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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 January 1981 - 28 April 1981
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in accordance with generally accepted scientific principles in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1981
Report Date:
1981
Reference Type:
study report
Title:
Unnamed
Year:
1983
Report Date:
1983

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
no
Remarks:
The study was subjected to auditing procedures
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid (undefined)
Details on test material:
- Description: orange amber liquid
- Storage: stored in a cool, dry place

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: CD (Sprague-Dawley derived).
- Age at study initiation: Approximately 6 weeks.
- Housing: Individually in elevated stainless steel cages.
- Diet: Standard laboratory diet ad libitum. Fresh food was presented once weekly.
- Water: ad libitum. By automated water system.
- Acclimation period: 14 Days (13 - 26 January 1981).

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle.

IN-LIFE DATES: From: To: 27 January 1981 - 28 April 1981

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Appropriate amounts of test substance were mixed with standard laboratory diet weekly.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each dietary admixture for each dose level were taken in duplicate, one sample for each dose level was analyzed for concentration of test material; the other was stored frozen for the first four weeks of the study. After four weeks, samples were analysed once every 2 weeks and the samples for off weeks were stored frozen.
Duration of treatment / exposure:
Males: 90 days
Females: 91 days
Frequency of treatment:
Daily: the test material was administered in the animals' feed, available ad libitum. Fresh food was presented once weekly.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 2000, 10000 and 50000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.0, 158.15, 775.74, 3865.67 mg/kg bw/day males
Basis:
actual ingested
Remarks:
Doses / Concentrations:
0.0, 183.89, 933.09, 4633.36 mg/kg bw/day females
Basis:
actual ingested
No. of animals per sex per dose:
- 2000 and 10000 ppm dose level: 20 animals per sex per dose.
- 0 and 50000 ppm dose level: 30 animals per sex per dose.
Control animals:
yes, plain diet
Details on study design:
The additional 10 animals in the 0 and 50000 ppm dose groups were held for a 1 month recovery period during which no test material was administered. The purpose of the recovery period was to assess the reversibility of any toxic effects noted.

Examinations

Observations and examinations performed and frequency:
OBSERVATIONS
- Mortality and gross signs of toxicologic or pharmacologic effects: twice daily.
- Detailed physical examination for signs of local or systemic toxicity, pharmacologic effects and palpation for tissue masses: weekly.
- Bodyweight: twice pre-test, weekly during treatment and terminally (after fasting).
- Food consumption: weekly, beginning one week prior to treatment.
- Test substance intake: calculated from weekly body weight and food consumption data. Based on nominal concentrations.

LABORATORY STUDIES
Blood was obtained via venipuncture of the orbital sinus (retrobulbar venous plexus) under light ether anaesthesia. Animals were selected randomly; the same animals were used at all intervals when feasible. Rats were fasted overnight prior to blood collections and were not dosed until after samples were collected.
10 males and 10 females were examined pre-test; 10 animals per sex per group were examined at 7 and 13 weeks.

PARAMETER EVALUATED
-Haematology
Haemoglobin haematocrit
Erythrocytes platelets
total leukocytes differential leukocytes

-Clinical Chemistry
serum glutamic oxaloacetic transaminase serum glutamic pyruvic transaminase
alkaline phosphatase blood urea nitrogen
fasting glucose total protein
cholesterol sodium
potassium chloride
calcium creatinine
total bilirubin direct bilirubin
* Due to a technical error Albumin was not evaluated for pre-test blood work, therefore Globulin and A/G Ratio could not be calculated.

-Urinalysis
gross appearance specific gravity
pH protein
glucose ketones
bilirubin occult blood
urobilinogen
Sacrifice and pathology:
POSTMORTEM
- Animals dying spontaneously or killed in a moribund condition: subject to complete gross postmortem examination.

NECROPSY
- All animals were subjected to necropsy.
- Sacrifice method: exsanguination under ether anaesthesia.
- Organs weighed and organ/bodyweight ratios calculated: (paired organs weighed separately)
kidneys ovaries
liver spleen
heart pituitary
brain adrenals
testes thyroid/parathyroid

TISSUES PRESERVED (numbers in parentheses indicate number of organs/sections preserved).
adrenal (2) aorta (abdominal)
bone (sternum, femur) bone marrow (sternum)
brain cecum
colon duodenum
epididymis (2) eye (2-with optic nerve)
heart ileum
jejunum kidney (2)
liver lungs (with mainstem bronchi)
lymph node (mesenteric) mammary gland (inguinal)
nerve (sciatic) oesophagus
ovaries pancreas
pituitary prostate/seminal vesicles
rectum salivary gland (submandibular)
skeletal muscle (right bicep femoris) skin (with mammary gland)
spinal cord (cervical) spleen
stomach testes
thymus thyroid/parathyroid
trachea urinary bladder
uterus
gross lesions (including a section of normal-appearing portion of same tissue)
tissue masses
entire head

- Preservatives: 10 % neutral buffered formalin used for all tissues.
- Stains: Haematoxylin and Eosin
- Tissues examined histopathologically: slides of all tissues listed were prepared and examined microscopically for control and high dose animals. In addition, the livers and kidneys from all dosed, control and recovery animals were examined microscopically. Livers from six randomly selected high dose animals were stained with Oil Red-O for lipids.
- As an addendum to the pathology report, microscopic examination of the thyroid glands from the 2000 and 10000 ppm dose groups, male and female, was performed.
Statistics:
If a standard error for one treated group is 0.0, or when N (number of animals) is less than or equal to two animals for any treated group, the variances of the two groups remaining were tested for equality using the F-test. If the N (number of animals) for the control group is less than or equal to two animals, no statistics are presented due to lack of variance.

Statistical evaluation of equality of means was made by the appropriate one way analysis of variance technique, followed by a multiple comparison procedure if needed. First, Bartlett's test was performed to determine if groups had equal variance. If the variances were equal, parametric procedures were used; if not, nonparametric procedures were used. The parametric procedures were the standard one way ANOVA using the F distribution to assess significance. If significant differences among the means were indicated, Dunnett's test was used to determine which means were significantly different from the control. If a nonparametric procedure for testing equality of means was needed, the Kruskal-Wallis test was used, and if differences were indicated a summed rank test (Dunn) was used to determine which treatments differed from control.

A statistical test for trend in the dose levels was also performed. In the parametric case (i.e. equal variance) standard regression techniques with a test for trend and lack of fit were used. In the nonparametric case Jonckheere's test for monotonic trend was used.
The test for equal variance (Bartlett's) was conducted at the 1 %, two-sided risk level. All other statistical tests were conducted at the 5 % and 1 %, two-sided risk level.

The variances of the two groups were tested for equality using the F test. If the variances were equal, a standard independent two sample t-test was used to determine equality of means. If the variances differed, Welch's t-test was used to determine equality of means. All tests were conducted at the 5 % and 1 %, two-sided risk level.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See below
Mortality:
mortality observed, treatment-related
Description (incidence):
See below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See below
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See below
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:
effects observed, treatment-related
Description (incidence and severity):
See below
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See below
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See below
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY
Four high dose males died during the course of this study. Two (Nos. 704 and 725) were found dead after 48 and 63 days on test. Neither animal showed any significant in-life observations which would account for their deaths. A further two animals (Nos. 719 and 727) showed decreased food consumption, red nasal discharge, and cool body temperature. One (No. 719) died spontaneously on test day 90, while the latter was euthanised on test day 63.
In addition, two females (No. 403 from the 2000 and No. 804 from the 50000 ppm dose groups) died following eye bleeding during Week 7. All remaining animals survived the duration of the study and were sacrificed either on test day 91 or on test day 120, following the one month recovery period.

CLINICAL OBSERVATIONS
Physical observations noted during the course of this study included excessive lacrimation, swollen salivary glands and alopecia. These observations occurred sporadically in both sexes of all groups, including the controls, in about 10 - 20 % of all animals. These findings are not considered to be related to the administration of the test substance.
One finding, however, did tend to occur more frequently in the treated animals compared to the controls. Red and/or mucoidal nasal discharge occurred in the high dose males at an incidence of approximately 10 - 20 %, compared to about 3 - 6 % in the control males. This observation occurred mainly during weeks 8 to 12 of the study and was not evident to any significant extent during the recovery period. The incidence of this finding in the 2000 and 10000 ppm dose group males was similar to the control group. Females were not significantly affected.

BODYWEIGHT
Mean bodyweights for both sexes in the 2000 and 10000 ppm dose groups were similar to the control values during the course of this study.
However, the high dose animals of both sexes exhibited a statistically significant reduction in mean bodyweights during the latter phase of the treatment period (p<0.01). At Week 13 there was about a 10 and 7 % reduction in group mean bodyweights (males and females respectively) as compared to their control groups. During the recovery period, these bodyweight effects were still evident in both sexes. However, it was only in the males that this was statistically significant; although this effect decreased as the recovery period progressed.

FOOD CONSUMPTION
Mean food consumption values for the treated groups of both sexes did not differ markedly from their respective control groups. During the latter phase of the treatment period, a slight trend toward increased food consumption occurred in both the treated males and females. This trend continued during the recovery period with consistently elevated values noted in both the high dose males and females, as compared to their respective controls. This trend was probably due to the high content of the test material in the treated diets with the resultant decrease in body weights in the animals, as noted above. This would tend to increase food consumption on a (g/kg/day) basis.

- Test Substance Intake
Since food consumption values were similar among the treated groups and controls for both sexes, test substance intake values for the treated groups were proportional to the nominal dietary concentrations of the test substance. As expected, during the first several weeks of the study the animals consumed more feed on a body weight basis and, therefore, test substance intake values were also higher, as compared to the latter weeks of the study.

HAEMATOLOGY
Pre-test values for both sexes were within normal limits for rats of this age and strain. At weeks 7 and 13 there was a trend toward slightly reduced haemoglobin levels in both sexes of the high dose group. However, these values were within the normal physiological range for this species and strain, as were all other haematology parameters evaluated.

CLINICAL CHEMISTRY
Pre-test clinical chemistry parameters were within the normal physiological range for rats of this age and strain. Relatively high alkaline phosphatase values were seen but these are expected in immature animals.
At week 7, there was a statistically significant increase in the SGPT level in the high dose males (p<0.01), while in the high dose females, alkaline phosphatase and cholesterol were increased (p<0.05).
At week 13 a trend toward increased BUN values was evident in the 10000 and 50000 ppm dose level males. Bilirubin values (both direct and total) were significantly lower in the high dose animals of both sexes at weeks 7 and 13.
Other statistically significant values were evident at both the 7 and 13 week intervals. However, these are considered to be biologically insignificant as the means of these values fall within the normal physiological range for the laboratory rat.
The effects on SGPT and BUN levels in the high dose males are suggestive of a toxic effect in the liver and kidney. The effects on cholesterol and alkaline phosphatase in the high dose females also suggest the liver as a target organ. The data indicate a minimal degree of damage in these organs in the high dose animals, as the relative changes in the above parameters are slight.

URINALYSIS
All urinalysis parameters measured were within normal limits at both the 7 and 13 week evaluations for all groups, both males and females.

ORGAN WEIGHTS AND ORGAN/BODY WEIGHT RATIOS
Among the males, a statistically significant decrease in the mean absolute brain weight occurred in the 50000 ppm dose group, while the spleen/body weight ratio was significantly increased (p<0.05). A trend toward increased absolute organ weight as well as organ/body weight ratios was evident for the kidneys, thyroid and liver in the treated males. These increases were statistically significant in the high dose males for the kidneys and liver and in all treatment groups for the thyroids (p<0.01).
Among the females, the brain and spleen organ/body weight ratios were increased in all treatment groups. These effects were statistically significant for the brain/body and spleen/body weight ratios in the 50000 ppm dose group. In addition, a trend toward increased ovary/body weight ratios was found for all the treatment groups. A trend toward increased absolute organ weight and organ/body weight ratios was evident in the females for the kidneys and liver in dose levels 10000 and 50000 ppm and for the thyroids in all treatment groups.
For the most part, the above increases in relative organ/body weight ratios are probably due to the reduced terminal body weights for the treated animals without a concomitant decrease in the absolute organ weight. However, for the livers and kidneys in both sexes, these parameters are fairly sensitive indicators of damage for both of these organs. Therefore, this data confirms the clinical chemistry data which indicated a slight toxic effect in the livers and kidneys in the high dose animals. The other organ weight changes noted above were not reflected in any unusual gross or microscopic changes.
At the end of the recovery period, the increase in the kidney/body weight ratio was still evident in the high dose males, while the increases in the liver and thyroid organ/body weight ratios were no longer present. Among the high dose females, all of the organ weight effects previously noted were no longer present.

PATHOLOGY
Gross postmortem findings in the animals of this study occurred sporadically in both sexes of the treated and/or control animals. They did not appear to be related to the administration of the test substance.

Microscopic evaluations of tissues from the terminal sacrifice animals revealed a significant incidence of hepatic cytoplasmic vacuolisation in the high dose group. Sections stained with Oil Red-O confirmed the presence of lipid in these vacuoles. The males were more severely affected than the females. After one month of recovery, this effect was still evident in the males, although to a lesser degree. Female rats were almost completely recovered. Minimal fatty change was evident in the 10000 ppm dose group, while the 2000 ppm dose group was unaffected.
Microscopic observation of the kidneys from high dose males indicated a minimal to moderate necrosis of tubular epithelial cells with protein accumulation in the lumen. After one month of recovery, 4 of 10 animals were still affected. In the high dose females, only 2 of 20 animals were affected. None of the female recovery animals showed evidence of renal damage. Animals from dose groups 2000 and 10000 ppm did not exhibit renal lesions.
Microscopic examination of the thyroid glands from all groups showed that there was no significant difference in the microscopic appearance of the thyroid glands between the treated and control groups of rats.
Other microscopic findings occurred sporadically in the treated and/or control groups and therefore, did not appear to be related to the administration of the test substance.

Effect levels

Dose descriptor:
NOEL
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 2000 ppm is equivalent to 158.15 mg/kg bodyweight/day for males and approximately 183.89 mg/kg bodyweight/day for females.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Key to Abbreviations and Symbols

SD = Standard Deviation                                                   N = Number of animals

SGPT = Serum Glutamic Pyruvic Transaminase                       ALKPHOS = Alkaline Phosphatase

BUN = Blood Urea Nitrogen                                              CHOL = Cholesterol

TBILI = Total Bilirubin                                                    DBILI = Direct Bilirubin

*Significantly different from the control at p≤0.05

**Significantly different from the control at p≤0.01

 

Table 2 Male Clinical Chemistry Summary Data

Time Period

Dose Level (ppm)

 

SGPT

(IU/L)

 

ALKPHOS

(IU/L)

 

BUN

(mg/dL)

 

CHOL

(mg/dL)

 

TBILI

(mg/dL)

 

DBILI

(mg/dL)

 

Pre-test

0

Mean

SD

N

25.0

4.0

9

386.0

96.0

9

17.7

1.2

9

75.5

13.1

7

0.08

0.03

9

0.06

0.02

3

 

 

 

 

Week 7

0

Mean

SD

N

17.0

1.0

1

123.0

29.0

10

16.5

2.1

10

57.0

8.0

10

0.04

0.03

10

0.03

0.03

10

2000

Mean

SD

N

17.0

2.0

10

124.0

28.0

10

15.7

2.0

10

43.0*

11.0

10

0.02

0.01

10

0.02

0.01

10

10000

Mean

SD

N

16.0

3.0

10

129.0

28.0

10

16.6

2.4

10

55.0

8.0

10

0.06

0.03

10

0.03

0.02

10

50000

Mean

SD

N

23.0**

12.0

10

118.0

29.0

10

17.9

2.5

10

59.0

16.0

10

0.03

0.01

10

0.01

0.03

10

 

 

 

 

Week 13

0

Mean

SD

N

19.0

2.0

10

81.0

19.0

10

15.9

1.4

10

57.0

10.0

10

0.08

0.03

9

0.05

0.03

9

2000

Mean

SD

N

19.0

5.0

10

82.0

15.0

10

15.4

1.5

10

49.0

18.0

10

0.06

0.03

10

0.03

0.02

10

10000

Mean

SD

N

16.0

4.0

10

87.0

17.0

10

17.4

1.3

10

63.0

11.0

10

0.08

0.03

9

0.09

0.04

9

50000

Mean

SD

N

29.0

13.0

10

82.0

18.0

10

18.1*

2.1

10

81.0

34.0

10

0.04**

0.01

10

0.04

0.01

10

 

Table 3 Female Clinical Chemistry Summary Data

Time Period

Dose level (ppm)

 

SGPT

(IU/L)

 

ALKPHOS

(IU/L)

 

BUN

(mg/dL)

 

CHOL

(mg/dL)

 

TBILI

(mg/dL)

 

DBILI

(mg/dL)

 

Pre-test

0

Mean

SD

N

17.0

2.0

9

319.0

88.0

9

17.1

3.2

9

69.3

22.1

6

0.11

0.03

9

0.02

0.01

4

 

 

 

 

Week 7

0

Mean

SD

N

23.0

4.0

9

54.0

13.0

9

18.8

2.6

9

68.0

16.0

9

0.06

0.10

8

0.05

0.03

8

2000

Mean

SD

N

22.0

4.0

9

66.0

11.0

9

20.3

5.9

9

58.0

13.0

9

0.03

0.01

9

0.02*

0.02

9

10000

Mean

SD

N

18.0

4.0

9

57.0

11.0

9

16.8

2.0

9

70.0

22.0

9

0.08

0.02

9

0.05

0.04

9

50000

Mean

SD

N

22.0

 8.0

9

78.0*

26.0

9

18.0

1.8

9

91.0*

18.0

9

0.05

0.04

9

0.02*

0.01

9

 

 

 

 

Week 13

0

Mean

SD

N

23.0

13.0

10

39.0

9.0

10

17.1

2.4

10

98.0

23.0

10

0.31

0.02

10

0.10

0.04

10

2000

Mean

SD

N

24.0

12.0

10

49.0

12.0

10

17.4

2.3

10

87.0

17.0

10

0.08*

0.02

10

0.06

0.01

10

10000

Mean

SD

N

21.0

6.0

10

37.0

9.0

10

16.6

1.6

10

77.0

17.0

10

0.08**

0.01

10

0.08

0.03

10

50000

Mean

SD

N

18.0

9.0

10

47.0

13.0

10

18.4

2.2

10

100.0

27.0

10

0.04**

0.02

10

0.04**

0.01

10

 

Table 4 Male Summary Data for Organ Weights and Organ/Bodyweight Ratios

Dose level (ppm)

 

Terminal Bodyweight

(g)

Right Kidney

Left Kidney

Liver

Thyroid

Weight (g)

Org/TBW

(x 1000)

Weight (g)

Org/TBW

(x 1000)

Weight (g)

Org/TBW

(x 100)

Weight (g)

Org/TBW

(x 100000)

0

Mean

SD

N

427.0

33.0

20

1.370

0.157

20

3.21

0.24

20

1.428

0.305

20

3.36

0.73

20

10.879

1.253

20

2.55

0.16

20

0.0129

0.0042

18

3.00

0.92

18

2000

Mean

SD

N

427.0

30.0

20

1.436

0.133

20

3.36

0.22

20

1.414

0.150

20

3.31

0.24

20

11.669

1.751

20

2.72

0.27

20

0.0202**

0.0028

20

4.75**

0.73

20

10000

Mean

SD

N

422.0

30.0

20

1.462

0.143

20

3.47*

0.35

20

1.425

0.129

20

3.39

0.33

20

11.869

1.155

20

2.81**

0.11

20

0.0238**

0.0030

20

5.67**

0.87

20

50000

Mean

SD

N

399.0*

33.0

16

1.571**

0.155

16

3.95**

0.33

16

1.516

0.164

16

3.81**

0.30

16

16.453**

2.098

16

4.12**

0.35

16

0.0176**

0.0056

15

4.41**

1.32

15

 

Table 5 Female Summary Data for Organ Weights and Organ/Bodyweight Ratios

Dose level (ppm)

 

Terminal Bodyweight

(g)

Right Kidney

Left Kidney

Liver

Thyroid

Weight (g)

Org/TBW

(x 1000)

Weight (g)

Org/TBW

(x 1000)

Weight (g)

Org/TBW

(x 100)

Weight (g)

Org/TBW

(x 100000)

0

Mean

SD

N

243.0

16.0

20

0.836

0.077

20

3.45

0.31

20

0.820

0.076

20

3.38

0.26

20

6.557

0.559

20

2.70

0.17

20

0.0108

0.0035

18

4.51

1.51

18

2000

Mean

SD

N

233.0

20.0

19

0.839

0.070

19

3.61

0.24

19

0.827

0.055

19

3.56

0.20

19

6.492

0.604

19

2.79

0.18

19

0.0139*

0.0021

18

5.98**

0.78

18

10000

Mean

SD

N

236.0

19.0

20

0.854

0.089

20

3.62

0.33

20

0.830

0.077

20

3.53

0.34

20

7.247**

0.654

20

3.07**

0.21

20

0.0180**

0.0032

18

7.71**

1.55

18

50000

Mean

SD

N

223.0*

19.0

19

0.876

0.097

19

3.92**

0.24

19

0.860

0.103

19

3.85**

0.29

19

9.111**

0.955

19

4.08**

0.33

19

0.0145**

0.0033

18

6.52**

1.46

18

Applicant's summary and conclusion

Conclusions:
The data indicate toxic effects in the liver and kidneys due to the administration of the test substance in the 50000 ppm dose group males and females.
These effects appear to be reversible based on microscopic evaluations of these organs in the recovery animals. The evidence is equivocal with regard to toxicity in the 10000 ppm dose group, while the 2000 ppm dose appears to be the NOEL. This nominal dose level is equivalent to an actual ingested dose level of 158.15 mg/kg bw/day in males and 183.89 mg/kg bw/day in females over the 13 week exposure period. Since the repeated dose toxicity of the test substance is >100 mg/kg day/bw classification is not required.
Executive summary:

A 90-day study was conducted to assess the toxicity of the test material broadly in accordance with OECD guideline 408.

 

The test material was administered via dietary admixture, 7 days per week to CD (Sprague-Dawley derived) rats for three months. Dose levels were 0, 2000, 10000 and 50000 ppm. Rats were randomly divided into four groups of 30 rats/sex/group in the 0 and 50000 ppm dose groups and 20 rats/sex/group in the 2000 and 10000 ppm dose groups.

All animals were subject to necropsy at the termination of the study. This was at 90 days for 20 males and 91 days for 20 females from all groups; the remaining 10 animals were subjected to a one month recovery period and necropsied when this was completed.

 

- Two females and four males died during the course of the study. A significant physical observation noted was red/mucoidal nasal discharge. This occurred more frequently in the high dose males during the latter phase of the treatment period. During the recovery phase, this observation was no longer evident.

- A significant bodyweight decrease was observed during the latter portion of the study in the high dose animals of both sexes. This decrease persisted throughout the recovery period.

- Clinical chemistry effects were also noted. At Week 7, serum glutamic pyruvic transaminase was elevated in the high dose males, while alkaline phosphatase and cholesterol were elevated in the high dose females. At Week 13, blood urea nitrogen values were increased in the 10000 and 50000 ppm dose level males. Direct and total bilirubin values were reduced in the high dose animals at both 7 and 13 weeks.

- At the 13 week necropsy, a trend toward increased absolute and relative (to body weight) organ weights was evident for the kidneys and liver for the males in the 10000 and 50000 ppm dose groups and for the thyroids in all treatment groups. Among the females, a trend toward increased absolute and relative organ weights were found for the liver, thyroids and kidneys in all treatment groups.

- Microscopic evaluation of selected tissues indicated slight cellular necrosis in the proximal tubules of the kidney and lipid deposition in livers of the high dose animals only. The males were more sensitive to these effects than the females. These findings were still evident in the recovery animals, although at a lesser incidence and degree.

 

The data indicate toxic effects in the liver and kidneys due to the administration of the test substance in the 50000 ppm dose group males and females.

These effects appear to be reversible based on microscopic evaluations of these organs in the recovery animals. The evidence is equivocal with regard to toxicity in the 10000 ppm dose group, while the 2000 ppm dose appears to be the NOEL. This nominal dose level is equivalent to an actual ingested dose level of 158.15 mg/kg bw/day in males and 183.89 mg/kg bw/day in females over the 13 week exposure period. The repeated dose toxicity of the test substance is therefore above the limits of classification.