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Diss Factsheets

Administrative data

Description of key information

• 90 -Day repeated dose oral toxicity (OECD 408)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
September 21, 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
NAME OF TEST MATERIAL (as stated in the study report): Trisisotridecylphosphite

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Valtris Specialty Chemicals
- Batch No. 330965
- Expiration date of the lot/batch: March 02, 2019
- Purity: 100% (UVCB substance)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in original container.
- Stability under test conditions: The stability of the test item in dose formulations was determined using a validated analytical method. The test item was found stable up to 24 hours under use conditions (i.e., room temperature). Dose formulations were prepared daily and used immediately after preparation.
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Jai Research Foundation
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: not more than 9 weeks
- Housing: 2 rats/cage/sex during the study period in sterile solid floor polypropylene rat cages (size: 410 × 282 × 150 mm). Each cage fitted with a stainless steel top grill (height: 30 mm) having provision for keeping rodent pellet feed and a polypropylene water bottle with stainless steel drinking nozzle.
- Diet (e.g. ad libitum): standard sterile rodent pellet feed (Teklad Certified Global 16% Protein Rodent Diet), ad libitum.
- Water (e.g. ad libitum): filtered drinking water (Reverse Osmosis water filter system) in polypropylene bottles, ad libitum.
- Acclimation period: minumum of 5 days.

DETAILS OF FOOD AND WATER QUALITY: Quality of feed (microbial contaminant) and water (microbial and chemical contaminant) is checked regularly.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 deg C
- Humidity (%): 50 +/- 20%
- Air changes (per hr): minimum of 15
- Photoperiod (hrs dark / hrs light): 12 h light and 12 h darkness, light hours being 06.00 - 18.00 h; light intensity of 130 to 325 LUX.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: dose formulations were mixed using a magnetic stirrer before dosing and with spatula/ glass rod/ cannula intermittently during dosing. The dose formulation were prepared on each day of dosing and used immediately after preparation.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility; the test item is known to hydrolyze in water.
- Dose volume: 10 mL/kg b.w.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration and homogeneity of dose formulations were analysed at JRF Department of Chemistry, using a validated analytical method (JRF study number 228-2-13-18511). Two set of samples (10 samples per set) were collected by sampling three aliquots (upper, middle and lower layers) from each concentration except vehicle control (only one aliquot). The sampling was done before initiation of the dosing (on day 1) and monthly intervals thereafter. One set of samples was used for analyses and other set of samples was stored at refrigerated condition. The additional set of samples can be used in case the satisfactory results are not obtained from the first set of samples. The mean concentration was determined and compared to the nominal value and the coefficient of variation calculated. The acceptance criteria used for analysis are ±15% from nominal value and %CV < 10.
Duration of treatment / exposure:
90 consecutive days
Frequency of treatment:
once daily
Dose / conc.:
0 mg/kg bw (total dose)
Dose / conc.:
42 mg/kg bw (total dose)
Dose / conc.:
125 mg/kg bw (total dose)
Dose / conc.:
375 mg/kg bw (total dose)
No. of animals per sex per dose:
10 per sex per group (main groups)
10 per sex (control and high dose, recovery groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a 28-day dose range finding study (JRF study number 410-1-02-18513).
- Post-exposure recovery period for satellite groups: 70 days
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day for mortality and signs of morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: all rats were observed for any clinical sign at least twice a day.

BODY WEIGHT: Yes
- Time schedule: at beginning of the treatment (Pre-treatment) and at least at weekly interval thereafter.

FOOD CONSUMPTION: Yes
- Time schedule: weekly and reported as g/rat/day.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the commencement of treatment and on the surviving rats prior to the terminal and recovery sacrifices.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment and recovery periods.
- Anaesthetic used for blood collection: Yes (under light isoflorane anaesthesia)
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked: Haematocrit, Haemoglobin, Mean Corpuscular Haemoglobin, Mean Corpuscular Haemoglobin Concentration, Mean Corpuscular Volume, Platelet Count, Total Erythrocyte Count (Red Blood Cells), Total Leukocyte Count (White Blood Cells), Differential Leukocyte Count, Reticulocyte Count, Prothrombin Time, Activated Partial Thromboplastin Time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment and recovery periods.
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked: Alanine Aminotransferase, Albumin, Albumin : Globulin ratio, Alkaline Phosphatase, Aspartate Aminotransferase, Bile Acids, Blood Urea Nitrogen, Calcium, Creatinine, Creatine Kinase, Gamma Glutamyl Transpeptidase, Globulin, Glucose, Inorganic Phosphorous, Lactate Dehydrogenase, Total Cholesterol, Total Protein, Total Bilirubin, Triglycerides, Urea, Chloride, Potassium, Sodium.

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the treatment and recovery periods.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Appearance, Colour, Volume, Sediment evaluation (Microscopic examination), Specific gravity, pH, Protein, Glucose, Ketone, Blood, Bilirubin, Urobilinogen.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to initiation of dosing and at weekly intervals thereafter.
- How many animals: all animals
- Battery of observations: home cage, handling, open field, functional observational battery, motor activity, sensory reactivity, grip strength, landing hindlimb foot splay.

SPERMATOGENESIS: Yes
- Time scheule: beginning and end of recovery period
- How many animals: 5 males (control and high dose)
- Parameters: sperm count, motility and morphology
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, full gross necropsy.

ORGAN WEIGHTS: Yes, Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Prostate, Seminal vesicles with coagulating glands, Spleen, Testes, Thymus, Thyroid with parathyroid, Uterus with cervix.

HISTOPATHOLOGY: Yes, Adrenals, Aorta, Bone with marrow (femur) and bone marrow smear* from femur, Brain, Cecum, Colon, Duodenum, Epididymides, Esophagus, Eyes, Gross lesions if any, Harderian gland, Heart, Ileum with Peyer’s patches, Jejunum, Kidneys, Liver, Lungs, Lymph nodes - mesenteric and prescapular, Mammary glands in females, Ovaries, Pancreas, Sciatic nerves, Pituitary gland, Prostate, Rectum, Salivary glands, Seminal vesicles with coagulating glands, Skeletal muscle, Skin, Spinal cord at three levels - cervical, mid thoracic and lumbar, Spleen, Stomach, Testes, Thymus, Thyroid with parathyroid, Trachea, Urinary bladder, Uterus with cervix, Vagina.
- How many animals: all control and high dose animals, all gross lesions, all animals for organs with significant weight effects.
Statistics:
The raw data are processed to get group means and standard deviations with significance between the vehicle control and treated groups evaluated using a validated statistical software. All the parameters characterised by continuous data such as body weight, body weight change, food consumption, NBO parameters (urination, defecation, rear and vocalisation), FOB parameters (motor activity, grip strength and foot splay), organ weight, relative organ weight and clinical pathology (haematology, clinical chemistry and some of the urinalysis parameters) data are subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. Where the data do not meet the homogeneity of variance, F-test are performed followed by the t-tests to calculate significance. Any other appropriate statistical analyses test can be used based on the available data and the details are included in the final report. The significance is calculated at least at the 5% levels between vehicle control groups and different dose levels of test item.
Clinical signs:
no effects observed
Description (incidence and severity):
Rats from all groups were normal throughout the study period.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean body weight and body weight change of male rats from treatment groups were comparable with that of the respective vehicle control group. Statistically significant decrease in mean body weight was observed in female rats of low dose group during week 1 when compared with that of the vehicle control group. Statistically significant increase in mean body weight was observed in female rats of high dose recovery group during weeks 9 to 11 and 17 when compared with that of the vehicle control recovery group. Statistically, significant increase in mean body weight change was also observed in female rats of high dose recovery group during weeks 2, 3, 9 to 11, 14 and 17 when compared with that of the vehicle control recovery group; these differences are not be considered as a treatment related effects due to inconsistency between intervals and absence of similar effects in high dose female animals of the main study group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean food consumption of male and female rats from treatment groups were comparable with that of the respective vehicle control group except statistically significant increase observed at week 7 in female rats of high dose recovery group, was considered as isocratic in nature.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmological examination of rats did not reveal any abnormality.
Haematological findings:
no effects observed
Description (incidence and severity):
Statistically significant increases in neutrophil counts in the mid-dose and high-dose females, and statistically significant decreases in prothrombin time in high dose females were not considered an effect as values were within historical control ranges. Statistical results in recovery animals were not considered an effect in the absence of similar results in main study animals.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In the high dose animals of both sexes, a statistically significant decrease was noted in serum chloride. Statistically significant decrease was noted in serum sodium in male rats of the high dose group, and statistically significant increase was noted in serum inorganic phosphorus in female rats of the high dose group. These changes were considered to be related to test item treatment. However, in the absence of other supporting findings (histopathological change in kidneys), these changes were considered as a treatment related non-adverse effect.

In the high dose group of both sexes, statistically significant increases were noted in serum BUN and urea. This was not considered to be treatment related adverse effect in the absence of histopathological change in kidneys, and values of treated groups were below upper historical value (Urea: 54.61 and 63.68; BUN: 25.50 and 29.74; in male and female rats, respectively; 97.5th percentile; N = 95). Another reason for not considered these results as treatment related adverse effects was that mean values of the vehicle control group (Urea: 26.24 ± 5.99 and 34.91 ± 3.56 in male and female rats, respectively) were at lower end of historical control values (Urea: 39.75 ± 7.21 and 47.10 ± 8.14 in male and female rats, respectively).

Statistically significant decrease was noted in serum ALT in low dose male rats, whereas statistically significant increase was noted in AST in low dose female rats. Due to lack of dose dependency, these changes were not considered as related to test item treatment.

Results noted above at the end of treatment were not observed after the 70-day recovery period. Statistically significant increase in serum creatinine was noted in high dose female rats at the end of the recovery period, which was considered unrelated to treatment due to absence of effect at the end of treatment in the main study animals.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Statistically significant decrease was noted in volume of urine in mid dose male rats, which was not considered an effect due to absence of dose dependency.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Neurobehavioral Observations (NBO)
Home Cage Observations: In home cage, rats from treatment and vehicle control groups revealed normal postures asleep (curled up often asleep), sitting C (sitting or standing alert, watching), and rearing. Clonic and tonic movements were absent in home cage during NBO.

Handling Observations: NBO performed during handling of rats did not reveal any abnormality related to the treatment. All rats revealed normal behaviour during removal (very easy - rats sit quietly) and handling (easy - alert, limbs put against the body). Eyelids were wide open in all rats. None of the rats showed lacrimation, salivation, and piloerection. Eye and skin examination did not reveal any abnormality in any rat.

Open Field Observations: In open field, rats from treatment and vehicle control groups showed normal gait, mobility and respiration during three minutes observation period. The clonic and tonic movements, stereotypy, and bizarre behavior were absent. Arousal level of male and female rats from treatment groups was comparable with that of the respective vehicle control group. No statistical significant change was observed in vocalization and defecation count of male and female rats from treatment groups when compared with that of the respective vehicle control group. Statistically significant increase in urination count was observed at week 17 in female rats of high dose recovery group when compared with that of the respective vehicle control recovery group. Statistically significant increase in rears count was observed at weeks 2, 4 to 7, 9, and 10 in male rats and decrease in rears count was observed at week 17 in female rats of high dose recovery group when compared with that of the respective vehicle control recovery group.
 
The significant changes observed in rears and urination counts could not be considered as treatment related effects due to inconsistency between intervals and absence of similar effects at same interval in high dose of main group.

Functional Observational Battery
Motor Activity: Motor activity counts of male and female rats from treatment groups were comparable with that of the respective vehicle control group.

Sensory Reactivity: Sensory reactivity parameters viz., approach response, touch response, click response, tail pinch response, pupil response and air righting reflex of male and female rats from treatment groups were comparable with that of the respective vehicle control group.

Grip Strength: The hindlimb and forelimb grip strength values of male and female rats from treatment groups were comparable with that of the respective vehicle control group.

Hindlimb Foot Splay: The hindlimb foot splay values of male and female rats from treatment groups were comparable with that of the respective vehicle control group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decrease was noted in absolute and relative weights of adrenals and absolute weight of spleen in mid dose group male and female rats respectively, which were not considered to be treatment related in the absence of a dose response.

Statistically significant increase was noted in relative liver weight in both sexes of the high dose group. Absolute liver weights were also significantly increased in high dose females. These liver findings were considered to be test item related non-adverse adaptive changes in the absence of corresponding clinical pathology indicators and lack of histopathological changes after the recovery period.

Statistically significant increase was observed in absolute and relative kidney weight in high dose male rats. These changes were considered to be test item related non-adverse adaptive changes in absence of histopathological lesions in kidneys.

Statistically significant decrease was noted in absolute and relative weights of testes in mid dose group male animals, which was not considered to be related to test item treatment as all individual animal values were within historical control ranges (testes: 3.074 to 4.736 g; 2.5th percentile to 97.5th percentile, N = 215).

Weights of testes and epididymides of 4 of 15 male rats of high dose groups (3 rats from main study group and 1 rat from 5-day recovery group) were below historical control ranges (testes: 3.074 to 4.736 g; epididymides: 1.112 to 1.795 g; 2.5th percentile to 97.5th percentile, N = 215), while vehicle control rats’ weights were within range. Testes and epididymides weights of the remaining 11 high dose male rats were comparable to that of the vehicle control group. Group mean values for the high dose animals were not statistically significantly different from the control group.

In high dose recovery group male rats sacrificed close to terminal (5 day recovery), statistically significant increase was noted in absolute and relative thyroid with parathyroid weights which could not be related to treatment due to absence of same alteration in high dose group male rats sacrificed at the end of treatment.

Statistically significant increase was noted in absolute weights of spleen, brain and thymus, and relative weight of thymus in high dose recovery female rats. These changes were not noted at the end of treatment, hence considered as not related to test item treatment.

Weights of testes and epididymides of 1 of 5 male rats each of vehicle control and high dose recovery group were below historical ranges (testes: 3.074 to 4.736 g; epididymides: 1.112 to 1.795 g; 2.5th percentile to 97.5th percentile, N = 215).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
External examination of male and female rats did not reveal any abnormality.

Internal examination of female rats did not reveal any lesions. Internal examination of high dose male rats sacrificed at the end of treatment or after 5-day recovery revealed reduced size of testes and epididymides in 3/10 rats and 1/5 rats, respectively. At the end of 70-day recovery, reduced size of testes and epididymides were noted in 1 of 5 male rats of the vehicle control and high dose groups. The overall incidence was therefore 5 of 20 in high dose males compared to 1 of 20 in vehicle control males. Further, historical control incidence rates for such adverse effects in the testes are 2 of 340. These findings in the testes and epididymides of high dose males were thus considered to be test item related adverse effects.
Neuropathological findings:
no effects observed
Description (incidence and severity):
Neuropathological examination was limited to routine histology of brain, spinal cord, and peripheral nerve. No effects were observed in these tissues.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Gross lesions observed in the testes and epididymides of 4 of 15 high-dose male animals were supported by tubular atrophy and luminal aspermia/hypospermia (4 rats), and leydig cell hyperplasia (2 rats). These lesions were considered treatment related as they were not noted in vehicle control rats. No histopathological changes were seen in the testes or epididymides of remaining 11 high dose male rats or in any of the mid-dose or low-dose male rat.

Tubular atrophy in testes and luminal aspermia was also observed in one male rat each of the control and high dose recovery groups sacrificed after the 70-day recovery period.

In female rats, the incidence of hepatocellular vacuolation at the end of the treatment period was 5/10 in controls, 5/10 in low dose, 7/10 in mid dose, and 9/10 in high dose group. This was considered as treatment related adaptive changes and observed to have recovered after the 4-week recovery period.

Other changes noted in organ weight (increase in kidneys and liver weight in high dose male rats) did not lead to histopathological lesions.

Other microscopic lesions were considered to be spontaneous or incidental in nature and not treatment related.

Independent histopathology review by Dr. Chirukandath Gopinath, BVSc, PhD, MVSc, FRCPath revealed no major differences between the independent peer review pathologist and the study pathologist.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Sperm Parameters: Sperm parameter abnormalities were observed in 1 of 5 male rats each from close to terminal sacrifice high dose recovery (5 days recovery), high dose recovery (70 days of recovery) and vehicle control recovery groups (70 days recovery). In all three rats, 0 to 40% motility, 180 to 200 abnormal sperm out of 200 observed sperm and very low cauda epididymal and testicular sperm count were observed compared to other male rats. These rats with reduced sperm parameters also had markedly reduced weights of absolute and relative testes and epididymides.

Overall, statistically significant changes were not observed in mean sperm parameter values of male rats from close to terminal sacrifice high dose (sacrificed after 5 days of recovery) and high dose recovery (sacrificed after 70 days of recovery) groups compared to that of the respective vehicle control recovery group as all of the other rats in both those vehicle control and high dose recovery groups had normal sperm parameters.
Dose descriptor:
NOAEL
Effect level:
375 mg/kg bw (total dose)
Based on:
test mat.
Sex:
female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male
Basis for effect level:
gross pathology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
other: Sperm parameters
Remarks on result:
other: A striking variation of response across high dose animals and the absence of dose response raises uncertainty about these findings
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
375 mg/kg bw (total dose)
System:
male reproductive system
Organ:
testes
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
not specified

Striking abnormalities of the testes and epididymides were observed in one vehicle control and five high dose male rats. Changes included severely reduced organ weights, altered sperm parameters and marked histopathology. No changes were observed in the accessory sex glands of the affected animals. The remaining fifteen high dose male rats were comparable to their respective vehicle controls. This striking variation of response within a dose group is unusual, although based on the increased incidence in the high dose rats it cannot be discounted and is considered, with some uncertainty, to be treatment related. Sperm data are tabulated below. The full pathology report for this study with organ weight and microscopic findings are attached.

The absence of a dose response together with the absence of any similar testicular / epididymides changes in the companion 28 -day dose-range finding study (JRF Study Number 410 -1 -02 -18513) at doses up to 1000 mg/kg/day contribute to the uncertainty about findings seen in this study.

Sperm Motility of Individual Rat

 

Group: G5 (Vehicle Control Recovery); 0 mg/kg b. wt.

Rat N°

Sperm Motility Grade

Rat N°

Sperm Motility Grade

81

3

86

4

82

4

87

1

83

3

88

4

84

3

89

3

85

4

90

3

 

Group: G6 (High Dose Recovery); 375 mg/kg b. wt.

Rat N°

Sperm Motility Grade

Rat N°

Sperm Motility Grade

101

2

106

4

102

3

107

1

103

3

108

2

104

4

109

4

105

4

110

4

 


 

Sperm Count of Individual Rat

 

Group: G5 (Vehicle Control Recovery); 0 mg/kg b. wt.

Cauda Epididymal Sperm Count

Rat N°

Neubauer Hemocytometer (RBC) Chamber N° 1

Neubauer Hemocytometer (RBC) Chamber N° 2

Average/

Sum

Right Cauda Epididymis Weight (g)

N° of Sperm (million/g)     

1

2

3

4

5

Total

1

2

3

4

5

Total

 

81

25

24

29

27

24

129

-

-

-

-

-

-

129.0

0.288

559.90

 

82

24

24

23

22

26

119

-

-

-

-

-

-

119.0

0.298

499.16

 

83

24

18

21

27

29

119

24

18

34

31

21

128

123.5

0.277

557.31

 

84

33

23

20

21

25

122

30

30

27

22

25

134

128.0

0.220

727.27

 

85

27

21

26

27

25

126

-

-

-

-

-

-

126.0

0.234

673.08

 

86

30

27

29

29

24

139

-

-

-

-

-

-

139.0

0.338

514.05

 

87

1

2

2

1

2

8

2

1

3

1

3

10

9.0

0.160

70.31

 

88

27

30

30

28

32

147

-

-

-

-

-

-

147.0

0.317

579.65

 

89

29

30

31

29

32

151

-

-

-

-

-

-

151.0

0.289

653.11

 

90

22

24

28

26

31

131

26

28

27

31

31

143

137.0

0.296

578.55

 

 

Group: G6 (High Dose Recovery); 375 mg/kg b. wt.

Cauda Epididymal Sperm Count

Rat N°

Neubauer Hemocytometer (RBC) Chamber N° 1

Neubauer Hemocytometer (RBC) Chamber N° 2

Average/

Sum

Right Cauda Epididymis Weight (g)

N° of Sperm (million/g)     

1

2

3

4

5

Total

1

2

3

4

5

Total

101

1

2

0

4

3

10

0

4

3

4

2

13

11.5

0.197

72.97

102

24

23

19

21

22

109

-

-

-

-

-

-

109.0

0.282

483.16

103

38

31

36

40

30

175

-

-

-

-

-

-

175.0

0.301

726.74

104

32

30

31

30

35

158

-

-

-

-

-

-

158.0

0.256

771.48

105

27

28

22

29

29

135

-

-

-

-

-

-

135.0

0.313

539.14

106

22

21

28

30

21

122

29

30

33

22

28

142

132.0

0.300

550.00

107

0

2

0

3

1

6

1

0

1

0

4

6

6.0

0.163

46.01

108

28

37

34

32

29

160

-

-

-

-

-

-

160.0

0.318

628.93

109

42

34

42

37

35

190

-

-

-

-

-

-

190.0

0.324

733.02

110

19

23

29

18

22

111

26

24

26

27

16

119

115.0

0.297

484.01

Key:- = Not applicable


 

 

Group: G5 (Vehicle Control Recovery); 0 mg/kg b. wt.

Testicular Sperm Count

Rat N°

Neubauer Hemocytometer (RBC) Chamber N° 1

Neubauer Hemocytometer (RBC) Chamber N° 2

Average/

Sum

Right Cauda Epididymis Weight (g)

N° of Sperm (million/g)     

1

2

3

4

5

Total

1

2

3

4

5

Total

81

27

27

27

29

29

139

-

-

-

-

-

-

139.0

2.068

84.02

82

21

20

29

25

30

125

22

19

21

19

16

97

111.0

2.150

64.53

83

25

27

27

30

23

132

-

-

-

-

-

-

132.0

2.055

80.29

84

18

19

30

20

32

119

20

18

20

20

26

104

111.5

1.969

70.78

85

11

13

16

20

18

78

14

19

18

19

16

86

82.0

1.715

59.77

86

24

22

21

18

22

107

-

-

-

-

-

-

107.0

2.012

66.48

87

2

1

2

0

2

7

1

0

1

2

1

5

6.0

0.827

9.07

88

18

20

18

20

18

94

-

-

-

-

-

-

94.0

2.015

58.31

89

18

20

23

24

20

105

-

-

-

-

-

-

105.0

1.959

67.00

90

26

26

24

28

24

128

-

-

-

-

-

-

128.0

2.102

76.12

 

Group: G6 (High Dose Recovery); 375 mg/kg b. wt.

Testicular Sperm Count

Rat N°

Neubauer Hemocytometer (RBC) Chamber N° 1

Neubauer Hemocytometer (RBC) Chamber N° 2

Average/

Sum

Right Cauda Epididymis Weight (g)

N° of Sperm (million/g)     

1

2

3

4

5

Total

1

2

3

4

5

Total

101

3

1

3

2

3

12

2

2

4

0

3

11

11.5

1.103

13.03

102

14

16

16

15

18

79

-

-

-

-

-

-

79.0

2.095

47.14

103

16

16

17

21

19

89

-

-

-

-

-

-

89.0

2.135

52.11

104

18

16

15

17

20

86

-

-

-

-

-

-

86.0

1.788

60.12

105

31

20

26

21

25

123

19

21

18

19

16

93

108.0

1.959

68.91

106

22

24

19

26

27

118

18

19

20

24

22

103

110.5

1.969

70.15

107

0

0

1

1

1

3

1

1

0

2

0

4

3.5

1.060

4.13

108

24

26

21

25

22

118

-

-

-

-

-

-

118.0

2.088

70.64

109

22

21

20

18

19

100

-

-

-

-

-

-

100.0

1.945

64.27

110

25

30

26

25

23

129

-

-

-

-

-

-

129.0

2.090

77.15

Key:- = Not applicable


 

Sperm Morphological (Number) of Individual Rat

 

Group: G5 (Vehicle Control Recovery); 0 mg/kg b. wt.    N° of Sperms Observed/Rat: 200

Rat N°

N° of Sperms with Abnormality Observed

N° of Abnormal Sperm

N° of Normal Sperm

Tail-

less

Head-

less

Coiled Tail

Detach Head

Pin Head

No Hook

No Hook and Tail

Balloon Head

Broken Tail

81

3

1

0

4

0

0

0

0

0

8

192

82

2

1

0

4

0

0

0

0

0

7

193

83

3

5

0

0

0

0

0

0

0

8

192

84

7

2

0

1

0

0

0

0

1

11

189

85

9

1

0

6

0

0

0

0

0

16

184

86

1

1

0

1

0

0

0

0

0

3

197

87

3

64

1

11

0

14

72

35

0

200

0

88

2

1

0

3

0

0

0

0

0

6

194

89

12

2

0

4

0

0

0

0

0

18

182

90

3

4

0

0

1

0

0

0

0

8

192

 

Group: G6 (High Dose Recovery); 375 mg/kg b. wt.         N° of Sperms Observed/Rat: 200

Rat N°

N° of Sperms with Abnormality Observed

N° of Abnormal Sperm

N° of Normal Sperm

Tail-

less

Head-

less

Coiled Tail

Bent Tail

Detach Head

Pin Head

No Hook

No Hook and Tail

Balloon Head

Blunt Head

101

4

55

0

0

4

1

7

75

9

27

182

18

102

6

3

0

2

0

0

0

0

0

0

11

189

103

11

1

0

0

3

0

0

0

0

0

15

185

104

5

1

0

0

4

1

2

0

0

0

13

187

105

11

3

1

0

6

2

1

0

0

0

24

176

106

10

0

0

1

0

1

0

0

0

0

12

188

107

35

42

0

0

7

1

30

72

8

3

198

2

108

9

0

0

0

0

4

1

0

0

0

14

186

109

11

5

0

0

4

1

0

0

0

0

21

179

110

4

3

0

0

3

1

0

0

0

0

11

189

 

Conclusions:
Based on the results of this 90-day GLP OECD 408 study, the subchronic NOAEL for tris isotridecyl phosphite by gavage in female Wistar rats is considered to be 375 mg/kg b.wt. The subchronic NOAEL for male Wistar rats is considered to be 125 mg/kg b. wt., based on an increased incidence of testes and epididymides abnormalities seen in the high dose (375 mg/kg b. wt.) group. The testes/epididymides findings seen in the high dose male animals are considered unusual, with 5/20 rats showing severe effects and 15/20 rats being comparable to concurrent controls.
Executive summary:

A 90 -day gavage study (OECD 408 by GLP) of tris isotridecyl phosphite (TiTDP) was conducted in Wistar rats. Groups of 10 male and 10 female animals received daily doses of 42, 125 or 375 mg/kg/day TiTDP, or corn oil (vehicle control). Additional groups of vehicle control and high dose animals were similarly treated and held after treatment to evaluate recovery (28 days for females; 5 or 70 days for males). The male recovery period was adjusted to evaluate findings in testes / epididymides at the end of treatment. Animals were evaluated for mortality, clinical signs, body weight, food consumption, neurobehavior, opthalmology, clinical pathology (hematology, clinical biochemistry, urinalysis), organ weights, gross and microscopic pathology. Sperm paramters were also evaluated in male recovery animals.

Dose formulations were prepared daily and were determined to be stable and homogeneous.

No mortality or morbidity occurred, and no abnormal clinical signs or treatment-related opthalmological or neurobehavioral effects were observed. Body weights and food consumption were comparable to controls.

Effects of treatment in female rats from the high-dose group consisted of decreased serum chloride, increased serum inorganic phosphorus, increased serum BUN and urea, and increased liver weight accompanied by an increased incidence of hepatocellular vacuolation. Findings in the female livers are considered adaptative and non-adverse in absence of clinical pathology indicators and similar histopathology to vehicle control rats at the end of the recovery period. Other clinical chemistry findings are considered as treatment related non-adverse effect due to either absence of histopathological change in kidneys or absence of similar effects at the end of recovery period.

Effects of treatment in male rats from the high dose group consisted of decreased serum chloride, decreased serum sodium, and increased liver and kidney weights. These findings are also considered to be adaptive and non-adverse due to absence of histopathological changes or absence of clinical pathology indicators. se findings are also considered to be adaptive and non-adverse due to absence of histopathological changes or absence of clinical pathology indicators.

Striking abnormalities of the testes and epididymides were observed in one vehicle control and five high dose male rats. Changes included severely reduced organ weights, altered sperm parameters and marked histopathology. No changes were observed in the accessory sex glands of the affected animals. The remaining fifteen high dose male rats were comparable to their respective vehicle controls. This striking variation of response within a dose group is unusual, although based on the increased incidence in the high dose rats it cannot be discounted and is considered, with some uncertainty, to be treatment related.

Based on the results of this 90-day GLP OECD 408 study, the subchronic NOAEL for tris isotridecyl phosphite by gavage in female Wistar rats is considered to be 375 mg/kg b.wt. The subchronic NOAEL for male Wistar rats is considered to be 125 mg/kg b. wt., based on an increased incidence of testes and epididymides abnormalities seen in the high dose (375 mg/kg b. wt.) group. The testes/epididymides findings seen in the high dose male animals are considered unusual and warrent further evaluation, with 5/20 rats showing severe effects and 15/20 rats being comparable to concurrent controls.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
October 03, 2008
Deviations:
yes
Remarks:
dose range finding design
Principles of method if other than guideline:
This study was designed to inform selection of dose levels for a 90-day repeat dose toxicity study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
NAME OF TEST MATERIAL (as stated in the study report): Trisisotridecylphosphite

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Valtris Specialty Chemicals
- Batch No. 330965
- Expiration date of the lot/batch: March 02, 2019
- Purity: 100% (UVCB substance)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in original container.
- Stability under test conditions: The stability of the test item in dose formulations was determined using a validated analytical method. The test item was found stable up to 24 hours under use conditions (i.e., room temperature). Dose formulations were used immediately after preparation.
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Jai Research Foundation
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 9 weeks
- Housing: in groups of 2 to 3 rats/cage/sex in sterilised solid floor polypropylene rat cages
- Diet (e.g. ad libitum): standard rodent pellet feed (Teklad Certified Global 16% Protein Rodent Diet), ad libitum.
- Water (e.g. ad libitum): clean and filtered drinking water (RO water filtration system) in autoclaved polypropylene bottles, ad libitum.
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY: checked periodically for microbial and chemical contaminants

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23
- Humidity (%): 64 - 66
- Photoperiod (hrs dark / hrs light): 12 h lighting and 12 h darkness; light hours were 06.00 - 18.00 h which was maintained by fluorescent tube lights using autotimer.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: The prepared dose formulations were thoroughly mixed using a magnetic stirrer before dosing and with cannula intermittently during dosing. The dose formulation was given to rats immediately after preparation.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility; the test material is expected to hydrolyze in water.
- Dose Volume: 10 mL/kg b.w.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability, concentration of test item and homogeneity in dose formulation were analysed at Department of Chemistry, during analytical method validation study (JRF study number 228-2-13-18511). Two set of samples (10 samples per set) were collected by sampling three aliquots (upper, middle and lower layers) from each concentration except vehicle control (only middle layer). The sampling was done before initiation of the dosing (day 1) and at the end of the dosing (day 28) for the dose formulation analyses. One set of samples were used for analyses and other set of samples were stored at -70 ± 10 °C. The second set of samples will be discarded during report finalization. The mean concentration was determined and compared to the nominal value and the coefficient of variation calculated. The acceptance criteria used for analysis was ±15% from nominal value and %CV < 10.
Duration of treatment / exposure:
28 consecutive days
Frequency of treatment:
once daily
Dose / conc.:
0 mg/kg bw (total dose)
Dose / conc.:
250 mg/kg bw (total dose)
Dose / conc.:
500 mg/kg bw (total dose)
Dose / conc.:
1 000 mg/kg bw (total dose)
No. of animals per sex per dose:
5 males and 5 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on experience with similar test materials.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day for mortality and signs of morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a day for clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations: at the beginning of treatment (day 1) and at weekly intervals thereafter. Rats were also weighed on the day of necropsy (fasted body weight).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: at weekly intervals for each cage, and calculated as g/rat/day.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Full gross necropsy under the direct supervision of a veterinary pathologist. Rats were examined carefully for external abnormalities. The thoracic and abdominal cavities were cut, opened and a thorough examination of the organs was carried out to detect abnormalities.

ORGAN WEIGHTS: Yes, liver, kidneys, adrenals, testes, epididymides, thymus, heart, brain, spleen, uterus with cervix and ovaries. The paired organs was weighed together and combined weight was presented. The organ weight ratios as percentage of body weight were determined.

HISTOPATHOLOGY: Yes. Due to presence of statistically significant alteration in absolute and relative weight of liver and kidneys of all three treated groups (G2, G3 and G4), histopathology was carried out of liver and kidneys of rats from vehicle control, low and mid dose groups of both sexes. At the request of the sponsor, testes of male rats from all groups were also examined microscopically.
Statistics:
All the raw data was recorded in standard formats and data was summarised in tabular form. The raw data was processed to get group means and standard deviations with significance among the vehicle control and treatment groups using a validated statistical software. All the parameters characterised by continuous data such as body weight, body weight change, food consumption, absolute organ weight and relative organ weight were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test (Gad, S.C. and Weil, C.S., 1994). Where the data do not meet the homogeneity of variance, F-test was performed followed by the t-tests to calculate significance. All analyses and comparisons were evaluated at the 5% (P<0.05) and 1% (P<0.01) levels.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant increase in absolute and relative weights of liver in mid dose and high dose groups of both sexes and low dose group of male rats, in dose dependent manner. The effect was also observed in low dose group of female rats though statistical significance was not observed. Significant increases was also observed in absolute and relative weights of kidneys in all treatment groups of male rats though statistical significance was not achieved for relative weights of low dose group male rats.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic examination of liver revealed treatment related hypertrophy of hepatocytes in low and mid dose group of both sexes. These lesions were considered as effect of test item and it was supported by increase in relative and absolute weight of liver. In low dose females, hepatocellular vacuolation (4/5) was noted with minimal severity. As this lesion was not observed with similar incidence in mid dose females (1/5) and not observed in any of males animals, it could not be confirmed as related to test item treatment.

Kidneys from the low- and mid- dose males and females did not reveal any alteration in microscopic examination. Livers and kidneys from the high dose animals were not examined microscopically in this dose range study.

Microscopic examination of testes from all groups showed sertoli cells only tubules in two rats (one control and one low-dose animal) and tubular atrophy in one mid-dose rat. These findings were considered incidental and not a treatment related effect.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Dose descriptor:
LOEL
Effect level:
250 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Remarks on result:
other: lowest dose level tested
Dose descriptor:
LOEL
Effect level:
250 mg/kg bw (total dose)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Remarks on result:
other: lowest dose level tested
Critical effects observed:
no
Conclusions:
This dose range finding study (OECD 407 inspired) was conducted to inform dose level selection for a 90-day repeat dose toxicity study. Rats were administered tris isotridecyl phosphite by gavage for 28 consecutive days and observed for mortality, clinical signs, body weights, food consumption, gross pathology, selected organ weights, and histopathology of liver and kidneys (control, low- and mid- dose groups) and testes (all male groups). Effects were limited to dose-related increases in liver weight in both males and females, and dose-related increases in kidney weight in males. These effects were noted in all test groups. Microscopic examination of liver revealed hypertrophy consistent with the liver weight increases. Microscopic changes were not observed in the kidneys or testes of male rats. Based on these findings, dose levels of 42, 125 and 375 were selected for the 90-day repeat dose toxicity study.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
125 mg/kg bw/day
Study duration:
subchronic
Species:
rat
System:
male reproductive system
Organ:
testes

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A 90 -day gavage study (OECD 408 by GLP) of tris isotridecyl phosphite (TiTDP) was conducted in Wistar rats. Groups of 10 male and 10 female animals received daily doses of 42, 125 or 375 mg/kg/day TiTDP, or corn oil (vehicle control). Additional groups of vehicle control and high dose animals were similarly treated and held after treatment to evaluate recovery (28 days for females; 5 or 70 days for males). The male recovery period was adjusted to evaluate findings in testes / epididymides at the end of treatment. Doses were selected based on results of a 28 -day dose range finding study of 250, 500 and 1000 mg/kg/day. Animals were evaluated for mortality, clinical signs, body weight, food consumption, neurobehavior, opthalmology, clinical pathology (hematology, clinical biochemistry, urinalysis), organ weights, gross and microscopic pathology. Sperm paramters were also evaluated in male recovery animals.

Dose formulations were prepared daily and were determined to be stable and homogeneous.

No mortality or morbidity occurred, and no abnormal clinical signs or treatment-related opthalmological or neurobehavioral effects were observed. Body weights and food consumption were comparable to controls.

Effects of treatment in female rats from the high-dose group consisted of decreased serum chloride, increased serum inorganic phosphorus, increased serum BUN and urea, and increased liver weight accompanied by an increased incidence of hepatocellular vacuolation. Findings in the female livers are considered adaptative and non-adverse in absence of clinical pathology indicators and similar histopathology to vehicle control rats at the end of the recovery period. Other clinical chemistry findings are considered as treatment related non-adverse effect due to either absence of histopathological change in kidneys or absence of similar effects at the end of recovery period.

Effects of treatment in male rats from the high dose group consisted of decreasedserum chloride, decreased serum sodium, andincreased liver and kidney weights. These findings are also considered to be adaptive and non-adverse due to absence of histopathological changes or absence of clinical pathology indicators. se findings are also considered to be adaptive and non-adverse due to absence of histopathological changes or absence of clinical pathology indicators.

Striking abnormalities of the testes and epididymides were observed in one vehicle control and five high dose male rats. Changes included severely reduced organ weights, altered sperm parameters and marked histopathology. The remaining fifteen high dose male rats were comparable to their respective vehicle controls. This striking variation of response within a dose group is unusual, although based on the increased incidence in the high dose rats it cannot be discounted and is considered, with some uncertainty, to be treatment related.

Based on the results of this 90-day GLP OECD 408 study, the subchronic NOAEL for tris isotridecyl phosphite by gavage in female Wistar rats is considered to be 375 mg/kg b.wt. The subchronic NOAEL for male Wistar rats is considered to be 125 mg/kg b. wt., based on an increased incidence of testes and epididymides abnormalities seen in the high dose (375 mg/kg b. wt.) group. The testes/epididymides findings seen in the high dose male animals are considered unusual, with 5/20 rats showing severe effects and 15/20 rats being comparable to concurrent controls. The absence of a dose response together with the absence of any similar testicular / epididymides changes in the companion 28 -day dose-range finding study (JRF Study Number 410 -1 -02 -18513) at doses up to 1000 mg/kg/day contribute to the uncertainty about findings seen in this study. Furthermore, studies of other isoalkyl phosphites and the isoalkyl hydrolysis product of TiTDP do not support a concern for male reproductive effects. Given the unusual nature of the results in the high dose male animals of the OECD 408 study of TiTDP, efforts are being proposed to further evaluate and attempt to replicate these findings.

Justification for classification or non-classification

In a 90 -day gavage study (OECD 408 by GLP) the NOAELs for TiTDP were 375 mg/kg/day (females) and 125 mg/kg/day (males). Classification of TiTDP for STOT-RE is therefore not justified according to the criteria described in Regulation (EC) No 1272/2008.