Registration Dossier

Administrative data

Description of key information

Repeated Dose Toxicity Oral - 90 day Rat: NOEL >20000 mg/kg in diet

Repeated Dose Toxicity Oral - 14 day Dog: NOEL 150,000 ppm in the diet.

Repated Dose Toxicity Oral - 90 day Dog: NOEL >20,000 ppm in the diet.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 27, 1980 to December 29, 1980.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Standard followed not detailed in the study report, however the report is robust.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: Not specified in the study report
Deviations:
not specified
Principles of method if other than guideline:
Phase I of this study was to assess the safety of TVCI R-8968 when administered to Sprague Dawley rats from weanling (F0) through sexual maturity, mating, gestation, lactation and weaning their offspring (F1).
Phase II of the study was to assess the safety of TVCI when administered to weanlings (F1) for 90 days.
GLP compliance:
no
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Animals and Housing:
One hundred and five male and one hundred and five female non-sibling Sprague Dawley rats, 21 days old arrived at WIL Research Laboratories, Inc. on August 19, 1980 from Harlan Industries, Indianapolis, Indiana. The animals were singly housed in stainless steel cages, elevated above the droppings in a laminar air-flow room. These animals were fed Certified Purina Rodent Meal #5002 and water -ad libitum and acclimated for one week.
Animal Assignment:
One hundred male and one hundred female rats were randomized into four groups. Randomization and assignment to dose groups were accomplished by a computer program based on stratified body weight and random number generation.

After all of the litters were weaned one hundred males and one hundred females were randomly selected from their litters, within the same dose level, for the same dose level for Phase II using a computer program based on stratified body weight and random number generation. The animals were singly housed in stainless steel cages. The cages were elevated above the droppings in a laminar airflow room. These animals continually received test material in the diet before selection, during selection and upon transferring to singly cage housing for and during the 90 day study. Of the 30 animals per sex chosen for control and high dose, 10 animals per sex were considered a satellite reversal group. The remaining unselected pups were terminated on January 2, 1981 by C02 inhalation.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Certified Purina Rodent Meal and the test material were mixed together, in the appropriate concentrations, using the Twin Shell Blender. The route of administration was by oral admixture into food rations.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Fresh diets were prepared weekly. Two sets of diet samples, each containing 25 grams from three layers, top-middle-bottom, of each dose level were tested for homogeneity analysis on weeks 0, 1, 2, 4, 6, 10, 12 and 18 of the study.
A 100 gram sample, from each dose level, was taken and stored in the test material archives at WIL Research as a retention sample for each week.

A satisfactory spectrophotometer was not available to carry out analysis of diet as proposed in the protocol of the study. Thus, a modification of the proposed method was made and was used for all diet analysis. The extraction portion of the JR Lane procedure was not changed so there was no need to determine recoveries. The TVCI in the extracts was determined using a Tracor HPLC as the means of quantitative measurement. No effort was made to resolve the TVCI peak from other components because of the need to modify the procedure rapidly. Thus, the response from the diet extracts with no added TVCI at the same elution time was used as a control for correction of peak height. Some resolution of TVCI from diet extractives was obtained so the method is an improvement over the spectrophotometric assay.
The liquid chromatograph used was assembled from the following Tracor (Austin, Texas) modules: a Model 995 isochromatographic pump, a Model 970A variable wave length UV detector and a Model TS-10 recorder. The 25 cm column (4.6 mm, ID) was packed with 3.5 grams of Partisil-10 PAC (Whatman). The chromatography was isocratic, with a mobile phase of chloroform-methanol (100-1). The mobile phase was pumped at a flow rate of 1.00 ml/min. A 20 ul sample was introduced to the column through a Rheodyne Model 7120 injector. Peak height was measured from the detector signal sent to the recorder. Full scale deflection on the recorder is 100 units and was read to 0.5 of a unit. The recorder was set to move the chart paper at 2.5 cm/min. The Tracor variable wave length detector has the capability of scanning the spectrum of a sample which is in the flow cell. Using that technique it was found that TVCI had a maximum absorbance at 270 nm with that particular detector. Thus the absorbance detector was set at 270 nm routinely. Under the conditions of the chromatography TVCI was eluted as a sharp peak with a retention time of about 1.58 min which is about the t0 of the column. Samples of the diet without TVCI added were processed thru the proposed modified procedures. The chromatogram showed three peaks which were not well resolved with retention times of 1.45, 1.55 and 1.70 min. The chromatography did allow for reduction of more than 60% of the UV absorbing interference from the extracts. Analysis of diet samples containing the lowest concentration of TVCI (2.0 grams per kg) showed that TVCI eluted between the second and third interfering components. Thus the height of the valley between the second and third peak in the control chromatograms was used as a correction in the calculation of the concentration of TVCI.
The analyses of the diet samples thus followed a routine format. In addition to the standard TVCI, at least three sub-samples of control diet (0.0 grams TVCI/kg) were analyzed with each set of subsamples containing TVCI. For analysis of the standard, the control diet and the low level diet (2.0 grams per kg) the absorbance was set at 0.16 AUFS. The average peak height from the three control chromatograms was used as a correction of peak height for unknown sub-samples. For analysis of the medium test level (6.3 grams per kg) the absorbance was set at 0.32 AUFS. For analysis of the high test level (20.0 grams/kg) the absorbance was set at 1.28 AUFS.
Calculation
h = peak height of an unknown
hc = peak height of controls
hs = peak height of standard
h - hc
concentration in grams per kg = ----------- x factor
hs
The standard contained 100 ug/ml. For the low level of diet the factor was one since the controls and low level samples were both analyzed at 0.16 AUFS. For the higher levels of test diet the factor was 2 or 8 when the absorbance was set at 0.32 or 1.28 AUFS. The hc was reduced appropriately for the analyses at 0.32 and 1.28 AUFS. That is, the correction value at 0.32 was hc/2 and at 1.28 it was hc/8.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily in feed
Remarks:
Doses / Concentrations:
2.0, 6.325, or 20.0 grams TVCI R-8968/kg
Basis:
nominal in diet
No. of animals per sex per dose:
30 males and 30 females in the control and high dose groups (120 animals)
20 males and 20 females in the low and medium dose groups (80 animals)
Control animals:
yes, plain diet
Details on study design:
See Any other informaton for animal assignments
Positive control:
Positive control not used in this study.
Observations and examinations performed and frequency:
Observations and Measurements:
All animals were observed at least once daily for pertinent abnormal behavior and toxic signs. Body weight and food consumption measurements were performed on a weekly basis throughout the study. Number of food consumption or body weight measurements made weekly may vary due to spillage or animal death.
Clinical Laboratory Tests:
Clinical laboratory tests were conducted on test weeks 6 and 12. Week 12 was a deviation from protocol due to the clinical laboratory demand. During weeks 6 and 12, clinical determinations were made on 20 animals (10 males and 10 females) from each dose level. The animals were selected by computer randomization and the same animals were used at both intervals.
The urine samples were collected one day before the blood samples. All animals selected for hematology and serum chemistry determinations were fasted approximately 16-18 hours prior to bleeding. Samples were obtained from the retro-orbital sinus. The following clinical parameters were measured (detailed in table for see Any other information).
Sacrifice and pathology:
Euthanasia:
All animals were sacrificed by CO2 inhalation.
At the termination of Phase II, a complete necropsy was performed on all animals. The following organs were weighed from each animal in Phase II:
Liver, Kidneys, Brain, Heart, Spleen, Gonads (with epididymis), Thyroid (with parathyroid), Pituitary, Adrenals
The following tissues and/or organs were collected, fixed in 10% neutral buffered formalin and a histological examination was performed on the following organs and tissues: (WIL-80149) control and high dose :
Adrenals, Brain, Kidneys, Aorta, Eye (with optic nerve), Rectum, Heart, Liver, Trachea, Esophagus, Stomach, Duodenum, Ileum, Jejunum, Cecum, Colon, Sciatic Nerve, Lung with Mainstem Bronchi, Mesenteric Lymph Node, Skeletal Muscle, Skin, Mammary Gland, Spleen, Pancreas, Pituitary, Prostate/Corpus, Cervix Uteri, Sternebra (Bone Marrow), Salivary Gland, Ovaries/Testes (with epididymis), Thymus, Thyroid (with parathyroid), Urinary Bladder, Any Other Tissues with Lesions

Processing:
The tissues were trimmed according to standard procedure and processed in an autotechnicon on a 16 hour schedule, dehydrated in Technicon brand dehydrant (D-29), cleared in Technicon brand clearant (NC-670) and infiltrated in Paraplast®. The tissues were embedded in blocks of Paraplast® according to standard procedures. The tissues were sectioned at 6 microns and stained with Harris's Hematoxylin and Eosin and examined.
Other examinations:
None specified
Statistics:
All food uptake values and body weight data were statistically analyzed using the Dunnett's Test.
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:
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:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Details on results:
Clinical Observations:
There was one animal, number 87, group 4 male, died in the 16th week. Sporadic clinical observation were noted, however, no consistent test material related effects were noted.
Body Weights:
No significant body weight differences were observed during Phase II of the study when compared to the controls. There were no test material related effects in Phase II.
Food Consumption:
There were sporadic differences noted, however, no consistent test material related effects were noted in Phase II.

Hematology:
In Phase II at week 6 in the group 4 females the hematocrit was statistically significantly higher (p < -0.01) than the control group females. However, this value was within normal biological limits for this laboratory and not considered to be biologically significant. All other parameters measured at 6 weeks and 12 weeks when compared to the controls were normal using the Dunnett's analysis. Therefore there were no consistent test material related effects noted in the hematology parameters.
Serum Chemistry:
There were sporadic changes in blood glucose, alkaline phosphatase and SGOT at 6 weeks and 12 weeks. However no consistent test material related effects were noted.
Urinalysis:
All parameters measured at 6 and 12 weeks in the test groups when compared to the controls were normal.
Diet Analyses - Homogeneity and Stability:
All samples were within acceptable limits for homogeneity.

Pathology:
There were no test material effects noted in the test animals when compared to the controls.
Dose descriptor:
NOEL
Effect level:
> 20 000 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test material effects noted
Critical effects observed:
not specified

Summary of Major Clinical Observations

 

Frequency/Number of Animals

Dose Group

Observation

1

2

3

4

MALES

Emaciation

32/8

19/3

27/6

47/6

Dehydration

7/5

8/2

14/3

26/8

Scruffy Haircoat

91/12

48/9

14/5

34/11

Alopecia – Forepaws

155/4

52/2

120/3

180/3

Alopecia – Forearms

132/2

52/2

25/2

211/3

Dried Red Nasal Discharge

446/30

550/20

395/20

752/29

Red Material Around One or Both Eyes

8/6

14/9

24/11

7/7

Urine Stains

7/6

18/8

26/11

35/21

Loose Faeces

118/28

108/20

70/20

124/28

White Worms Around Anal Opening

103/27

70/18

63/18

60/26

Salivation

59/17

34/12

47/14

91/22

FEMALES

Emaciation

205/13

184/10

67/8

71/8

Dehydration

9/3

9/3

9/5

10/6

Scruffy Haircoat

27/8

20/4

9/6

3/2

Alopecia – Forepaws

73/3

79/2

73/3

204/5

Alopecia – Forearms

52/3

74/2

63/1

201/4

Dried Red Nasal Discharge

474/29

393/20

335/20

534/30

Red Material Around One or Both Eyes

20/6

13/7

16/11

38/9

Urine Stains

3/3

12/2

17/9

36/13

Loose Faeces

40/19

26/14

19/13

63/25

White Worms Around Anal Opening

25/17

24/12

14/9

22/16

Salivation

71/22

22/12

35/13

60/21

 

Body Weight (g) – Summary of Means

Sex:

MALE

FEMALE

Dose Grp.:

1

2

3

4

1

2

3

4

Week 0

121.5

118.2

115.3

121.6

107.6

98.2

101.4

108.1

Week 1

168.6

167.3

163.8

172.4

136.6

128.3

133.7

139.0

Week 2

213.1

217.7

206.9

208.4

158.4

151.3

154.2

152.7

Week 3

256.1

265.8

257.3

262.5

177.3

170.9

177.7

178.7

Week 4

288.2

296.0

290.9

296.7

192.1

186.5

191.3

195.7

Week 5

311.7

320.3

317.1

322.5

203.3

198.8

206.1

207.3

Week 6

328.4

336.5

335.0

339.1

210.5

206.1

214.1

214.3

Week 7

345.3

352.5

352.0

356.5

219.3

212.4

220.9

222.6

Week 8

362.2

372.1

370.2

373.5

226.1

220.5

230.6

231.5

Week 9

377.4

387.1

385.4

388.8

231.8

226.1

237.7

236.3

Week 10

387.2

395.9

396.5

400.0

235.3

231.7

242.0

241.4

Week 11

397.8

408.0

407.6

409.9

240.7

236.9

246.9

245.0

Week 12

410.3

421.7

421.5

424.1

245.8

243.1

253.1

252.1

Week 13

415.7

411.4

416.4

436.1

235.6

236.6

2243.7

237.4

 

Hematology Values – Summary Of Means Week 6

Sex:

MALE

FEMALE

Dose Grp.:

1

2

3

4

1

2

3

4

WBC

THOUS/UL

10.45

10.79

9.51

9.74

7.46

7.70

8.57

7.15

RBC

MIL/UL

8.11

8.01

8.03

7.93

7.51

7.47

7.54

7.76

HGB

G/DL

17.49

17.16

17.50

17.15

16.54

16.59

16.73

17.10

HCT

%

43.34

41.85

44.30

42.34

41.16

41.67

41.75

43.93**

MCV

CUBIC U

53.40

52.40

55.23

53.70

54.90

56.00

55.70

56.60

MCH

UUG

21.58

21.44

21.81

21.69

22.04

22.24

22.24

22.07

MCHC

G/DL

40.35

41.05

39.59

40.52

40.20

39.80

40.12

38.96

PLAT

THOUS/UL

764.40

742.00

735.00

764.00

698.70

678.70

731.10

794.20

** = Significantly Different from Control Group at .01 Level using Dunnett’s Test

Unit Code:

THOUS/UL = Thousands/Microliter

MIL/UL = Million/Microliter

G/DL = Grams/Deciliter

CUBIC U = Cubic Microns

UUG = Micro Micro Gram

 

Hematology Values – Summary Of Means Week 12

Sex:

MALE

FEMALE

Dose Grp.:

1

2

3

4

1

2

3

4

WBC

THOUS/UL

8.20

8.41

7.84

7.10

5.75

5.37

6.25

4.70

RBC

MIL/UL

8.44

8.67

8.70

8.34

7.94

7.97

8.11

7.97

HGB

G/DL

17.25

17.26

17.65

17.10

17.03

17.06

17.26

16.90

HCT

%

44.72

46.20

46.68

45.68

45.72

44.56

45.97

46.03

MCV

CUBIC U

53.10

53.60

53.70

54.90

57.50

56.00

56.80

57.80

MCH

UUG

20.48

19.94

20.30

20.55

21.48

21.48

21.30

21.23

MCHC

G/DL

38.68

37.47

37.87

37.48

37.39

38.48

37.59

36.87

PLAT

THOUS/UL

703.80

709.90

683.40

705.10

650.50

630.00

650.90

737.5-

Unit Code:

THOUS/UL = Thousands/Microliter

MIL/UL = Million/Microliter

G/DL = Grams/Deciliter

CUBIC U = Cubic Microns

UUG = Micro Micro Gram

 

Gross Pathology Observation – Organ Summary

Sex:

MALE

FEMALE

Dose Grp.:

1

2

3

4

1

2

3

4

BONE

1

0

0

1

0

0

0

0

CECUM

0

0

0

1

0

0

0

0

COLON

0

0

0

1

0

0

0

0

EPIDIDYMIS

0

1

0

0

0

0

0

0

EYES/OPTIC N.

1

1

0

0

0

0

0

0

KIDNEYS

0

0

3

1

0

0

1

1

LIVER

0

0

0

0

0

1

0

0

SKIN

1

0

0

3

0

0

0

0

SPLEEN

0

0

0

0

0

0

1

0

STOMACH

1

2

0

1

0

0

2

3

TESTES

0

0

0

1

0

0

0

0

UTERUS

0

0

0

0

2

1

1

0

Conclusions:
TVCI R-8968 when fed continuously for 90 days at a dose level of 20.0 grams/kg diet or less did not produce toxicity.
Executive summary:

Introduction:

The test material TVCI R-8968 is a proposed indirect food additive. The test material was administered orally in the feed as this is consistent with the expected route of human exposure.

Objective:

Phase II of the study was to assess the safety of TVCI when administered to weanlings F1 for 90 days.

Phase II, WIL-80148 (pup body weights of pups selected after culling litters to 8/litter on day 4 of life). WIL-80149 (PI), WIL-R0149 (F1) Reversal, started on January 1, 1981 and ended in April 2, 1981.

Summary:

TVCI R-8968 when fed continuously to (F1) for 90 days at a dosage level of 20.0 gram per kg diet or less did not produce toxicity in the (F1) generation.

The dosage levels of 2.0, 6.325, or 20.0 grams TVCI R-8968/kg of diet were administered in the feed throughout the study.

Samples from weekly prepared diets from all dosage level were taken periodically analyzed for homogeneity and all samples were homogeneous for that specific dose level. The feed mixture was tested for TVCI R-8968 stability in the diet and found to be stable for at least 25 days.

There were sporadic differences in the treatment group in body weights food consumption, body weight of offspring, hematology, and serum chemistries when compared to the controls. However, no consistent test material related effects were noted.

TVCI R-8968 when fed continuously for 90 days at a dose level of 20.0 grams/kg diet or less did not produce toxicity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
20 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat

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

Repeated Dose Toxicity: Oral - 90 day Rat

The test material TVCI R-8968 is a proposed indirect food additive. The test material was administered orally in the feed as this is consistent with the expected route of human exposure.

Summary:

The dosage levels of 2.0, 6.325, or 20.0 grams TVCI R-8968/kg of diet were administered in the feed throughout the study.

Samples from weekly prepared diets from all dosage level were taken periodically analyzed for homogeneity and all samples were homogeneous for that specific dose level. The feed mixture was tested for TVCI R-8968 stability in the diet and found to be stable for at least 25 days.

There were sporadic differences in the treatment group in body weights food consumption, body weight of offspring, hematology, and serum chemistries when compared to the controls. However, no consistent test material related effects were noted.

TVCI R-8968 when fed continuously for 90 days at a dose level of 20.0 grams/kg diet or less did not produce toxicity.

Repeated Dose Toxicity: Oral - 14 day Dog

The purpose of the study was to evaluate the potential toxicity of TVCI to the dog. Ten purebred beagle dogs were assigned to five groups (1/sex/group) and fed TVCI in the diet during a fourteen day period at levels of 25,000 – 250,000 ppm.

The dietary administration of TVCI at levels of 25,000-150,000 ppm demonstrated that these doses can be tolerated for a 14-day period. This conclusion is based on the food consumption, gross necropsy and body weight data. Levels higher than 150,000 ppm were found to have a limited palatability and a detrimental effects on body weight. 

Repeated Dose Toxicity: Oral - 90 Day Dog

The purpose of the study is to evaluate the sub-chronic toxicity effects of TVCI R-8968 when administered in the diet to beagle dogs for a period of 90 consecutive days. The dose levels if TVCI R-8968 used were 20,000, 6,300 and 2,000 ppm. Body weight and food consumption were measured weekly, animals were observed daily, blood sample for hematology and clinical chemistry determinations were collected prior to the initiation of the study and at monthly intervals thereafter. Urine was collected and analysed prior to the start of the study and again at the termination of the study. At the termination of the study (after 90 days of feeding), gross necropsy and histopathologic examinations were performed on all animals.

During the 13 week feeding period, no significant differences in body weight, food consumption or urine analysis were noted among groups, nor were there any overt external signs of toxicity or behavioural abnormality. Intermittent significant variations in mean hematology and clinical chemistry parameters were noted among some groups. However, since these changes were not dose related and were not supported by any histopathological findings, the changes were not regarded as toxicologically significant.

At terminal sacrifice, mean absolute and relative organ weights were comparable among groups. No test article-related histopathological findings were noted in any organs. 

Justification for classification or non-classification