Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study, not conducted according to GLP.

Data source

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

Materials and methods

Principles of method if other than guideline:
This is a two year oral repeated dose study
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Fyrol FR-2
- Physical state: Clear, viscous fluid
- Purity: 95%
- Storage condition of test material: Room Temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 53 days
- Weight at study initiation: Males 237g (mean) and ranging from 182 - 271g. Females 154g (mean) and ranging from 126 - 186g.
- Housing: Individually in elevated stainless steel cages
- Diet (e.g. ad libitum): Standard laboratory diet (Purina Lab Chow) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 21 days (23 March to 13 April 1978)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2
- Humidity (%): 55 +/- 15
- Air changes (per hr): NK
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle


IN-LIFE DATES: From: April 1978 To: April 1980

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Appropriate amounts of test substance (adjusted by most recent weekly body weight and food consumption data) and standard laboratory diet were mixed weekly.
Samples of diets prepared at each dose level were assayed for homogenity and stability of Fyrol FR-2 in feed prior to the initiation of the study. Samples of each diet prepared during the study were saved and samples selected at intervals were assayed for content of Fyrol FR-2. Results not provided, but presented in report no. 78005 (Biodynamics dept. of metabolism and analytical chemistry).

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Standard laboratory diet
- Storage temperature of food: NK
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Pubication not available, report no. 78005.
Duration of treatment / exposure:
24 Months
Frequency of treatment:
Continuously in the diet through the day prior to necropsy.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
5 mg/kg bw/day
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
20 mg/kg bw/day
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
80 mg/kg bw/day
Basis:
nominal in diet
No. of animals per sex per dose:
60 Males and 60 Females per dose group
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Doses selected on the basis of results from an 8 week pilot study (Biodynamics report no. 77-1898). Diets were adjusted after each body weight and food consumption measurement to achieve indicated doses.
- Rationale for animal assignment (if not random): More animals than required for the study were purchased and equilibrated. Animals considered unsuitable for the study on the basis of pretest physical and ophthalmoscopic examinations were eliminated prior to random selection for group assignment.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily animals were checked for mortality and gross signs of toxicologic or pharmacologic effects.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Twice pretest, weekly through 13 weeks, biweekly 14 through 26 weeks, monthly thereafter and terminally (after fasting).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was measured pretest, weekly through 14 weeks, biweekly 14 through 26 weeks and monthly thereafter.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Intake calculated from food consumption data, based on nominal concentrations.

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Examinations on all animals conducted at pretest, month 6, month 12, month 18 and month 24.
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was obtained via venipuncture of the orbital sinus under light anesthesia at month 3, 6 and 18. Blood was obtained from the abdominal aorta at necropsy intervals (months 12 and 24).
- Anaesthetic used for blood collection: Yes (identity) / No / No data
- Animals fasted: Yes
- How many animals: 10/sex at pretest, 10/sex/group at months 3, 6, 12 and 18 and 20/sex/group at month 24.
- Parameters examined: Hemoglobin, hematocrit, erythrocytes, reticulocytes, prothrombin time, partial thromboplastin time, total and differential leukocytes.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was obtained via venipuncture of the orbital sinus under light anesthesia at month 3, 6 and 18. Blood was obtained from the abdominal aorta at necropsy intervals (months 12 and 24).
- Animals fasted: Yes
- How many animals: 10/sex at pretest, 10/sex/group at months 3, 6, 12 and 18 and 20/sex/group at month 24.
- Parameters examined: Serum glutamic pyruvic transaminase, alkaline phosphataase, blood urea nitrogen, fasting glucose, total protein, albumin, globulin and A/G ratio.

URINALYSIS: Yes
- Time schedule for collection of urine: Pretest, months 3, 6, 12, 18 and 24.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters examined: Gross appearance, specific gravity, pH, protein, glucose, ketones, bilirubin, occult blood and microscopic analysis.

NEUROBEHAVIOURAL EXAMINATION: No

POSTMORTEM: Yes
- Time schedule: Complete gross postmortem examination conducted if animals died spontaneously or were killed in a moribund condition, at month 12 and month 24.
- How many animals: 10/sex/group at month 12 and all survivors at month 24.
- Organs weight and organ/body weight ratios collected: Brain, pituitary, adrenals, testes, heart, thyroid, spleen, kidneys and liver.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Body weight, food consumption, hematology and clinical chemistry parameters, organ weights and organ/body weight ratios were analyzed. Mean values of all dose groups were compared to control at each time interval. Statistically significant differences from control are indicated in appendices.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
effects observed, treatment-related
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY
Mortality rates in all groups were low during the first 12 months of the study with no remarkable difference in incidence between control groups and groups receiving TDCP. Mortality remained low in most groups from 12 through 17 months; however a slight increase in the number of deaths in the high dose males over that in control males was apparent during this interval. After month 17, the mortality rate increased in all groups and remained high until the end of the study (this can be expected in ageing animals). Total mortality in low- and mid-dose males and in all TDCP-treated females was considered comparable to that of the controls. Significantly greater mortality (p<0.05) was recorded for high dose males, (38/60 animals died in highest treatment group compared to 26/60 in controls).

BODY WEIGHT AND WEIGHT GAIN
There was a clear adverse effect on body weight in the 80 mg/kg/day groups, throughout the study, with body weights at termination >20 % lower than control. Slight decreases (most differences did not exceed 5 %) in male body weights in the 20 mg/kg/day at some intervals of the study may also have related to treatment. Food consumption for controls and high dose animals was generally comparable except for slight increases in values for the high dose groups during the last few months of the study.

OPHTHALMOCSOPIC
At six months one high-dose female exhibited thrombosis of the orbital blood vessels in the right eye with consequent arteriolar attenuation and venous congestion. This is an unusual lesion and its relationship to the Fyrol FR-2 administration is unclear. However, in the opinion of the ophthalmologist, the presence of this abnormality in a single animal is probably not related to the test material administration.
Examinations at 18 and 24 months revealed sacculations along the course of the retinal arterioles in one mid-dose male, four high-dose males and four high-dose females, primarily at 24 months. These changes may not be morphologic ones; similar sacculations have been observed by the ophthalmologist to disappear when the vessels are congested for any reason. This type of lesion is observed occasionally in old untreated rats. However, in this study, there appears to have been an acceleration of this abnormal arteriolar process in these treated animals.

HAEMATOLOGY
Hemoglobin, hematocrit and total erythrocyte values for high-dose statistically significant degree. Differences in males were more pronounced than those in females. Values for low- and mid-dose animals were generally comparable to or slightly lower then control values with only occasional statistically significant differences. In general, no concomitant increase in numbers of reticulocytes occured. Reticulocyte values for Fyrol FR-2 treated groups were generally similar to or lower than control values with no dose-related differences.
Total and differential leukocyte values and erythrocyte morphology evaluations revealed no differences among groups which were considered related to the administration of Fyrol FR-2.
Prothrombin times (PT) and partial thromboplatsin times (PTT) exhibited considerable variability from interval to interval; no consistent dose-related pattern of differences between values for control and treated groups was apparent over the course of the study. At month 24, statistically significant increases were noted for PT and PTT values of high-dose males and for PTT values for all treated groups of females, when compared to concurrent control values. Differences were slight, however, and no dose-relationship occured in values for the females.

CLINICAL CHEMISTRY
Serum AKP values for high-dose animals were lower than control values at most intervals throughout the study, frequently to a statistically significant degree. Values for low- and mid-dose groups were generally comparable to control values; at month 24 however, values for mid-dose males and females were statistically significantly lower than control values. The biological significance if any, of a reduction in AKp is unclear; this observation does not appear to represent a toxic effect of the test chemical.
BUN values were generally comparable among groups with no statistically significant differences. A few individual animals in the mid- and high-dose groups exhibited marked elevations in BUN values at 18 or 24 months; this was consistent with microscopic evidence of renal pathology in these animals.
Evaluation of serum glutamic pyruvic transaminase, fasting glucose, total protein, albumin and globulin values revealed no consistent dose-related differences between control and treated animals which were considered related to Fyrol FR-2 administration.
Plasma cholinesterase activity, measured at 18 and 24 months, was lower in high-dose females than in control females at both intervals; the difference at 18 months was statistically significant. In males, plasma cholinesterase activity in low- and high-dose groups was significantly lower than control activity at 18 months, but activity for the mid-dose was higher than the control value. At 24 months, plasma cholinesterase values for all groups of males was considered comparable. Erythrocyte cholinesterase activity measured at 18 and 24 months was similar among groups woth no dose- or test material-related differences.

URINALYSIS
Urinalysis data were generally comparable among groups.

ORGAN WEIGHTS
Liver weights and liver/body weight ratios of high-dose males and females were higher than concurrent control values, usually to a statistically significant degree, at both 12 and 24 months. Statistically signifcant increases also occured at the mid-dose level on some occasions. Mean liver weights and liver/body weight ratios for low-dose animals were comparable to or slightly higher than control weights, with no statistically significant differences. Comparsion of organ weight of Fyrol FR-2-treated animals to those control animals revealed dose-related increases in kidney weights and kidney/body weight ratios at the mid- and high-dose levels (both sexes) at 12 and 24 months. Differences at the high dose at both intervals and at the mid dose at month 24 were statistically significant at p<=0.05. Mean kidney weights and kidney/body ratios for low-dose animals were comparable or slightly higher than control weights, with no statistically significant differences.
Thyroid weights and thyrodid/body weight ratios of high-dose males and females at 12 and 24 months were higher than concurrent controls, usually to a statistically significant degree. Thyroid/body weight ratios for mid-dose animals were higher than control values, while low-dose thyroid weights were generally comparable to that of control animals.
In most cases these increases in kidney, liver and thyroid weights occured in the presence of significantly decreased body weights (high-dose males - months 12 and 24, mid-dose males and high-dose females - month 24).
Statistically significant increases in heart/body weight ratios occured in high-dose males and females at both 12 and 24 months and in the mid-dose males at 24 months. Absolute heart weights were generally comparable to those of controls even though body weights were, in most cases, significantly below control weights.
A few other statistically significant differences between organ weights of control and treated animals occured sporadically or appeared to reflect low terminal body weights. Because of morphologic abnormalities in the testes of several animals at 24 months, testis weights were obtained for a limited number of animals at this interval.

GROSS PATHOLOGY
Some abnormalities of the liver, kidneys, testes and seminal vesicles were seen at a higher incidence in Fyrol FR-2-treated groups than in the control groups. Unusual observations in the liver which occured more frequently in treated than in control groups included masses, nodules, ,and raised areas (high-dose group) and various discolourations (all treated groups). In the kidneys, enlargement was seen more frequently in mid-dose males and high-dose males and females than in control animals and there was a higher incidence of discolourations, surface irregularities (pitting), masses, nodules and cysts in Fyrol FR-2-treated groups than in the control group. Mid- and high-dose males exhibited a higher incidence of small seminal vesicles and testicular enlargement, massesm nodules, flaccidity and discolourations than did control males.
Other abmormalities seen grossly ocured with similar frequency in control and treated animals or were noted sporadically. There were no differences in type or distribution of these findings which were considered to represent an effect of Fyrol FR-2 administration.

HISTOPATHOLOGY NON-NEOPLASTIC
An increased incidence of parathyroid hyperplasia was also observed in high-dose animals. This finding may be associated with renal changes as discussed below.
A number of non-neoplastic abnormalities of the male reproductive system also occured more frequently in treated than in control animals. These included oligospermia and accumulation of degenerated seminal products in the epididymal tubular lumens in high-dose animals; sperm stasis and germinal epithelial atrophy with associated oligospermia in the testes of mid- and high-dose animals; and accumulation of eosinophilic material in the lumens of the seminiferous tubules, periarteritis nodosa in the testes, and atrophy and decreased secretory product in the seminal vesicles in animals in all treated groups.
Additional non-neoplastic alterations which occured more frequently in high-dose males and females than in control animals were erythroid/myeloid hyperplasia of the rib marrow and erythroid/myeloid metaplasia of the spleen.
HISTOPATHOLOGY: NEOPLASTIC (if applicable)
In the kidney, the incidence of renal cortical adenomas at 24 months in males was 1/45 (2 %), 3/49 (6 %), 9/48 (19 %) and 32/46 (70 %) in control, low, mid and high dose animals respectively (reaching statistical significance from the mid dose group, 20 mg/kg/day). At 24 months in females, the corresponding percentages were 0 %, 2 %, 17 % and 58 %, respectively, with statistical significance again from 20 mg/kg/day. There was no reported incidence at 12 months. In addition to the tumours, there was an increase in the incidence of hyperplasia of the convoluted tubule epithelium at 24 months in females at the high dose and in males in all treatment groups when compared to control animals.

Histological abnormalities were identified at a higher incidence in the livers of treated rats, as described in the repeated dose toxicity section, 4.1.2.6. This was associated with evidence of neoplastic alterations. In the livers of male animals at 24 months, the incidence of hepatocellular adenomas was 2/45 (4 %), 7/48 (14.5 %), 1/48 (2 %) and 13/46 (28 %) in control, low-, mid- and high-dose animals respectively, with statistical significance reached at the highest dose. In females, the corresponding percentages were 2 %, 2 %, 9 % and 16 %, respectively, with statistical significance again at the highest dose. At the 12 month interim sacrifice, the incidence of hepatocellular adenomas was 3/14 and 1/10 for males and females respectively at the highest dose only compared to none in control animals.
At 24 months, the incidence of hepatocellular carcinoma was also increased in males and females, with the incidence in males being 1/45 (2 %), 2/48 (4 %), 3/48 (6 %) and 7/46 (15 %) in control, low, mid- and high dose animals respectively, although this did not reach statistical significance. The corresponding values in females were 0/49, 2/47 (4 %), 2/46 (4 %) and 4/50 (8 %). There was no reported incidence at 12 months.

The incidence of interstitial cell tumours of the testes (benign tumours) at 24 months was 7/43 (16 %), 8/48 (17 %), 23/47 (49 %) and 36/45 (80 %) in the control, low-dose, mid-dose and high-dose animals, respectively. The effects were statistically significant in the mid-dose and high dose groups. At 12 months, 3/13 mid dose animals and 3/11 high dose animals were observed to have interstitial cell tumours; no tumours were observed in control animals at 12 months.

There was also an increased incidence of adrenal cortical adenomas in high dose females. The incidence of this finding at 24 months was 8/48 (17 %) in control females and 19/49 (39 %) in high-dose females; the difference being statistically significant. At 12 months the incidence was in females was 5/11 (45 %) and 1/10 (10 %) for control and high dose groups, respectively.

Effect levels

Dose descriptor:
LOAEL
Effect level:
5 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Histopathological observations in the male reproductive organs in Sprague Dawley Rats fed TDCP in a 2 year Bioassay

Tissue/

mg/kg/day b.w.

Males

0

5

20

80

Testes:

Seminiferous tubules – germinal epithelial atrophy with associated Oligospermia

12 months

24 months

5/14

30/43

2/12

29/48

3/13

42/47

7/11

44/45

Tubular lumens – Amorphous Eosinophilic material:

24 months only

2/43

4/48

12/47

11/45

Sperm stasis:

24 months only

5/43

5/48

11/47

14/45

Periarteritis nodosa:

24 months only

5/43

10/48

19/47

16/45

Epididymes:

Oligospermia:

12 months

24 months

0/14

11/41

#

9/32

#

7/13

1/11

35/44

Degenerated seminal product:

24 months only

 8/41

7/32

3/13

 22/44

Seminal vesicle:

Decreased secretory product:

12 months

24 months

0/15

1/41

#

11/13

#

17/19

1/10

22/42

Atrophy:

24 months only

0/41

4/13

6/19

10/42

# Animals not evaluated at 12 months.

Applicant's summary and conclusion

Conclusions:
The LOAEL obtained for this study is 5mg/kg bw/day under the given experimental conditions.
Executive summary:

In a 2-year carcinogenicity study in which groups of 60 male and 60 female rats were fed diets containing TDCP to achieve dose levels of 0, 5, 20 and 80 mg/kg bw/day for 24 months, significantly greater mortality was recorded for high dose males. There was a clear adverse effect on body weight in the 80 mg/kg/day groups throughout the study, with body weights at termination >20 % lower than controls. A significant reduction in red blood cell parameters was noted for high-dose animals. Absolute and relative kidney, liver and thyroid weights were also increased in mid- and high-dose animals.

A LOAEL of 5 mg/kg bw/day (based on the hyperplasia, considered a pre-neoplastic lesion, observed in the kidneys in all treated groups and the testicular effects observed at this dose) can be derived from this study.