice, and there were no clinical signs attributable to the test item. Post-exposure, all animals were normal throughout the experimental period. No clinical signs were noted during the recovery period.
Mortality:
no mortality observed
Description (incidence):
All animals survived to scheduled sacrifice, and there were no mortality attributable to the test item. Post-exposure, all animals were normal throughout the experimental period. No mortalities were noted during the recovery period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No statistically significant treatment-related variations were observed in all the tested dose groups when compared with control group.
Food efficiency:
no effects observed
Description (incidence and severity):
No statistically significant treatment-related variations were observed in all the tested dose groups when compared with control group.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ocular abnormalities were observed during ophthalmoscopic examination.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant treatment related changes in haematology parameters were noted. However, the following statistically significant variations were noted.
In main group males, decrease in HCT (G2) was noted. In females decrease in APTT (G2, G3 and G4), MCV and MCH (G2 and G4) was noted.
In recovery males decrease in Percent monocytes (G4R) was noted. However, in female no significance observed.
All the noted variations in main group are considered incidental in the absence of dose responsiveness. Variations noted at the end of recovery period are considered incidental as no such variations were noted in recovery females.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant changes were observed in clinical chemistry parameters when compared to the vehicle control group. However, the following statistically significant variations were noted.
In main group males, increase in chloride (G4) was noted; In females no significance was noted.
In recovery group no significance was observed.
The observed variations noted are considered incidental due to lack of dose responsiveness and/or could be due to random biological variation.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No adverse treatment-related effects were observed in urinalysis parameters when compared to the control group. However, a single incidence of a statistically significant decrease in specific gravity was observed in G4R females. This effect, which was minimal in nature, lacked dose-dependency and was therefore not attributed to the test item.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no adverse effects during neurological/functional examinations were observed in main group (G1 and G4) and recovery group (G1R and G4R), however in females (G4R) excessive grooming was noted, this is considered as incidental due to lack of dose response.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
In main group males, decrease in absolute weight of testis in G2; increase in absolute and relative weight of lungs in G2 and G4 was noted. In females decrease in absolute and relative weight of spleen in G3 was noted.
In recovery group, increase in absolute and relative weight of thymus (G4R); decrease in absolute and relative weight in spleen (G4R) was noted. In females, increase in absolute and relative weight of ovaries (G4R) were noted.
In main group males, statistically significant increase in relative weight of lungs in G2 and G4 was noted. In females, decrease in relative weight of spleen (G3) and liver (G2); increase in relative weight of lungs (G4) were noted.
In recovery group males, decrease in relative weight of spleen (G4R); increase in relative weight of thymus (G4R) was noted. In females increase in relative weight of ovaries (G4R) was noted.
All the observed changes are considered incidental in the absence of gross and histopathological changes in the high dose group animals and also lack dose dependency.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related gross pathological lesions were observed in main group and recovery group animals.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related, microscopic findings in animals evaluated after terminal and recovery period in the study.
Few microscopic findings observed in this study such as ultimobranchial cyst in thyroid gland, epithelial cysts in thymus and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected in laboratory rats.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
BALF Fluid: The bronchio-alveolar lavage fluid measured at termination revealed the following statistically significant changes: In main group males, increase in percent eosinophils and basophils (G3) was noted; In females, increase in percent monocytes (G4) was noted.
In recovery group, females increase in LDH (G4R) was noted; however, in males no significance was noted.
There were no test item-related microscopic findings in animals evaluated after terminal and recovery period in the study and the noted variations are considered non adverse effect.
Key result
Dose descriptor:
NOAEC
Effect level:
>= 0.03 - ca. 0.03 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food efficiency
gross pathology
haematology
histopathology: non-neoplastic
mortality
ophthalmological examination
organ weights and organ / body weight ratios
urinalysis
other: BALF findings
Key result
Critical effects observed:
no

TABLE 1.   SUMMARY OF CLINICAL SIGNS AND MORTALITY RECORD

Group, Sex & Dose

(mg/L)

No. of

Animals

Clinical Signs of Toxicity

Mortality

(No. of Incidences/

No. of Animals)

G1, M & 0

5

N

0/5

G2, M & 0.001

5

N

0/5

G3, M & 0.003

5

N

0/5

G4, M & 0.03

5

N

0/5

G1R, M & 0

5

N

0/5

G4R, M & 0.03

5

N

0/5

M: Male; N: None.

TABLE 1 (Contd…). SUMMARY OF CLINICAL SIGNS AND MORTALITY RECORD

Group, Sex & Dose

(mg/L)

No. of

Animals

Clinical Signs of Toxicity

Mortality

(No. of Incidence/

No. of Animals)

G1, F & 0

5

N

0/5

G2, F & 0.001

5

N

0/5

G3, F & 0.003

5

N

0/5

G4, F & 0.03

5

N

0/5

G1R, F & 0

5

N

0/5

G4R, F & 0.03

5

N

0/5

F: Female; N: None.

TABLE 2.  SUMMARY OF BODY WEIGHTS (g) RECORD

Group, Sex & Dose (mg/L)

Body Weight (g) on Days

1

5

8

12

15

22

26

G1, M & 0

Mean

187.88

191.93

205.03

209.22

220.95

234.65

241.23

±SD

14.19

14.38

13.69

13.43

13.45

13.00

12.55

n

5

5

5

5

5

5

5

G2, M & 0.001

Mean

183.58

187.41

199.18

203.48

214.50

228.10

235.87

±SD

14.65

14.59

13.65

13.86

13.30

14.17

14.78

n

5

5

5

5

5

5

5

G3, M & 0.003

Mean

182.16

185.60

197.45

201.89

214.35

228.81

235.88

±SD

15.60

15.80

14.39

13.60

13.57

14.20

13.89

n

5

5

5

5

5

5

5

G4, M & 0.03

Mean

180.15

183.99

196.54

201.16

213.32

227.63

235.65

±SD

18.66

18.08

17.15

16.54

15.36

14.94

14.81

n

5

5

5

5

5

5

5

G1, F & 0

Mean

152.09

155.06

161.34

163.66

172.01

182.69

189.18

±SD

12.49

12.10

10.71

10.09

10.81

11.06

10.92

n

5

5

5

5

5

5

5

G2, F & 0.001

Mean

153.99

157.68

163.82

166.47

175.30

184.89

190.90

±SD

11.36

11.61

11.46

11.06

11.32

11.43

11.75

n

5

5

5

5

5

5

5

G3, F & 0.003

Mean

149.86

153.07

160.44

162.55

170.62

180.12

186.94

±SD

11.55

12.04

11.07

10.79

10.78

10.64

11.15

n

5

5

5

5

5

5

5

G4, F & 0.03

Mean

151.90

154.59

160.20

162.81

170.66

180.51

186.39

±SD

8.04

7.91

8.06

7.73

7.95

9.00

9.00

n

5

5

5

5

5

5

5

M: Male; F: Female; SD: Standard Deviation; n: Number of animals.

TABLE 2 (Contd...). SUMMARY OF BODY WEIGHTS (g) RECORD

Group, Sex & Dose (mg/L)

Body Weight (g) on Days

1

5

8

12

15

22

26

29

36

43

50

57

64

71

78

84

G1R, M & 0

Mean

184.58

188.92

201.70

205.86

217.92

232.09

242.91

250.80

272.02

294.25

317.10

339.64

362.85

386.35

411.61

432.52

±SD

10.63

11.00

10.49

9.66

9.90

9.61

9.14

9.32

9.71

10.66

10.84

10.19

10.54

10.56

10.87

10.69

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

182.85

186.62

199.13

203.34

215.44

230.12

243.82

251.75

272.84

295.09

317.85

340.56

363.70

386.94

412.13

433.06

±SD

12.36

12.69

11.96

13.79

13.22

13.17

12.42

12.49

11.83

12.80

13.47

13.68

13.72

13.78

14.37

14.48

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

151.42

154.17

159.60

162.05

170.53

180.30

187.50

192.72

208.02

223.72

239.22

254.79

271.47

288.34

305.89

321.44

±SD

12.00

11.75

10.93

10.98

10.61

8.85

8.38

8.72

9.15

8.87

9.39

9.33

9.70

9.90

9.79

9.86

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

151.49

154.31

160.21

162.55

173.23

182.00

189.45

194.62

209.88

225.43

241.03

256.53

273.35

290.30

307.74

323.36

±SD

11.10

11.31

10.58

9.78

11.74

11.05

10.57

10.69

10.98

10.90

10.94

10.91

11.02

10.83

10.45

10.36

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: Number of animals.

TABLE 3.  SUMMARY OF PERCENT CHANGE IN BODY WEIGHT WITH RESPECT TO DAY 1 RECORD

Group, Sex & Dose (mg/L)

Percent Change in Body Weight (%) during Days

1 to 5

1 to 8

1 to 12

1 to 15

1 to 22

1 to 26

G1, M & 0

Mean

2.16

9.19

11.44

17.71

25.05

28.59

±SD

0.52

1.38

1.95

2.09

2.93

3.31

n

5

5

5

5

5

5

G2, M & 0.001

Mean

2.10

8.58

10.93

16.97

24.38

28.62

±SD

0.42

1.46

1.86

2.19

2.28

2.25

n

5

5

5

5

5

5

G3, M & 0.003

Mean

1.89

8.50

10.98

17.87

25.84

29.76

±SD

0.53

1.67

2.17

3.16

3.61

4.10

n

5

5

5

5

5

5

G4, M & 0.03

Mean

2.18

9.25

11.86

18.72

26.75

31.25

±SD

0.62

1.77

2.42

3.76

4.74

5.28

n

5

5

5

5

5

5

G1, F & 0

Mean

1.99

6.19

7.75

13.25

20.30

24.59

±SD

0.53

1.70

2.28

2.33

2.66

3.28

n

5

5

5

5

5

5

G2, F & 0.001

Mean

2.40

6.41

8.15

13.90

20.15

24.06

±SD

0.28

0.53

1.01

1.18

2.05

1.85

n

5

5

5

5

5

5

G3, F & 0.003

Mean

2.13

7.12

8.54

13.96

20.34

24.89

±SD

0.72

1.16

1.75

2.21

3.07

2.90

n

5

5

5

5

5

5

G4, F & 0.03

Mean

1.78

5.48

7.22

12.39

18.86

22.74

±SD

0.29

0.59

1.35

1.44

0.90

1.30

n

5

5

5

5

5

5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: Number of animals.

TABLE 3 (Contd...). SUMMARY OF PERCENT CHANGE IN BODY WEIGHT WITH RESPECT TO DAY 1 RECORD

Group, Sex & Dose (mg/L)

Percent Change in Body Weight (%) during Days

1 to 5

1 to 8

1 to 12

1 to 15

1 to 22

1 to 26

1 to 29

1 to 36

1 to 43

1 to 50

1 to 57

1 to 64

1 to 71

1 to 78

1 to 84

G1R, M & 0

Mean

2.35

9.31

11.59

18.14

25.84

31.73

36.01

47.54

59.58

71.99

84.25

96.85

109.61

123.32

134.68

±SD

0.30

0.61

1.51

1.92

2.46

3.09

3.31

3.90

3.97

4.48

5.40

5.85

6.55

7.11

7.80

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

2.06

8.94

11.20

17.86

25.92

33.48

37.82

49.42

61.60

74.08

86.54

99.24

111.99

125.80

137.28

±SD

0.45

0.81

0.61

0.99

1.38

3.24

3.40

4.36

4.67

5.39

5.93

6.81

7.54

8.13

8.78

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

1.84

5.48

7.11

12.75

19.33

24.13

27.57

37.71

48.14

58.41

68.75

79.81

90.99

102.65

112.97

±SD

0.41

1.23

1.71

2.42

4.43

4.95

4.92

5.32

6.27

6.72

7.66

8.36

9.11

10.22

11.06

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

1.87

5.81

7.39

14.41

20.25

25.20

28.63

38.75

49.05

59.39

69.67

80.81

92.06

103.64

113.99

±SD

0.23

1.11

1.55

2.31

2.97

3.11

3.53

4.27

4.67

5.22

6.01

6.67

7.64

8.66

9.19

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: Number of animal.

TABLE 4.  SUMMARY OF AVERAGE FEED CONSUMPTION (g/rat/day) RECORD

Group, Sex & Dose (mg/L)

Week 1

Week 2

Week 3

Week 4

G1, M & 0

Mean

18.74

18.84

18.68

32.63

±SD

2.77

2.75

2.72

4.74

n

2

2

2

2

G2, M & 0.001

Mean

18.60

18.66

18.49

32.58

±SD

2.42

2.74

2.71

4.67

n

2

2

2

2

G3, M & 0.003

Mean

18.63

18.74

18.55

32.63

±SD

2.60

2.99

2.92

4.79

n

2

2

2

2

G4, M & 0.03

Mean

18.67

18.48

18.56

32.65

±SD

2.62

2.67

2.59

4.99

n

2

2

2

2

G1, F & 0

Mean

14.20

14.55

14.39

32.70

±SD

1.66

1.72

1.61

4.72

n

2

2

2

2

G2, F & 0.001

Mean

14.46

14.37

14.33

32.70

±SD

1.72

1.44

1.54

4.86

n

2

2

2

2

G3, F & 0.003

Mean

14.22

14.39

14.35

32.66

±SD

1.37

1.57

1.41

4.71

n

2

2

2

2

G4, F & 0.03

Mean

14.12

14.22

14.36

32.57

±SD

1.47

1.64

1.60

4.74

n

2

2

2

2

 M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: number of cages.

TABLE 4 (Contd...). SUMMARY OF AVERAGE FEED CONSUMPTION (g/rat/day) RECORD

Group, Sex & Dose (mg/L)

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

G1R, M & 0

Mean

18.58

18.64

18.42

18.94

18.81

19.28

19.29

19.31

19.34

19.25

19.39

22.32

±SD

2.54

2.78

2.63

3.14

3.13

3.09

3.30

3.52

3.64

3.55

3.65

4.15

n

2

2

2

2

2

2

2

2

2

2

2

2

G4R, M & 0.03

Mean

18.63

18.68

18.50

18.63

18.66

19.20

19.21

19.32

19.45

19.33

19.46

22.34

±SD

2.65

2.89

2.76

2.78

2.98

3.14

3.30

3.21

3.30

3.24

3.29

3.79

n

2

2

2

2

2

2

2

2

2

2

2

2

G1R, F & 0

Mean

14.24

14.26

14.25

14.15

14.30

15.14

15.26

15.30

15.57

15.58

15.67

18.02

±SD

1.57

1.28

1.39

1.84

1.88

1.82

1.84

2.09

2.21

2.01

1.97

2.33

n

2

2

2

2

2

2

2

2

2

2

2

2

G4R, F & 0.03

Mean

14.07

14.19

14.19

14.15

14.36

14.74

14.93

15.16

15.33

15.27

15.43

17.69

±SD

1.48

1.88

1.67

1.78

1.94

2.25

2.19

2.27

2.43

2.11

2.13

2.41

n

2

2

2

2

2

2

2

2

2

2

2

2

 M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: number of cages.

TABLE 5.  SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB)

Week 4

Parameters↓

Group & Sex

G1 & M

G4 & M

G1 & F

G4 & F

Dose (mg/L)

0

0.03

0

0.03

Number of Animals

5

5

5

5

Home cage observations

Home Cage posture

1

1

1

1

Respiratory pattern

1

1

1

1

Clonic involuntary movements

1

1

1

1

Tonic involuntary movements

1

1

1

1

Vocalization

1

1

1

1

Palpebral Closure

1

1

1

1

Handling observation

 

 

Ease of removal from the cage

2

2

2

2

Ease of handling animal in hand

2

2

2

2

Red or crusty deposits

Eyes

1

1

1

1

Nose

1

1

1

1

Mouth

1

1

1

1

Lacrimation

1

1

1

1

Salivation

1

1

1

1

Fur Appearance

1

1

1

1

Piloerection

1

1

1

1

Eye Prominence

1

1

1

1

Muscle Tone

1

1

1

1

Home cage observations: Home cage posture- 1=Normal,Respiratory Pattern -1=Normal,Clonic involuntary movements -1=None/Normal,Tonic involuntary movements -1=None/Normal,Vocalization -1=Absent (Normal),Palpebral closure -1=Eyelids wide open (Normal),Handling observation : Ease of removal from the cage -2=Normal,Ease of handling animal in hand -2=Normal,Red/Crusty deposits -1=Absent,Lacrimation- 1=None,Salivation-1=Absent (Normal),Fur Appearance -1=Normal,Piloerection -1=None, Eye Prominence -1=Normal,Muscle tone -1=Normal

M: Male; F: Female.

TABLE 5 (Contd...). SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB) RECORD

Week 4

Parameters↓

Group & Sex

G1 & M

G4 & M

G1 & F

G4 & F

Dose (mg/L)

0

0.03

0

0.03

Number of Animals

5

5

5

5

Open field Observation

Mobility

1

1

1

1

Gait

1

1

1

1

Arousal

3

3

3

3

Number of Rearing

Mean

7.4

6.6

7.8

7.6

±SD

1.5

0.9

1.9

1.1

Numbers of Urination

Mean

1.4

1.4

1.4

1.6

±SD

0.5

0.5

0.5

0.5

Number of Defecation

Mean

1.0

1.0

1.0

0.8

±SD

0.7

0.7

0.7

0.4

Clonic involuntary movement

1

1

1

1

Tonic involuntary movement

1

1

1

1

Stereotype Behaviour

1

1

1

1

Excessive Grooming

Mean

3.8

4.0

4.2

4.4

±SD

0.4

1.0

0.8

0.5

Sensory Observations

 

Approach Response

2

2

2

2

Auditory Response

2

2

2

2

Touch Response

2

2

2

2

Pupil Reflex

2

2

2

2

Tail Pinch Response

2

2

2

2

Righting Reflex

1

1

1

1

Physiological observation

 

Body temperature (°F)

Mean

98.2

98.2

98.5

98.5

±SD

0.2

0.2

0.1

0.1

Open field Observation: Mobility -1=Normal,Gait -1=Normal,Arousal -3=Normal,Clonic involuntary movements -1=None/Normal,Tonic involuntary movements -1=None/Normal,Stereotype Behaviour -1=Absent, Sensory Observations: Approach response -2=Normal, Auditory Response -2=Normal,Touch Response -2=Normal,Pupil Reflex -2=Normal,Tail Pinch Response -2=Normal,Righting Reflex -1=Present

TABLE 5 (Contd...). SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB) RECORD

Week 12

Parameters↓

Group & Sex

G1R & M

G4R & M

G1R & F

G4R & F

Dose (mg/L)

0

0.03

0

0.03

Number of Animals

5

5

5

5

Home cage observations

Home Cage posture

1

1

1

1

Respiratory pattern

1

1

1

1

Clonic involuntary movements

1

1

1

1

Tonic involuntary movements

1

1

1

1

Vocalization

1

1

1

1

Palpebral Closure

1

1

1

1

Handling observation

 

 

Ease of removal from the cage

2

2

2

2

Ease of handling animal in hand

2

2

2

2

Red or crusty deposits

Eyes

1

1

1

1

Nose

1

1

1

1

Mouth

1

1

1

1

Lacrimation

1

1

1

1

Salivation

1

1

1

1

Fur Appearance

1

1

1

1

Piloerection

1

1

1

1

Eye Prominence

1

1

1

1

Muscle Tone

1

1

1

1

Home cage observations: Home cage posture- 1=Normal,Respiratory Pattern -1=Normal,Clonic involuntary movements -1=None/Normal,Tonic involuntary movements -1=None/Normal,Vocalization -1=Absent (Normal),Palpebral closure -1=Eyelids wide open (Normal),Handling observation : Ease of removal from the cage -2=Normal,Ease of handling animal in hand -2=Normal,Red/Crusty deposits -1=Absent,Lacrimation- 1=None,Salivation-1=Absent (Normal),Fur Appearance -1=Normal,Piloerection -1=None, Eye Prominence -1=Normal,Muscle tone -1=Normal

M: Male; F: Female; R: Recovery.

TABLE 5 (Contd...). SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB) RECORD

Week 12

Parameters↓

Group & Sex

G1R & M

G4R & M

G1R & F

G4R & F

Dose (mg/L)

0

0.03

0

0.03

Number of Animals

5

5

5

5

Open field Observation

Mobility

1

1

1

1

Gait

1

1

1

1

Arousal

3

3

3

3

Number of Rearing

Mean

7.8

7.6

8.8

9.0

±SD

0.8

1.7

0.8

1.0

Numbers of Urination

Mean

1.2

1.2

1.0

1.4

±SD

0.8

0.4

0.7

0.5

Number of Defecation

Mean

1.0

1.0

1.2

1.0

±SD

0.7

0.7

0.4

0.7

Clonic involuntary movement

1

1

1

1

Tonic involuntary movement

1

1

1

1

Stereotype Behaviour

1

1

1

1

Excessive Grooming

Mean

4.2

3.4

4.0

5.4*

±SD

0.8

0.9

0.7

0.5

Sensory Observations

 

Approach Response

2

2

2

2

Auditory Response

2

2

2

2

Touch Response

2

2

2

2

Pupil Reflex

2

2

2

2

Tail Pinch Response

2

2

2

2

Righting Reflex

1

1

1

1

Physiological observation

 

Body temperature (°F)

Mean

97.9

97.9

98.7

98.7

±SD

0.2

0.2

0.2

0.2

Open field Observation: Mobility -1=Normal,Gait -1=Normal,Arousal -3=Normal,Clonic involuntary movements -1=None/Normal,Tonic involuntary movements -1=None/Normal,Stereotype Behaviour -1=Absent, Sensory Observations: Approach response -2=Normal, Auditory Response -2=Normal,Touch Response -2=Normal,Pupil Reflex -2=Normal,Tail Pinch Response -2=Normal,Righting Reflex -1=Present

M: Male; F: Female; R: Recovery; *: Statistically significant (p<0.05).

TABLE 6.  SUMMARY OF HIND LIMB FOOT SPLAY (cm) RECORD

# Week 4 & 12

Group, Sex & Dose (mg/L)

Hind Limb Foot Splay (cm)

G1, M & 0

Mean

6.0

±SD

0.4

n

5

G4, M & 0.03

Mean

5.7

±SD

0.2

n

5

G1, F & 0

Mean

6.4

±SD

0.5

n

5

G4, F & 0.03

Mean

5.9

±SD

0.6

n

5

G1R, M & 0

Mean

8.3

±SD

0.4

n

5

G4R, M & 0.03

Mean

7.8

±SD

0.5

n

5

G1R, F & 0

Mean

6.5

±SD

0.4

n

5

G4R, F & 0.03

Mean

5.8

±SD

0.8

n

5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: number of animals.

# Week 4 for main group animals and week 12 for recovery group animals.

TABLE 7. SUMMARY OF ACTIMETER READING RECORD

# Week 4 & 12

Group, Sex & Dose (mg/L)

Movement Counts (no.)

G1, M & 0

Mean

2114.4

±SD

61.2

n

5

G4, M & 0.03

Mean

2067.8

±SD

75.0

n

5

G1, F & 0

Mean

2179.2

±SD

71.7

n

5

G4, F & 0.03

Mean

2164.4

±SD

26.9

n

5

G1R, M & 0

Mean

2074.0

±SD

54.0

n

5

G4R, M & 0.03

Mean

2117.0

±SD

26.3

n

5

G1R, F & 0

Mean

2213.2

±SD

27.6

n

5

G4R, F & 0.03

Mean

2221.8

±SD

9.4

n

5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: number of animals.

# Week 4 for main group animals and week 12 for recovery group animals.

TABLE 8. SUMMARY OF GRIP STRENGTH (kgf) MEASUREMENT RECORD

# Week 4 & 12

Group, Sex & Dose (mg/L)

Fore limb

Hind limb

G1, M & 0

Mean

1.352

0.330

±SD

0.032

0.021

n

5

5

G4, M & 0.03

Mean

1.373

0.354

±SD

0.034

0.017

n

5

5

G1, F & 0

Mean

1.358

0.316

±SD

0.020

0.018

n

5

5

G4, F & 0.03

Mean

1.364

0.337

±SD

0.009

0.019

n

5

5

G1R, M & 0

Mean

1.563

0.536

±SD

0.036

0.020

n

5

5

G4R, M & 0.03

Mean

1.550

0.531

±SD

0.029

0.025

n

5

5

G1R, F & 0

Mean

1.418

0.371

±SD

0.053

0.023

n

5

5

G4R, F & 0.03

Mean

1.399

0.351

±SD

0.056

0.017

n

5

5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: number of animals.

# Week 4 for main group animals and week 12 for recovery group animals.

TABLE 9.     SUMMARY OF OPHTHALMOSCOPIC EXAMINATION RECORD

Week 4

Week 12

Group, Sex & Dose

(mg/L)

G1, M & 0

G4, M & 0.03

G1R, M & 0

G4R, M & 0.03

Number of Animals

05

05

05

05

Observations

Eye Parameters ↓

Eye

L

R

L

R

L

R

L

R

Eye Lids

N

N

N

N

N

N

N

N

Cornea

N

N

N

N

N

N

N

N

Iris

N

N

N

N

N

N

N

N

Aqueous Humour

N

N

N

N

N

N

N

N

Lens

N

N

N

N

N

N

N

N

Vitreous Humour

N

N

N

N

N

N

N

N

Retina/Optic disc

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N: No Abnormality Detected; L: Left; R: Right; M: Male; F: Female.

TABLE 9 (Contd…).  SUMMARY OF OPHTHALMOSCOPIC EXAMINATION RECORD

Week 4

Week 12

Group, Sex & Dose

(mg/L)

G1, F & 0

G4, F & 0.03

G1R, F & 0

G4R, F & 0.03

Number of Animals

05

05

05

05

Observations

Eye Parameters ↓

Eye

L

R

L

R

L

R

L

R

Eye Lids

N

N

N

N

N

N

N

N

Cornea

N

N

N

N

N

N

N

N

Iris

N

N

N

N

N

N

N

N

Aqueous Humour

N

N

N

N

N

N

N

N

Lens

N

N

N

N

N

N

N

N

Vitreous Humour

N

N

N

N

N

N

N

N

Retina/Optic disc

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N: No Abnormality Detected; L: Left; R: Right; F: Female.

TABLE 10.     SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose (mg/L)

Total Leucocyte

Count

Total Erythrocyte

Count

Hemoglobin

Haematocrit

Mean Corpuscular

Volume

Mean Corpuscular

Hemoglobin

Mean Corpuscular

Hemoglobin Concentration

Platelet Count

Mean Platelet

Volume

(WBC)

(RBC)

(HGB)

(HCT)

(MCV)

(MCH)

(MCHC)

(PLT)

(MPV)

(103cells/µL)

(106cells/µL)

(g/dL)

(%)

(fL)

(pg)

(g/dL)

(103cells/µL)

(fL)

G1, M & 0

Mean

10.44

8.93

17.14

53.50

60.02

19.22

32.06

972.00

5.90

±SD

2.75

0.41

0.82

2.05

3.62

1.14

0.84

131.79

0.25

n

5

5

5

5

5

5

5

5

5

G2, M & 0.001

Mean

9.54

8.57

15.86

50.28*

58.84

18.54

31.54

865.20

6.08

±SD

3.23

0.51

0.51

1.88

4.30

1.15

0.33

28.49

0.41

n

5

5

5

5

5

5

5

5

5

G3, M & 0.003

Mean

10.98

8.30

16.30

52.12

63.28

19.72

31.24

760.80

7.22

±SD

3.97

0.81

0.87

1.58

5.95

1.10

1.25

348.58

1.47

n

5

5

5

5

5

5

5

5

5

G4, M & 0.03

Mean

12.13

8.88

16.24

52.10

58.76

18.30

31.18

877.80

6.66

±SD

3.91

0.56

0.92

1.78

2.46

0.60

0.76

118.40

1.02

n

5

5

5

5

5

5

5

5

5

G1R, M & 0

Mean

9.06

8.85

16.46

50.18

56.66

18.60

32.86

835.00

6.78

±SD

2.95

0.22

0.74

2.10

2.28

0.89

0.48

171.39

0.22

n

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

8.17

9.08

16.00

48.82

53.80

17.62

32.78

757.20

6.70

±SD

1.98

0.33

0.85

2.43

2.94

1.04

0.55

350.57

0.22

n

5

5

5

5

5

5

5

5

5

M: Male; R: Recovery; SD: Standard Deviation; n: number of animals; *: Statistically significant (p<0.05).

TABLE 10 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose (mg/L)

Reticulocyte Count

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

Absolute Reticulocyte Count

(Retic)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(Retic)

(%)

(%)

(%)

(%)

(%)

(%)

(109cells/L)

G1, M & 0

Mean

4.03

20.72

72.38

4.02

0.66

1.02

356.90

±SD

1.89

4.02

3.94

1.89

0.42

0.38

160.41

n

5

5

5

5

5

5

5

G2, M & 0.001

Mean

4.09

27.06

65.18

5.10

0.68

0.80

342.64

±SD

1.83

2.48

3.20

0.62

0.13

0.10

124.07

n

5

5

5

5

5

5

5

G3, M & 0.003

Mean

7.29

21.04

71.66

4.18

0.94

1.06

571.88

±SD

5.52

6.75

7.23

1.17

0.27

0.55

374.97

n

5

5

5

5

5

5

5

G4, M & 0.03

Mean

4.66

21.72

71.72

4.16

0.88

0.62

406.30

±SD

2.03

6.20

6.74

0.88

0.58

0.22

156.82

n

5

5

5

5

5

5

5

G1R, M & 0

Mean

2.75

24.76

68.60

3.74

1.36

0.48

243.48

±SD

0.51

6.03

5.00

0.65

0.41

0.16

49.00

n

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

2.24

26.74

68.40

2.26*

1.68

0.28

203.34

±SD

0.23

5.60

4.67

0.61

0.71

0.13

17.59

n

5

5

5

5

5

5

5

 M: Male; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05).

TABLE 10 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose (mg/L)

Absolute

Neutrophils

Absolute

Lymphocytes

Absolute

Monocytes

Absolute

Eosinophils

Absolute

Basophils

Prothrombin

Time

Activated

Prothrombin Time

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(PT)

(APTT)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(Seconds)

(Seconds)

G1, M & 0

Mean

2.14

7.57

0.42

0.06

0.10

15.72

20.68

±SD

0.63

2.10

0.20

0.03

0.03

1.40

0.77

n

5

5

5

5

5

5

5

G2, M & 0.001

Mean

2.55

6.25

0.48

0.06

0.08

16.12

20.48

±SD

0.84

2.21

0.13

0.03

0.02

0.56

2.41

n

5

5

5

5

5

5

5

G3, M & 0.003

Mean

2.21

8.01

0.43

0.10

0.11

16.94

26.86

±SD

0.78

3.43

0.12

0.03

0.07

2.33

8.44

n

5

5

5

5

5

5

5

G4, M & 0.03

Mean

2.75

8.57

0.52

0.10

0.07

16.32

23.94

±SD

1.41

2.46

0.23

0.07

0.03

1.11

4.54

n

5

5

5

5

5

5

5

G1R, M & 0

Mean

2.12

6.32

0.36

0.13

0.04

15.32

21.20

±SD

0.29

2.46

0.17

0.06

0.02

1.49

1.80

n

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

2.14

5.63

0.19

0.13

0.03

17.96

26.26

±SD

0.43

1.63

0.09

0.05

0.02

5.13

6.31

n

5

5

5

5

5

5

5

M: Male; R: Recovery; SD: Standard Deviation; n: Number of animals.

TABLE 10 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose (mg/L)

Total

Leucocyte Count

Total Erythrocyte

Count

Hemoglobin

Haematocrit

Mean Corpuscular

Volume

Mean Corpuscular

Hemoglobin

Mean Corpuscular

Hemoglobin Concentration

Platelet

Count

Mean Platelet

Volume

(WBC)

(RBC)

(HGB)

(HCT)

(MCV)

(MCH)

(MCHC)

(PLT)

(MPV)

(103cells/µL)

(106cells/µL)

(g/dL)

(%)

(fL)

(pg)

(g/dL)

(103cells/µL)

(fL)

G1, F & 0

Mean

9.06

8.85

16.60

49.98

56.50

18.84

33.30

1023.80

6.02

±SD

2.15

0.71

0.75

3.61

1.04

1.19

2.14

93.66

0.13

n

5

5

5

5

5

5

5

5

5

G2, F & 0.001

Mean

9.75

9.77

17.18

52.46

53.74*

17.58*

32.74

1092.80

6.16

±SD

4.30

1.21

2.04

6.09

1.24

0.39

0.21

213.31

0.30

n

5

5

5

5

5

5

5

5

5

G3, F & 0.003

Mean

9.40

9.63

17.08

52.46

54.48

17.76

32.58

980.00

6.38

±SD

3.58

0.96

1.70

5.61

1.70

0.51

0.57

262.16

0.49

n

5

5

5

5

5

5

5

5

5

G4, F & 0.03

Mean

9.93

9.26

16.10

49.62

53.60*

17.38*

32.44

992.60

6.60

±SD

2.52

0.32

0.60

1.67

1.50

0.72

0.62

290.58

0.52

n

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

8.38

8.72

16.10

47.82

54.82

18.50

33.70

711.40

6.58

±SD

1.44

0.65

1.11

3.32

0.93

0.51

0.73

276.36

0.29

n

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

8.96

8.55

15.82

47.44

55.50

18.52

33.36

854.20

6.66

±SD

1.56

0.41

0.91

3.04

1.58

0.56

0.54

202.83

0.23

n

5

5

5

5

5

5

5

5

5

F: Female; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05).

TABLE 10 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose (mg/L)

Reticulocyte Count

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

Absolute Reticulocyte Count

(Retic)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(Retic)

(%)

(%)

(%)

(%)

(%)

(%)

(109cells/L)

G1, F & 0

Mean

2.43

21.12

72.62

3.46

1.26

0.64

214.50

±SD

0.43

4.43

4.70

0.96

0.68

0.24

40.96

n

5

5

5

5

5

5

5

G2, F & 0.001

Mean

1.72

27.40

66.90

2.64

1.32

0.84

166.78

±SD

0.56

6.67

7.47

0.66

0.34

0.61

47.63

n

5

5

5

5

5

5

5

G3, F & 0.003

Mean

2.15

24.56

70.40

2.38

1.06

0.76

204.00

±SD

0.54

5.33

5.11

1.22

0.43

0.62

37.56

n

5

5

5

5

5

5

5

G4, F & 0.03

Mean

1.91

26.70

67.40

2.78

1.58

0.44

176.24

±SD

0.61

3.96

5.05

0.88

0.64

0.11

51.51

n

5

5

5

5

5

5

5

G1R, F & 0

Mean

1.84

22.58

71.16

2.90

2.10

0.38

161.28

±SD

0.29

5.49

6.51

1.01

1.42

0.04

33.92

n

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

1.95

23.14

71.68

2.38

1.64

0.34

165.02

±SD

0.54

5.94

6.51

0.31

0.32

0.09

40.29

n

5

5

5

5

5

5

5

F: Female; R: Recovery; SD: Standard Deviation; n: Number of animals.

TABLE 10 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose (mg/L)

Absolute Neutrophils

Absolute Lymphocytes

Absolute Monocytes

Absolute Eosinophils

Absolute Basophils

Prothrombin Time

Activated Prothrombin Time

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(PT)

(APTT)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(Seconds)

(Seconds)

G1, F & 0

Mean

1.94

6.55

0.31

0.12

0.06

16.82

25.28

±SD

0.68

1.48

0.09

0.07

0.04

1.78

1.76

n

5

5

5

5

5

5

5

G2, F & 0.001

Mean

2.51

6.70

0.25

0.13

0.07

17.50

20.36*

±SD

0.74

3.44

0.09

0.06

0.03

3.15

1.60

n

5

5

5

5

5

5

5

G3, F & 0.003

Mean

2.24

6.66

0.25

0.09

0.08

16.44

19.46*

±SD

0.82

2.75

0.21

0.01

0.06

2.49

2.35

n

5

5

5

5

5

5

5

G4, F & 0.03

Mean

2.66

6.69

0.28

0.15

0.04

17.50

17.86*

±SD

0.80

1.70

0.13

0.06

0.01

4.63

2.02

n

5

5

5

5

5

5

5

G1R, F & 0

Mean

1.89

5.97

0.24

0.18

0.03

16.12

22.70

±SD

0.54

1.25

0.08

0.12

0.01

0.82

4.20

n

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

2.10

6.39

0.21

0.15

0.03

16.94

24.66

±SD

0.81

1.07

0.05

0.05

0.01

1.48

5.92

n

5

5

5

5

5

5

5

F: Female; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05).

TABLE 11. SUMMARY OF BALF RECORD

Group, Sex & Dose (mg/L)

Total Leucocyte Count

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

Absolute Neutrophils

(WBC)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(Neut)

(103cells/µL)

(%)

(%)

(%)

(%)

(%)

(103cells/µL)

G1, M & 0

Mean

0.36

17.16

73.14

2.00

0.24

1.60

0.06

±SD

0.16

5.63

7.02

1.27

0.29

1.21

0.03

n

5

5

5

5

5

5

5

G2, M & 0.001

Mean

0.54

22.56

67.02

2.66

0.70

3.46

0.14

±SD

0.42

4.76

7.90

0.82

0.93

2.57

0.12

n

5

5

5

5

5

5

5

G3, M & 0.003

Mean

0.15

23.48

65.82

3.22

4.22*

8.06*

0.02

±SD

0.13

20.92

26.49

4.01

3.80

3.11

0.02

n

5

5

5

5

5

5

5

G4, M & 0.03

Mean

0.15

25.36

66.18

2.70

1.96

2.16

0.04

±SD

0.06

9.32

12.11

2.65

2.09

1.27

0.02

n

5

5

5

5

5

5

5

G1R, M & 0

Mean

0.34

17.08

57.66

1.10

0.90

5.56

0.06

±SD

0.12

3.07

20.72

0.49

1.33

3.29

0.03

n

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

0.40

17.76

56.22

1.78

0.88

3.66

0.07

±SD

0.22

9.61

12.23

1.80

0.75

2.14

0.06

n

5

5

5

5

5

5

5

M: Male; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05).

TABLE 11 (Contd…). SUMMARY OF BALF RECORD

Group, Sex & Dose (mg/L)

Absolute Lymphocytes

Absolute

Monocytes

Absolute Eosinophils

Absolute

Basophils

Lactate

Dehydrogenase

Total Protein

(Lymph)

(Mono)

(Eos)

(Baso)

(LDH)

(TPR)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(u/L)

(g/dL)

G1, M & 0

Mean

0.26

0.01

0.00

0.01

63.76

322.20

±SD

0.11

0.00

0.00

0.01

27.81

108.59

n

5

5

5

5

5

5

G2, M & 0.001

Mean

0.34

0.01

0.00

0.02

83.96

291.48

±SD

0.24

0.01

0.00

0.01

53.17

142.06

n

5

5

5

5

5

5

G3, M & 0.003

Mean

0.12

0.00

0.00

0.01

52.34

306.36

±SD

0.10

0.00

0.00

0.01

45.59

105.45

n

5

5

5

5

5

5

G4, M & 0.03

Mean

0.10

0.00

0.00

0.00

34.26

281.34

±SD

0.05

0.01

0.00

0.00

9.63

105.25

n

5

5

5

5

5

5

G1R, M & 0

Mean

0.21

0.00

0.00

0.02

72.74

253.26

±SD

0.12

0.00

0.00

0.02

29.08

178.66

n

5

5

5

5

5

5

G4R, M & 0.03

Mean

0.22

0.01

0.00

0.01

149.78

200.61

±SD

0.11

0.01

0.00

0.01

122.02

89.26

n

5

5

5

5

5

5

M: Male; R: Recovery; SD: Standard Deviation; n: Number of animals.

TABLE 11 (Contd…). SUMMARY OF BALF RECORD

Group, Sex & Dose (mg/L)

Total Leucocyte

Count

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

Absolute Neutrophils

(WBC)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(Neut)

(103cells/µL)

(%)

(%)

(%)

(%)

(%)

(103cells/µL)

G1, F & 0

Mean

0.25

22.28

64.02

1.18

3.18

2.42

0.05

±SD

0.13

1.84

8.09

0.62

2.18

1.52

0.03

n

5

5

5

5

5

5

5

G2, F & 0.001

Mean

0.21

24.72

58.52

1.88

3.64

7.48

0.05

±SD

0.09

11.62

21.00

0.67

4.05

6.51

0.03

n

5

5

5

5

5

5

5

G3, F & 0.003

Mean

0.32

23.14

65.12

2.02

1.72

1.94

0.08

±SD

0.17

6.81

9.88

0.69

1.53

0.98

0.06

n

5

5

5

5

5

5

5

G4, F & 0.03

Mean

0.23

16.04

71.72

3.52*

3.48

4.42

0.04

±SD

0.17

8.52

11.69

1.93

2.34

4.54

0.03

n

5

5

5

5

5

5

5

G1R, F & 0

Mean

0.22

21.06

53.16

0.98

1.72

6.22

0.04

±SD

0.09

6.39

7.11

1.31

1.64

2.01

0.02

n

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

0.21

17.64

61.56

1.86

0.90

4.18

0.03

±SD

0.07

6.61

16.43

1.10

0.53

1.54

0.02

n

5

5

5

5

5

5

5

F: Female; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05).

TABLE 11 (Contd…). SUMMARY OF BALF RECORD

Group, Sex & Dose (mg/L)

Absolute Lymphocytes

Absolute

Monocytes

Absolute Eosinophils

Absolute

Basophils

Lactate

Dehydrogenase

Total Protein

(Lymph)

(Mono)

(Eos)

(Baso)

(LDH)

(TPR)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(u/L)

(g/dL)

G1, F & 0

Mean

0.16

0.00

0.01

0.00

40.70

272.13

±SD

0.10

0.00

0.00

0.01

23.61

89.97

n

5

5

5

5

5

5

G2, F & 0.001

Mean

0.13

0.00

0.01

0.02

63.46

302.96

±SD

0.08

0.00

0.01

0.02

32.58

89.90

n

5

5

5

5

5

5

G3, F & 0.003

Mean

0.21

0.01

0.00

0.00

51.52

355.10

±SD

0.10

0.01

0.01

0.01

21.60

108.66

n

5

5

5

5

5

5

G4, F & 0.03

Mean

0.16

0.01

0.00

0.01

46.32

346.50

±SD

0.11

0.01

0.01

0.01

23.88

251.82

n

5

5

5

5

5

5

G1R, F & 0

Mean

0.12

0.00

0.00

0.01

46.52

235.94

±SD

0.06

0.00

0.00

0.01

12.39

96.20

n

5

5

5

5

5

5

G4R, F & 0.03

Mean

0.13

0.00

0.00

0.01

91.78*

151.51

±SD

0.05

0.00

0.00

0.00

26.14

37.86

n

5

5

5

5

5

5

F: Female; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05).

 TABLE 12. SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex & Dose (mg/L)

Glucose

Urea

Creatinine

Total

Cholesterol

Triglycerides

Total

Protein

Albumin

Alanine

aminotransferase

Aspartate

aminotransferase

(GLU)

(CRE)

(CHO)

(TRI)

(TPR)

(ALB)

(ALT)

(AST)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(g/dL)

(U/L)

(U/L)

G1, M & 0

Mean

91.60

31.90

0.41

42.00

44.60

6.82

3.09

53.20

94.20

±SD

8.88

2.88

0.03

8.40

8.85

0.48

0.07

13.94

10.66

n

5

5

5

5

5

5

5

5

5

G2, M & 0.001

Mean

85.00

31.50

0.40

49.60

41.20

7.00

3.15

57.40

103.20

±SD

8.51

1.64

0.01

6.43

12.76

0.20

0.15

7.57

12.77

n

5

5

5

5

5

5

5

5

5

G3, M & 0.003

Mean

97.00

32.28

0.39

47.60

49.20

6.90

3.10

53.40

108.20

±SD

15.70

4.40

0.02

11.01

8.23

0.30

0.11

6.19

7.76

n

5

5

5

5

5

5

5

5

5

G4, M & 0.03

Mean

86.60

35.32

0.38

50.40

48.00

6.64

2.99

71.00

109.00

±SD

9.71

11.73

0.03

12.30

13.67

0.43

0.17

15.89

21.51

n

5

5

5

5

5

5

5

5

5

G1R, M & 0

Mean

77.20

34.62

0.52

53.60

29.80

7.52

3.37

62.20

106.60

±SD

10.08

6.83

0.05

7.47

4.71

0.29

0.18

7.26

17.56

n

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

89.00

36.06

0.53

52.60

33.60

7.66

3.50

62.00

104.40

±SD

6.71

1.99

0.05

8.68

9.02

0.15

0.33

13.62

14.06

n

5

5

5

5

5

5

5

5

5

M: Male; R: Recovery; SD: Standard Deviation; n: number of animals.

TABLE 12 (Contd…). SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex & Dose (mg/L)

Alkaline

phosphatase

Total

Bilirubin

Calcium

Phosphorous

Globulin

Blood

Urea Nitrogen

Sodium

Potassium

Chloride

(ALP)

(BIT)

(CAL)

(PHO)

(GLO)

(BUN)

(Na)

(K)

(CLO)

(U/L)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(mg/dL)

(mmol/L)

(mmol/L)

(mmol/L)

G1, M & 0

Mean

202.00

0.01

9.78

7.30

3.73

14.89

142.10

3.87

109.74

±SD

79.50

0.02

0.33

0.63

0.45

1.34

0.83

0.18

0.91

n

5

5

5

5

5

5

5

5

5

G2, M & 0.001

Mean

178.80

0.04

9.92

7.24

3.85

14.70

142.12

3.97

110.44

±SD

21.75

0.02

0.29

0.43

0.15

0.76

0.89

0.16

0.73

n

5

5

5

5

5

5

5

5

5

G3, M & 0.003

Mean

184.40

0.04

9.94

7.14

3.80

15.06

141.14

3.96

109.42

±SD

47.26

0.05

0.21

0.55

0.33

2.05

0.91

0.29

1.27

n

5

5

5

5

5

5

5

5

5

G4, M & 0.03

Mean

188.80

0.03

9.80

7.44

3.65

16.48

142.92

3.94

111.54*

±SD

57.73

0.02

0.12

0.36

0.27

5.47

0.91

0.09

0.52

n

5

5

5

5

5

5

5

5

5

G1R, M & 0

Mean

142.60

0.05

9.60

6.08

4.15

16.16

144.46

3.54

105.24

±SD

31.91

0.01

0.28

0.47

0.26

3.19

0.27

0.34

1.42

n

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

163.80

0.05

9.52

5.36

4.16

16.83

144.52

3.55

106.68

±SD

44.64

0.01

0.36

0.92

0.30

0.93

0.64

0.12

0.63

n

5

5

5

5

5

5

5

5

5

M: Male; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05).

TABLE 12 (Contd…). SUMMARY OF CLINICAL CHEMISTRY

Group, Sex & Dose (mg/L)

Glucose

Urea

Creatinine

Total

Cholesterol

Triglycerides

Total

Protein

Albumin

Alanine

aminotransferase

Aspartate

aminotransferase

(GLU)

(CRE)

(CHO)

(TRI)

(TPR)

(ALB)

(ALT)

(AST)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(g/dL)

(U/L)

(U/L)

G1, F & 0

Mean

95.80

29.54

0.39

53.60

48.60

7.08

3.35

42.60

91.20

±SD

9.86

4.95

0.04

11.74

5.50

0.36

0.25

7.44

8.76

n

5

5

5

5

5

5

5

5

5

G2, F & 0.001

Mean

100.20

28.98

0.37

54.40

50.80

7.32

3.33

44.20

96.00

±SD

2.68

2.77

0.01

6.62

14.79

0.22

0.13

6.61

6.63

n

5

5

5

5

5

5

5

5

5

G3, F & 0.003

Mean

102.20

30.38

0.35

59.60

37.40

7.28

3.34

46.80

105.40

±SD

5.45

5.89

0.03

14.59

5.94

0.28

0.11

12.19

10.31

n

5

5

5

5

5

5

5

5

5

G4, F & 0.03

Mean

108.00

32.98

0.37

58.20

45.20

7.20

3.16

38.20

92.00

±SD

11.98

7.09

0.07

14.75

14.13

0.61

0.18

9.98

27.06

n

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

90.80

40.48

0.59

73.60

37.40

8.14

3.92

41.60

88.60

±SD

8.64

2.53

0.04

27.56

10.43

0.58

0.32

5.03

14.03

n

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

98.00

36.66

0.57

72.40

38.20

8.14

3.95

40.80

85.60

±SD

13.69

4.61

0.06

19.49

5.36

0.42

0.26

5.89

15.04

n

5

5

5

5

5

5

5

5

5

 F: Female; R: Recovery; SD: Standard Deviation; n: number of animals.

TABLE 12 (Contd…). SUMMARY OF CLINICAL CHEMISTRY

Group, Sex & Dose (mg/L)

Alkaline

phosphatase

Total

Bilirubin

Calcium

Phosphorous

Globulin

Blood

Urea Nitrogen

Sodium

Potassium

Chloride

(ALP)

(BIT)

(CAL)

(PHO)

(GLO)

(BUN)

(Na)

(K)

(CLO)

(U/L)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(mg/dL)

(mmol/L)

(mmol/L)

(mmol/L)

G1, F & 0

Mean

105.20

0.02

10.40

7.02

3.73

13.79

140.90

4.12

111.32

±SD

23.99

0.02

0.31

0.54

0.24

2.31

0.99

0.29

2.77

n

5

5

5

5

5

5

5

5

5

G2, F & 0.001

Mean

96.00

0.03

10.14

6.98

3.99

13.53

141.66

4.05

111.20

±SD

12.41

0.01

0.32

0.87

0.34

1.29

1.60

0.70

1.14

n

5

5

5

5

5

5

5

5

5

G3, F & 0.003

Mean

140.60

0.04

10.28

6.80

3.94

14.18

141.68

3.82

111.46

±SD

71.25

0.01

0.36

0.94

0.33

2.75

1.44

0.23

1.94

n

5

5

5

5

5

5

5

5

5

G4, F & 0.03

Mean

129.80

0.03

10.34

7.20

4.04

15.39

142.58

4.07

112.06

±SD

26.81

0.02

0.30

0.35

0.46

3.31

1.00

0.20

0.86

n

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

80.00

0.06

10.02

5.12

4.22

18.89

143.94

3.56

106.40

±SD

19.27

0.01

0.49

0.79

0.32

1.18

0.88

0.41

1.02

n

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

62.20

0.07

10.06

4.72

4.19

17.11

143.88

3.17

106.60

±SD

10.69

0.02

0.26

0.85

0.47

2.15

1.39

0.29

1.04

n

5

5

5

5

5

5

5

5

5

F: Female; R: Recovery; SD: Standard Deviation; n: Number of animals.

TABLE 13.  SUMMARY OF URINALYSIS

Examination

Group, Sex & Dose (mg/L)

G1, M & 0

G2, M & 0.001

G3, M & 0.003

G4, M & 0.03

G1R, M & 0

G4R, M & 0.03

Number of Animals

5

5

5

5

5

5

Physical

Colour

Pale Yellow

4

3

3

4

5

5

Yellow

1

2

2

1

-

-

Appearance

Clear

4

3

2

3

5

5

Turbid

1

2

3

2

-

-

Volume (mL)

Mean

5.5

5.9

5.7

5.4

6.5

6.5

±SD

0.9

0.7

1.3

0.4

0.6

0.8

Chemical

pH

Mean

6.4

6.0

6.8

6.1

7.5

7.4

±SD

1.6

0.9

1.3

1.0

0.6

0.5

Specific Gravity

Mean

1.009

1.007

1.007

1.006

1.018

1.016

±SD

0.002

0.004

0.003

0.002

0.007

0.007

Urobilinogen (mg/dL)

Mean

0.2

0.2

0.2

0.2

0.2

0.4

±SD

0.0

0.0

0.0

0.0

0.0

0.4

Bilirubin (mg/dL)

Neg

5

5

5

5

5

5

Ketones (mg/dL)

Neg

2

4

2

2

-

2

5

2

1

3

3

5

2

15

1

-

-

-

-

1

Blood (Ery/µL)

Neg

-

-

-

-

1

1

Ca10

1

-

-

-

-

1

Ca25

3

2

1

-

2

2

Ca80

1

3

4

5

1

1

>=Ca200

-

-

-

-

1

-

Proteins (mg/dL)

Neg

-

-

-

2

-

1

Trace

2

4

3

1

-

1

30

3

1

1

2

2

3

100

-

-

1

-

2

-

>=300

-

-

-

-

1

-

Nitrite

Neg

3

5

4

5

4

5

Pos

2

-

1

-

1

-

Leucocytes (Leu/µL)

Neg

2

3

-

1

-

1

Ca15

3

2

5

4

2

1

Ca70

-

-

-

-

3

2

Ca125

-

-

-

-

-

1

Glucose (mg/dL)

Neg

5

5

5

5

5

5

Micro albumin (MALB) (mg/dL)

Neg

-

-

-

2

-

-

>15

5

5

5

3

5

4

15

-

-

-

-

-

1

Microscopic

Epithelial cells

0

1

-

1

1

4

3

0-1

2

4

3

3

1

2

0-2

1

-

-

-

-

-

1-2

1

1

1

1

-

-

Casts

Absent

5

5

5

5

5

5

Crystals

Absent

2

1

1

2

-

-

Present

3

4

4

3

5

5

M: Male; Neg: Negative; Ca: Calculated Approximately; Pos: Positive; SD: Standard Deviation; R: Recovery.

TABLE 13 (Contd…). SUMMARY OF URINALYSIS

Examination

Group, Sex & Dose (mg/L)

G1, F & 0

G2, F & 0.001

G3, F & 0.003

G4, F &

0.03

G1R, F &

0

G4R, F

&

0.03

Number of Animals

5

5

5

5

5

5

Physical

Colour

Pale Yellow

5

4

4

4

5

5

Yellow

-

1

1

1

-

-

Appearance

Clear

5

3

4

4

5

5

Turbid

-

2

1

1

-

-

Volume (mL)

Mean

6.1

6.6

7.6

6.6

6.6

6.9

±SD

2.3

0.8

2.3

2.9

1.0

1.3

Chemical

pH

Mean

6.6

7.1

6.8

6.4

7.0

7.6

±SD

1.7

1.3

1.2

1.0

0.4

0.7

Specific Gravity

Mean

1.006

1.005

1.005

1.005

1.013

1.009*

±SD

0.002

0.000

0.000

0.000

0.003

0.002

Urobilinogen (mg/dL)

Mean

0.2

0.2

0.2

0.2

0.2

0.2

±SD

0.0

0.0

0.0

0.0

0.0

0.0

Bilirubin (mg/dL)

Neg

5

5

5

5

5

5

Ketones (mg/dL)

Neg

5

5

5

5

5

5

Blood (Ery/µL)

Neg

4

1

1

-

4

4

Ca10

-

-

-

3

1

-

Ca25

-

3

4

2

-

1

Ca80

1

1

-

-

-

-

Proteins (mg/dL)

Neg

1

3

2

4

5

3

Trace

3

2

3

1

-

1

30

1

-

-

-

-

-

100

-

-

-

-

-

1

Nitrite

Neg

4

4

4

5

3

5

Pos

1

1

1

-

2

-

Leucocytes (Leu/µL)

Neg

1

-

-

-

4

4

Ca15

4

5

5

5

1

-

Ca70

-

-

-

-

-

1

Glucose (mg/dL)

Neg

5

5

5

5

5

5

Micro albumin (MALB) (mg/dL)

Neg

1

3

2

4

3

3

>15

4

2

3

1

-

2

15

-

-

-

 

2

-

Microscopic

Epithelial cells

0

1

1

2

2

5

4

0-1

3

1

1

3

-

1

1-2

1

3

2

-

-

-

Casts

Absent

4

5

4

5

5

5

Present

1

-

1

-

-

-

Crystals

Absent

2

1

-

3

-

-

Present

3

4

5

2

5

5

F: Female; Neg: Negative; Ca: Calculated Approximately; Pos: Positive; SD: Standard Deviation; R: Recovery.

*: Statistically significant (p<0.05).

TABLE 14.      SUMMARY OF ABSOLUTE ORGAN WEIGHTS (g)

Group, Sex & Dose (mg/L)

Adrenals

Thymus

Spleen

Testes

Heart

Kidneys

Brain

Liver

Lungs

G1, M & 0

Mean

0.0546

0.3164

1.0157

3.2814

0.9708

1.9636

1.9756

8.1699

0.5472

±SD

0.0079

0.0341

0.0992

0.2444

0.0559

0.1238

0.1009

0.6837

0.0516

n

5

5

5

5

5

5

5

5

5

G2, M & 0.001

Mean

0.0507

0.2643

1.1574

2.8052*

1.0410

1.9459

1.9383

8.4021

0.7314*

±SD

0.0057

0.0492

0.2344

0.2205

0.0990

0.1978

0.1199

0.5872

0.0658

n

5

5

5

5

5

5

5

5

5

G3, M & 0.003

Mean

0.0525

0.2908

1.1117

2.9190

0.9237

1.7474

1.8780

7.0657

0.5565

±SD

0.0037

0.0359

0.1191

0.1732

0.0443

0.0918

0.0720

0.4982

0.0609

n

5

5

5

5

5

5

5

5

5

G4, M & 0.03

Mean

0.0542

0.2789

0.9743

3.4094

1.0249

1.9576

1.9070

7.4683

0.7014*

±SD

0.0049

0.0236

0.0965

0.4453

0.0507

0.1801

0.0340

0.9456

0.0711

n

5

5

5

5

5

5

5

5

5

G1R, M & 0

Mean

0.0609

0.2117

1.3692

3.6598

1.4328

2.8445

2.0602

11.2475

0.7140

±SD

0.0042

0.0517

0.0633

0.3034

0.1040

0.5484

0.0512

1.7149

0.0767

n

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

0.0631

0.2829*

1.1172*

3.3671

1.3194

2.3350

2.0685

10.1517

0.6762

±SD

0.0048

0.0365

0.1003

0.2875

0.0756

0.1815

0.0608

0.0759

0.0505

n

5

5

5

5

5

5

5

5

5

 M: Male; R: Recovery; SD: Standard Deviation, n: Number of animals; *: Statistically significant (p<0.05).

TABLE 14 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHTS (g)

Group, Sex & Dose (mg/L)

Adrenals

Thymus

Spleen

Ovaries

Uterus

Heart

Kidneys

Brain

Liver

Lungs

G1, F & 0

Mean

0.0623

0.3702

0.6049

0.1205

0.4089

0.7567

1.4280

1.7656

6.2227

0.5146

±SD

0.0037

0.0701

0.1064

0.0129

0.0518

0.0741

0.1903

0.0929

0.4228

0.0453

n

5

5

5

5

5

5

5

5

5

5

G2, F & 0.001

Mean

0.0594

0.3682

0.4791

0.1181

0.5253

0.7450

1.3006

1.8402

5.5576

0.5163

±SD

0.0080

0.0627

0.1005

0.0094

0.1533

0.0599

0.1535

0.0842

0.3985

0.0639

n

5

5

5

5

5

5

5

5

5

5

G3, F & 0.003

Mean

0.0561

0.2862

0.3462*

0.0987

0.3390

0.7369

1.2861

1.7616

5.6863

0.5077

±SD

0.0070

0.0741

0.0372

0.0138

0.0602

0.1018

0.1160

0.0763

0.3377

0.0388

n

5

5

5

5

5

5

5

5

5

5

G4, F & 0.03

Mean

0.0560

0.2707

0.4679

0.1156

0.3450

0.7066

1.2147

1.7537

6.0287

0.6040

±SD

0.0056

0.0276

0.0857

0.0180

0.0603

0.0985

0.1357

0.0714

0.6349

0.0824

n

5

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

0.0809

0.2637

0.6039

0.1321

0.7511

0.9010

1.5550

1.8811

7.9632

0.4801

±SD

0.0111

0.0441

0.0799

0.0168

0.2097

0.0950

0.0643

0.0875

0.4811

0.0316

n

5

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

0.0778

0.2352

0.5206

0.1647*

0.7788

0.9655

1.5697

1.8020

7.2701

0.4812

±SD

0.0035

0.0293

0.0647

0.0077

0.2014

0.0968

0.1387

0.1353

0.9500

0.0610

n

5

5

5

5

5

5

5

5

5

5

 F: Female, R: Recovery; SD: Standard Deviation, n: Number of animals; *: Statistically significant (p<0.05).

TABLE 15. SUMMARY OF ORGAN WEIGHT RELATIVE TO BODY WEIGHT(%) RECORD

Group, Sex & Dose (mg/L)

Fasting Body Weight (g)

Adrenals

Thymus

Spleen

Testes

Heart

Kidneys

Brain

Liver

Lungs

G1, M & 0

Mean

229.55

0.0237

0.1385

0.4480

1.4392

0.4259

0.8641

0.8683

3.5879

0.2396

±SD

20.95

0.0018

0.0167

0.0808

0.1679

0.0480

0.1288

0.1105

0.4917

0.0270

n

5

5

5

5

5

5

5

5

5

5

G2, M & 0.001

Mean

225.52

0.0225

0.1174

0.5200

1.2504

0.4629

0.8621

0.8604

3.7368

0.3261*

±SD

15.25

0.0022

0.0207

0.1333

0.1495

0.0523

0.0501

0.0388

0.3325

0.0437

n

5

5

5

5

5

5

5

5

5

5

G3, M & 0.003

Mean

228.80

0.0230

0.1278

0.4869

1.2795

0.4045

0.7658

0.8230

3.1008

0.2440

±SD

14.36

0.0012

0.0204

0.0550

0.1057

0.0231

0.0593

0.0548

0.3321

0.0305

n

5

5

5

5

5

5

5

5

5

5

G4, M & 0.03

Mean

229.21

0.0236

0.1220

0.4252

1.4888

0.4480

0.8539

0.8344

3.2547

0.3068*

±SD

15.64

0.0009

0.0117

0.0314

0.1825

0.0220

0.0491

0.0454

0.3036

0.0339

n

5

5

5

5

5

5

5

5

5

5

G1R, M & 0

Mean

423.01

0.0144

0.0499

0.3236

0.8657

0.3385

0.6744

0.4873

2.6686

0.1692

±SD

11.01

0.0008

0.0116

0.0088

0.0748

0.0194

0.1410

0.0183

0.4805

0.0227

n

5

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

418.89

0.0151

0.0676*

0.2670*

0.8047

0.3150

0.5574

0.4939

2.4239

0.1613

±SD

6.76

0.0013

0.0089

0.0268

0.0797

0.0187

0.0407

0.0156

0.0366

0.0098

n

5

5

5

5

5

5

5

5

5

5

 M: Male; R: Recovery; SD: Standard Deviation, n: Number of animals; *: Statistically significant (p<0.05).

TABLE 15 (Contd…). SUMMARY OF ORGAN WEIGHT RELATIVE TO BODY WEIGHT (%) RECORD

Group, Sex & Dose (mg/L)

Fasting Body

Weight (g)

Adrenals

Thymus

Spleen

Ovaries

Uterus

Heart

Kidneys

Brain

Liver

Lungs

G1, F & 0

Mean

183.73

0.0340

0.2021

0.3302

0.0659

0.2228

0.4117

0.7780

0.9621

3.3918

0.2801

±SD

10.58

0.0031

0.0399

0.0616

0.0093

0.0267

0.0288

0.0967

0.0446

0.2396

0.0176

n

5

5

5

5

5

5

5

5

5

5

5

G2, F & 0.001

Mean

184.83

0.0323

0.1989

0.2584

0.0639

0.2833

0.4037

0.7078

0.9991

3.0179

0.2812

±SD

11.27

0.0057

0.0301

0.0468

0.0023

0.0773

0.0323

0.1163

0.0846

0.3169

0.0489

n

5

5

5

5

5

5

5

5

5

5

5

G3, F & 0.003

Mean

181.71

0.0308

0.1592

0.1918*

0.0546

0.1868

0.4045

0.7093

0.9707

3.1331

0.2800

±SD

11.37

0.0027

0.0487

0.0310

0.0094

0.0339

0.0379

0.0690

0.0360

0.1629

0.0240

n

5

5

5

5

5

5

5

5

5

5

5

G4, F & 0.03

Mean

180.35

0.0312

0.1505

0.2599

0.0643

0.1925

0.3923

0.6760

0.9740

3.3548

0.3360

±SD

8.36

0.0040

0.0179

0.0485

0.0111

0.0407

0.0568

0.0940

0.0590

0.4502

0.0522

n

5

5

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

313.71

0.0258

0.0840

0.1930

0.0423

0.2400

0.2871

0.4958

0.5996

2.5426

0.1531

±SD

9.95

0.0035

0.0141

0.0286

0.0065

0.0704

0.0272

0.0176

0.0176

0.2084

0.0095

n

5

5

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

315.75

0.0247

0.0746

0.1648

0.0522*

0.2452

0.3060

0.4971

0.5706

2.2994

0.1527

±SD

10.67

0.0011

0.0098

0.0187

0.0037

0.0568

0.0317

0.0403

0.0362

0.2605

0.0212

n

5

5

5

5

5

5

5

5

5

5

5

 F: Female, R: Recovery; SD: Standard Deviation, n: Number of animals; *: Statistically significant (p<0.05).

 TABLE 16. SUMMARY OF ORGAN WEIGHT RELATIVE TO BRAIN WEIGHT (%) RECORD

Group, Sex & Dose (mg/L)

Adrenals

Thymus

Spleen

Testes

Heart

Kidneys

Liver

Lungs

G1, M & 0

Mean

2.7788

16.0226

51.3579

166.1722

49.2449

99.5469

413.9069

27.8307

±SD

0.4854

1.5547

3.4144

10.1365

3.9319

7.2237

33.9594

3.9036

n

5

5

5

5

5

5

5

5

G2, M & 0.001

Mean

2.6222

13.6994

60.4701

145.6095

53.8465

100.3085

435.2792

37.9309*

±SD

0.3220

2.6851

15.6939

19.4023

5.9632

6.4199

47.2753

5.0597

n

5

5

5

5

5

5

5

5

G3, M & 0.003

Mean

2.7980

15.4923

59.2262

155.4821

49.2030

93.0155

376.2989

29.7121

±SD

0.2108

1.8488

6.3794

8.1774

2.0680

1.8959

23.2915

3.8733

n

5

5

5

5

5

5

5

5

G4, M & 0.03

Mean

2.8385

14.6201

51.0374

178.7947

53.7343

102.6157

391.1467

36.7839*

±SD

0.2285

1.1804

4.1897

23.3721

2.2678

8.8050

44.0085

3.7514

n

5

5

5

5

5

5

5

5

G1R, M & 0

Mean

2.9629

10.3105

66.5051

177.9219

69.6012

137.7537

545.6673

34.6491

±SD

0.2716

2.6969

3.8161

17.8584

5.7242

24.4075

79.4291

3.5038

n

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

3.0525

13.6603*

53.9801*

162.9183

63.8029

112.8276

491.1223

32.6866

±SD

0.2426

1.5808

4.1095

15.1124

3.4811

7.0959

14.6572

2.1792

n

5

5

5

5

5

5

5

5

M: Male; R: Recovery; SD: Standard Deviation, n: Number of animals; *: Statistically significant (p<0.05).

TABLE 16 (Contd…). SUMMARY OF ORGAN WEIGHT RELATIVE TO BRAIN WEIGHT (%) RECORD

Group, Sex & Dose (mg/L)

Adrenals

Thymus

Spleen

Ovaries

Uterus

Heart

Kidneys

Liver

Lungs

G1, F & 0

Mean

3.5292

20.9151

34.2411

6.8497

23.1571

42.7947

80.6817

352.3827

29.1361

±SD

0.1907

3.3835

5.4867

0.8875

2.5721

2.2916

7.4942

13.8956

1.8218

n

5

5

5

5

5

5

5

5

5

G2, F & 0.001

Mean

3.2258

20.0988

26.2348

6.4392

28.7329

40.5900

70.6182

302.3913*

28.0970

±SD

0.4068

3.9272

6.4878

0.7038

9.0904

4.1657

6.8405

23.1828

3.5362

n

5

5

5

5

5

5

5

5

5

G3, F & 0.003

Mean

3.1860

16.3556

19.7471*

5.6262

19.2871

41.7060

73.1094

323.1543

28.8421

±SD

0.3606

4.8290

2.9899

0.9687

3.6096

4.1403

7.2204

21.4424

2.1647

n

5

5

5

5

5

5

5

5

5

G4, F & 0.03

Mean

3.1976

15.4181

26.5831

6.6137

19.7461

40.2033

69.2865

343.4061

34.3678*

±SD

0.3393

1.1681

3.8553

1.1811

3.8810

4.5301

7.6436

28.5407

3.6477

n

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

4.2971

14.0269

32.2042

7.0595

40.0258

47.8431

82.7173

424.6109

25.5294

±SD

0.4989

2.4052

4.8172

1.1360

11.8751

3.5805

2.8224

39.7625

1.4101

n

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

4.3383

13.1595

29.1223

9.1710*

43.1295

53.6468

87.3998

403.2624

26.7929

±SD

0.3998

2.2296

5.0961

0.7043

10.4359

4.6557

8.9537

40.2997

3.4916

n

5

5

5

5

5

5

5

5

5

 F: Female, R: Recovery; SD: Standard Deviation, n: Number of animals; *: Statistically significant (p<0.05).

TABLE 17.      SUMMARY OF CHAMBER (EXPOSURE) CONDITIONS


Group & Conc.

(mg/L)

 

Temp.

 (°C)

Rh

(%)

O2

(%)

CO2

 (mg/L)

Air inlet flow rate

(L/min)*

BZC

(mg/L)

G1/G1R & 0

Mean

22.55

55.52

20.39

617.48

20.00

-

±SD

0.31

1.90

0.19

5.43

0.00

-

G2 & 0.001

Mean

22.66

55.95

20.47

619.67

20.00

0.0011

±SD

0.31

1.35

0.21

4.84

0.00

0.0001

G3 & 0.003

Mean

22.64

55.88

20.50

619.73

20.00

0.0032

±SD

0.31

1.32

0.23

5.31

0.00

0.0001

G4/G4R & 0.03

Mean

22.59

55.10

20.38

617.74

20.00

0.032

±SD

0.31

1.59

0.20

5.12

0.00

0.001

    *: Values were constant throughout the exposure; hence standard deviation is zero, Temp.: Temperature; Rh: Relative Humidity; O2: Oxygen Concentration;

     CO2: Carbon Dioxide Concentration;BZC: Breathing Zone Concentration:

     SD: Standard Deviation.

TABLE 18.    SUMMARY OF NOMINAL CONCENTRATION

Day

Group

Test item Used (mg) (a)

Air flow rate

(L/minute)(b)

Minute

(min)

(c)

Nominal Concentration

(mg/L)

(a)    / (b) × (c)

1

G1/G1R 

(Air Only)

-

20

    360

-

G2, G3 and G4/G4R 

(Test Item)

1150

20

360

0.16

4370

0.61

5230

0.73

2

G1/G1R

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1120

20

360

0.16

4440

0.62

5110

0.71

 3

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1200

20

360

0.17

4380

0.61

5140

0.71

4

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1160

20

360

0.16

4350

0.60

5160

0.72

5

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1180

20

360

0.16

4430

0.62

5200

0.72

6

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1090

20

360

0.15

4340

0.60

5180

0.72

7

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1170

20

360

0.16

4430

0.62

5190

0.72

TABLE 18 (Contd…). SUMMARY OF NOMINAL CONCENTRATION

Day

Group

Test item Used (mg) (a)

Air flow rate

(L/minute)(b)

Minute

(min)

(c)

Nominal Concentration

(mg/L)

(a)    / (b) × (c)

8

G1/G1R 

(Air Only)

-

20

    360

-

G2, G3 and G4/G4R 

(Test Item)

1140

20

360

0.16

4570

0.63

5200

0.72

9

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1150

20

360

0.16

4330

0.60

5280

0.73

 10

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1190

20

360

0.17

4410

0.61

5110

0.71

11

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1130

20

360

0.16

4440

0.62

5210

0.72

12

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1170

20

360

0.16

4580

0.64

5290

0.73

13

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1190

20

360

0.17

4320

0.60

5150

0.72

14

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1160

20

360

0.16

4440

0.62

5120

0.71

TABLE 18 (Contd…). SUMMARY OF NOMINAL CONCENTRATION

Day

Group

Test item Used (mg) (a)

Air flow rate

(L/minute)(b)

Minute

(min)

(c)

Nominal Concentration

(mg/L)

(a)    / (b) × (c)

15

G1/G1R 

(Air Only)

-

20

    360

-

G2, G3 and G4/G4R 

(Test Item)

1170

20

360

0.16

4400

0.61

5170

0.72

16

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1170

20

360

0.16

4460

0.62

5210

0.72

 17

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1190

20

360

0.17

4400

0.61

5270

0.73

18

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1140

20

360

0.16

4360

0.61

5200

0.72

19

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1190

20

360

0.17

4590

0.64

5230

0.73

20

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1160

20

360

0.16

4590

0.64

5270

0.73

TABLE 19.     SUMMARY OF GROSS PATHOLOGY FINDINGS

Sex

Male

Route of administration

Inhalation

Treatment

Air only

Low Dose

Mid Dose

High Dose

Air only

High Dose Recovery

Group

G1

G2

G3

G4

G1R

G4R

Nominal Target Concentration (mg/L)

0

0.001*

0.003*

0.03*

0

0.03*

Number of animals

5

5

5

5

5

5

No. of dead rats during treatment

-

-

-

-

-

-

No. of moribund sacrificed rats

-

-

-

-

-

-

No. of terminally sacrificed rats

5

5

5

5

5

5

No. of rats showing gross pathology

-

-

-

-

-

-

Sex

Female

Route of administration

Inhalation

Treatment

Air only

Low Dose

Mid Dose

High Dose

Air only

High Dose Recovery

Group

G1

G2

G3

G4

G1R

G4R

Nominal Target Concentration (mg/L)

0

0.001*

0.003*

0.03*

0

0.03*

Number of animals

5

5

5

5

5

5

No. of dead rats during treatment

-

-

-

-

-

-

No. of moribund sacrificed rats

-

-

-

-

-

-

No. of terminally sacrificed rats

5

5

5

5

5

5

No. of rats showing gross pathology

-

-

1

-

-

-

Uterus– small in size

-

-

1

-

-

-

R: Recovery; *: Above doses were provided by the sponsor; - : No incidence.

TABLE 20.      SUMMARY OF HISTOPATHOLOGY FINDINGS – MAIN GROUP

Route of administration

Inhalation

Treatment

Air only

High Dose

Nominal Target Concentration (mg/L)

0

0.03

Group

G1

G4

Sex

M

F

M

F

Number of Animals

5

5

5

5

Adrenals

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Brain

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Cervical lymph nodes

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Esophagus

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Eyes with optic nerve and eye lids

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Heart

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Hilar lymph nodes

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Kidneys

Number examined

5

5

5

5

Within normal limits

4

4

3

4

Cyst

Present

1

1

2

-

Cast

Minimal

1

-

-

-

Basophilia, tubules

Minimal

-

-

1

-

Dilatation, tubules

Minimal

-

-

-

1

Larynx

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Liver

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Lungs(left lobe)

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Mandibular lymph nodes

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Mesenteric lymph nodes

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Nasopharyngeal tissue

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Olfactory bulb</