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Administrative data

Description of key information

Oral: NOAEL: Chronic study, 30 ppm (1.14 mg/kg bw/day), OECD 453, Gerspach 1998
Dermal: NOAEL: 1000 mg/kg bw/day, OECD 410, Gerspach 1996

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 January 1996 to 17 February 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed to GLP and in line with the standardised guidelines OECD 453, EPA OPP 83-5, EU Method B. 33 and Japanese MAFF with no deviations thought to impact the reliability of the presented results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)
Deviations:
yes
Qualifier:
according to guideline
Guideline:
other: EPA OPP 83-5 (Combined Chronic Toxicity / Carcinogenicity)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Guidance on Toxicology Study data for Application of Agricultural Chemical Registration, 28 January 1985.
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Tif: RAIf (Spraque-Dawley derived)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approximately 5 weeks old.
- Weight at study initiation: Males 191.5 to 294.1 g; Females 153.7 to 223.1 g.
- Housing: Housed in groups of 5 by sex in 1800 cm² cages with soft wood bedding.
- Diet: Pelleted, certified standard diet, provided ad libitum. All batches were assayed for composition and contaminant levels by the manufacture.
- Water: Tap water, provided ad libitum. The results of the routine chemical examination of water showed that contaminant levels were within the acceptable limits and were not considered to have affected the conduct of the study or the interpretation of the data.
- Acclimation period: 18 days for males and 17 for females.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 ºC
- Humidity (%): 55 ± 10%
- Air changes (per hr): 16 to 20 air charges/ hour
- Photoperiod (hrs dark / hrs light): 12 hours light per day.

IN-LIFE DATES: From: 18 January 1996 To: 17 February 1998.
Route of administration:
oral: feed
Vehicle:
water
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet: Diets were prepared at about monthly intervals.
- Mixing appropriate amounts with: The test material was weighed on a calibrated balance. The pulverized food was then homogenously mixed with the appropriate concentrations of the test material and about 25% water was added before pelleting to ensure the necessary pellet quality. The pellets were subsequently air dried. The test material was used without adjustment for purity.
- Storage temperature of food: Food was stored in stainless steel containers at room temperature in a separate area.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Pellet samples (about 200 g each) were collected during the pelleting process (for homogeneity analyses at beginning, middle, and end of pelleting process) and dispatched deep frozen for further analysis.
Duration of treatment / exposure:
24 months
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 10, 30, 100 and 300 mg/kg food
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 9.92, 29.6, 99.0 and 296.0 mg/kg food
Basis:
other: analytical conc.
Remarks:
Doses / Concentrations:
0.39, 1.14, 3.76 and 11.4 mg/kg bw/day (males)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
0.44, 1.30, 4.43 and 13.0 mg/kg bw/day (females)
Basis:
actual ingested
No. of animals per sex per dose:
Eighty per sex per dose, divided into the following groups:
> Group 1 consisted of fifty rats per sex per dose for carcinogenicity observations.
> Group 2 consisted of ten rats per sex per dose, used as a satellite group for haematology observations.
> Group 3 consisted of ten rats per sex per dose, used for blood chemistry and urine parameters.
> Group 4 consisted of ten rats per sex per dose, used for interim sacrifice at month 12.
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels were based on the results of the following previously conducted study:
Conditions: 3 month dietary toxicity study in rats, conducted at 20, 100, 300, 1000 and 4000 mg/kg food in 10 males and 10 females per dose group. The calculated average of daily doses were 1.21, 6.12, 18.8, 62.3, and 243 mg/kg bw in males, and 1.42, 7.07, 20.6, 69.3 and 282mg/kg bw in females, respectively.
Results: Body weight gains were decreased by 41% (males) and 20% (females) at 4000 mg/kg and 17% (males) and 12% (females) at 1000 mg/kg. In addition, red blood cell parameters were affected at these upper dose levels. The liver was a target organ as indicated by the incidence of changes in males and females at feeding levels of 1000 and 4000 mg/kg. The liver lesions were characterized by minimal or moderate necrosis of single hepatocytes in animals at 1000 and 4000 mg/kg, areas of modest or marked necrosis in three males at 4000 mg/kg which died during the study; minimal to marked inflammatory cell infiltration in males at 1000 mg/kg and in males and females at 4000 mg/kg, minimal to moderate pigmentation (most likely hemosiderin) in the Kupffer cell of the liver in males and females at 4000 pmg/kg and in the hepatocytes of three males which died; and minimal to marked cytoplasmic vacuolization in females at 1000 mg/kg and in males and females at 4000 mg/kg. Additionally, in males at 4000 mg/kg, minimal to moderate hypertrophy of hepatocytes and minimal to moderate hypertrophy of hepatocytes and minimal to moderate extramedullary hematopoiesis were observed.
Thus, body weight gain data, laboratory investigations, and histopathology in the liver showed that the maximum tolerated dose (MTD) criteria were clearly exceeded at 4000 and 1000 mg/kg.
- Rationale for selecting satellite groups: Individuals assigned to satellite groups were performed at random.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily, twice on weekdays and once at weekends.
- Cage side observations included: Mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly for the first 3 months and then weekly thereafter.
- Bodyweight gain: Determined as the difference in body weight between that and the previous measurement.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Weekly for the first 3 months and then monthly thereafter.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Beginning of treatment and then at 6, 12, 18 and 24 months.
- Dose groups that were examined: Both males and females from group 1 of the 300 mg/kg dose groups and the control were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected from the orbital plexus on weeks 13, 27, 53, 78 and 105.
- Anaesthetic used for blood collection: Yes, light ether anaesthesia.
- Animals fasted: Yes, overnight before sampling.
- How many animals: Twenty animals per sex per dose.
- Parameters checked in Table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected from the orbital plexus on weeks 13, 27, 53, 78 and 105.
- Animals fasted: Yes, overnight before sampling.
- How many animals: Ten animals per sex per dose.
- Parameters checked in Table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected overnight weeks 13, 27, 53, 78 and 105.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, both food and water were withheld overnight before sampling.
- How many animals: Ten animals per sex per dose.
- Parameters checked in Table 3 were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, see Table 4.
HISTOPATHOLOGY: Yes, see Table 5.
Statistics:
For each time point and parameter an univariate statistical analysis was performed. Nonparametric methods (Lehmann, 1975) were applied, to allow for non normal as well as normal data distribution.
Each treated group was compared to the control group either by Lepage's (Lepage, 1971) or Wilcoxon's two-sample test and tested for increasing or decreasing trends from control up to the respective dose group by Jonckheere's test for ordered alternatives (Jonckheere, 1954). The Lepage test is a combination of Wilcoxon and Ansari-Bradley statistics, i.e. a combined test for location and dispersion. The Lepage test has a good power against the more general alternative that the distributions differ not only in location but also in dispersion. The Jonckheere test is sensitive to dose related monotonic trends.
Survival analysis was performed by the regression model (partial likelihood) introduced by Cox (Cox, 1972) in order to compare survival time of treated animals (experimental group 1) with control animals.
All microscopic findings of animals of the carcinogenicity subgroup were subjected to a statistical analysis performed by a computer program.. Neoplastic lesions were analyzed by Peto's mortality-prevalence test (Peto et al., 1980), non-neoplastic lesions by Cochran-Armitage's linear trend test (Armitage, 1955). One-sided tests for positive trend using the group numbers (1,2,...,5) as scoring coefficents were performed. In all cases where an effect was found (p<=0.05), further analyses were carried out in order to determine the highest non-significant dose group, by deleting the highest dose group and re-performing the above analysis until a non-significant result was obtained. This is a so-called "closed testing procedure" which guarantees the observance of the nominal significance level of 5%. The p-values were computed using standard normal approximations.
Statistical significance, however, does not necessarily imply biological relevance.
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment related effects.
Mortality:
no mortality observed
Description (incidence):
No treatment related effects.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No treatment related effects.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment related effects.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No treatment related effects.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment related effects.
Haematological findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant effects.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment related effects.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment related effects.
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No toxicologically relevant effects.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related effects.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver effects in both males and females at 300 mg/kg.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No toxicologically relevant effects.
Details on results:
CLINICAL SIGNS AND MORTALITY
The incidence and distribution of clinical signs recorded during the study gave no indication of a treatment-related effect on the appearance and behaviour of the animals.
Considering animals of experimental group 1 (carcinogenicity group) only there were no adverse effects on survival as a results of treatment. The number and percentage of animals surviving to terminal necropsy are presented in Table 6.

BODY WEIGHT AND WEIGHT GAIN
Throughout the study, the body weight development of the treated animals was comparable to that of the control group. Cumulative mean body weight gain can be seen in Table 7. Statistical significances that were obtained for female groups during the initial study period, were considered to reflect the range of biological variability.

FOOD CONSUMPTION AND COMPOUND INTAKE
The mean food consumption of the treated animals was comparable to that of the control group. Cumulative food consumptions (CFC) and time weighted average (TWA) can be seen in Table 8.
No experimental relevance was given to the few isolated statistical significances which were considered to reflect the values' range of normal biological variability.
Daily male compound intake was calculated to be 0.39, 1.14, 3.76 and 11.4 mg/kg bw/day, for the nominal treatment groups 10, 30, 100 and 300 mg/kg.
Daily female compound intake was calculated to be 0.44, 1.30, 4.43 and 13.0 mg/kg bw/day, for the nominal treatment groups 10, 30, 100 and 300 mg/kg.

WATER CONSUMPTION
No experimental relevance was given to the few isolated statistical significances which were considered to reflect the values' range of normal biological variability.

OPHTHALMOSCOPIC EXAMINATION
The examinations of the eyes (lids and surrounds, conjunctiva, pupillary reflex, cornea, sclera, anterior chamber, lens, vitreous, and fundus) at pre-test and months 6, 12, 18, and 24 did not indicate an effect of the test material on the eyes.

The incidences of the findings in cornea, lens, vitreous, or fundus were not dose dependently distributed and occurred in comparable numbers in the control group. The corneal findings were overwhelmingly considered to reflect artifacts due to the mydriasis procedure. Occurrence of turbid lens is considered an age-dependent process. The fundus findings at 18 and 24 months were consequent to the examiners impaired sight onto the fundus due to turbid lens of the animal.

HAEMATOLOGY
No treatment-related changes were identified.
Myeloid leukemia was observed in one male (no. 292) of the 100 mg/kg group and contributed to the high mean values for white blood cell count, neutrophil count and large unstained cell count in this group at week 53. No toxicological relevance is attached to this finding which occasionally occurs spontaneously in this colony of rats.
A number of differences between the means achieved a level of statistical significance or showed a significant positive or negative trend. However, none of these differences was considered to be related to treatment as they were not consistent across time, did not form a dose relationship and/or were of insufficient magnitude to be of toxicological relevance.
In males, these differences included lower mean values for erythrocyte counts at weeks 13, 27, 53 and 78 for the 30 mg/kg dose group and at weeks 27 and 53 for 100 mg/kg; a lower hematocrit value at week 53 for 100 mg/kg; higher mean cell volume and mean corpuscular hemoglobin at week 53 for 30 mg/kg; a higher red cell volume distribution width for 30 mg/kg at week 78; a higher mean corpuscular hemoglobin concentration at week 27 for 300 mg/kg; higher hemoglobin concentration distribution width at weeks 13 and 53 for 30 mg/kg; higher reticulocyte counts for 30 mg/kg at weeks 53 and 78 and for 100 mg/kg at weeks 53 and 105; higher absolute and relative neutrophil counts for 100 mg/kg at week 53; lower absolute basophil count at week 13 for 300 mg/kg, and a higher platelet count for 100 mg/kg at week 78. Furthermore, there were a few changes in relative values of differential white blood cell counts (basophils, lymphocytes, monocytes, large unstained cells) without effects on the absolute values, which therefore, were considered to be without toxicological relevance.
In females, these differences included lower mean values for erythrocytes, hemoglobin and hematocrit at week 105 for 100 and 300 mg/kg; higher hemoglobin concentration and hematocrit values at week 13 for 10 mg/kg; a lower hemoglobin concentration distribution width for 10 mg/kg at week 78; higher absolute and relative neutrophil counts for 100 mg/kg and higher dispersion of neutrophil counts (individual values) for 300 mg/kg at week 27, and a lower mean prothrombin activity at week 27 for 100 mg/kg. Furthermore, there were a few changes in relative values of differential white blood cell counts (neutrophils, lymphocytes, monocytes) without effects on the absolute values, which therefore, were considered to be without toxicological relevance.

CLINICAL CHEMISTRY
No treatment-related changes to blood chemistry parameters were apparent.
A small number of differences between the means achieved a level of statistical significance or showed significant positive or negative trends. However, these differences were neither related to the dose administered, nor to the duration of treatment, and/or the change was of insufficient magnitude to be toxicologically relevant.
In males, these differences included a lower dispersion of glucose values at week 13 for 10 mg/kg, a lower mean value for glucose at week 53 for 100 mg/kg; a higher bilirubin value at week 13 for 30 mg/kg; a lower albumin value at week 27 for 100 mg/kg and a higher albumin value at week 13 for 300 mg/kg; a higher cholesterol value at week 13 and a higher dispersion of cholesterol values at week 105 for 30 mg/kg; lower sodium values at week 13 for 30 and 100 mg/kg and at week 27 for 30, 100 and 300 mg/kg; a higher potassium value at week 53 for 30 mg/kg; lower chloride values at week 27 for 30, 100 and 300 mg/kg, and at week 78 for 300 mg/kg higher phosphate values at weeks 53 and 105 for 300 mg/kg, and a higher mean value for alkaline phosphatase at week 78 for 300 mg/kg.
In females, these differences included lower values for glucose at week 13 for 30, 100 and 300 mg/kg and at week 53 for 10 and 30 mg/kg; a higher globulin value for 30 mg/kg at week 78; a lower albumin to globulin ratio at week 13 for 30 mg/kg; a lower value for cholesterol and a higher value for sodium at week 53 for 10 mg/kg; a lower triglyceride value for 300 mg/kg at week 105; higher potassium values at weeks 53 and 78 for 300 mg/kg; a higher calcium value for 30 mg/kg at week 78; higher phosphate values for 30 mg/kg at week 105 and for 300 mg/kg at weeks 27 and 105, and a higher alkaline phosphatase value for 300 mg/kg at week 78.

URINALYSIS
The quantitative and qualitative tests performed on urine samples collected at intervals during the study revealed no evidence of a treatment related effect on urine parameters investigated.
A small number of differences between the means achieved a level of statistical significance or showed significant positive or negative trends. However, these differences showed no dose relationship and/or the change was of insufficient magnitude to be toxicologically relevant. In males, these differences included lower mean values for relative density at week 53 in 100 and 300 mg/kg; higher pH values at week 13 for 30, 100 and 300 mg/kg; lower protein content at weeks 53 and 78 for 10 mg/kg, and higher bilirubin at week 13 for 100 mg/kg. Differences in females were limited to a higher urine volume at week 105 and higher pH values at weeks 13 and 105 for 100 mg/kg.

ORGAN WEIGHTS
The carcass weights and the organ weights of the treated animals were comparable to those of the control group at both the 12 and 24 month necropsy.
No toxicological relevance was attributed to the testes to body weight ratios (300 mg/kg) and the ovaries to body weight ratios (30 and 100 mg/kg) which reached statistical significances at the 12 month necropsy. The absolute mean testes weight value was less than control, and lay well within the range of reference values for our rat strain. The increase in testes weight ratio was very slight, approximately 3%, and there was no statistical significance at terminal necropsy. Additionally, there were no corroborative histological findings indicative of a treatment related effect. The absolute mean ovary weights in groups 30 and 100 mg/kg lay well within the historical reference range of our rat strain. There were no other corroborative findings, no consistent trend over the whole dose range and also no effect after two years of treatment.

GROSS PATHOLOGY
Positive results for the terminal necropsy can be seen in Table 9.
> Interim sacrifice (12 months)
At the 12 months interim sacrifice, all animals of experimental group 4 were examined macroscopically. A variety of macroscopic findings was observed in this study. They occurred in comparable numbers in all experimental groups and were similar to those occurring spontaneously in our colony of rats. Thus, no experimental relevance is attributed to these findings. In particular, masses on the body surface (chest wall, abdominal wall) microscopically reflecting neoplasias of the mammary gland, all were incidentally distributed among the dose groups.
> Final sacrifice (24 months) (including intercurrent deaths)
At the terminal sacrifice, all animals were examined macroscopically, including animals which died intercurrently.
No treatment related findings were observed during examination at final sacrifice. A variety of findings, as listed in Table 9, occurred in slightly increased incidences in treated animals. The histological counterparts of these gross lesions, occurring normally in aged rats, were not indicative of an effect due to the treatment. Therefore, these findings are considered devoid of toxicological relevance. They included cysts in the liver of males, corresponding histologically with biliary cyst, spongiosis and peliosis hepatis and cholangioma, enlarged spleens, corresponding with extramedullary hematopoiesis, congestion and/or infiltration by systemic neoplasia, cysts in the kidneys of males, corresponding histologically with simple cysts and an abscess, fluid in the tunica albuginea of testes in males, corresponding histologically with interstitial edema of testes, and deformation of the brain in females, corresponding histologically with hydrocephalus and/or pressure atrophy.
All other findings occurred in comparable numbers in all dose groups and were similar to those occurring spontaneously in our colony of rats. Thus no experimental relevance is attributed to these findings. In particular, masses on the body surface (head, back, chest wall, abdominal wall, inguinal region), all reported under 'skin/subcutis', microscopically reflecting neoplasias of the skin, subcutaneous tissue and the mammary gland, are all incidentally distributed among the dose groups.

HISTOPATHOLOGY: NON-NEOPLASTIC
> Interim sacrifice (12 months)
Microscopic findings are presented in Table 10.
Liver: Histopathology at 100 and 300 mg/kg confirmed the liver as a target organ. In animals sacrificed at 12 months, there was a minimal increase in incidence and/or severity of liver necrosis in males at 100 and 300 mg/kg, when compared to controls. In females, liver necrosis was seen only at 100 and 300 mg/kg, not in controls. Single cell necrosis (necrosis of single hepatocytes or minute clusters of hepatocytes) was seen only in 300 mg/kg females. There was an increase in severity of cholangiofibrosis in females at 300 mg/kg. Eight of 10 high dose females had an average severity grade of 2.3 compared with an average grade of 1.4 from 5 of 10 controls. The incidence appeared to be slightly increased in females at 100 and 300 mg/kg (8 of 10), although 5 of 10 controls were also noted with cholangiofibrosis. Neither the incidence nor the severity was affected by treatment in males; there was an apparent decrease in high dose (3 of 10) compared with the controls (6 of 10).
Additionally, a variety of other changes were found in the animals scheduled for interim sacrifice. They commonly occur in our colony of rats of this age, and, neither their incidences nor their distribution and morphologic appearance gave any indication of a treatment related association.
> Final sacrifice (24 months) (including intercurrent deaths)
Microscopic findings are presented in Table 11.
Liver: The following treatment related findings were observed: Compared to the controls, statistically significant higher numbers of females with fatty change were observed in the 300 mg/kg treatment group. The severity of this lesion was mainly minimal to slight and the distribution diffused. Compared to the control animals, the severity was slightly increased. Furthermore, slightly increased incidences of minimally to slightly increased mitotic activity of hepatocytes were recorded in high dose females. Compared to the controls, the severity was slightly increased.
In contrast to the 12-month data, the findings for cholangiofibrosis were generally comparable at final sacrifice. The incidences of this common finding in aged rats were greater than 70% in all female groups, although a significant positive trend was noted. There were no differences in average grade, 2.1 - 2.2, for all groups. In males, the incidences were greater than 50% in all groups and there appeared to show a slight decrease in average grade at 300 mg/kg (high dose 2.1 versus control 2.5). Therefore, cholangiofibrosis is considered of no toxicological relevance after 24 months of treatment. Slightly increased incidences of chronic congestion of the liver in high dose females proved to be significant on statistical examination. This lesion occurred in animals found dead or killed moribund and was in relation with their deteriorated general condition. Due to its low incidence of only 3/50 animals affected, this finding is considered devoid of toxicological relevance.

HISTOPATHOLOGY: NEOPLASTIC
> Interim sacrifice (12 months)
There was no evidence of an increased occurrence of neoplastic lesions. Microscopic findings are presented in Table 10.
> Final sacrifice (24 months) (including intercurrent deaths)
Microscopic findings are presented in Table 11.
No treatment induced neoplastic changes were observed after two years of treatment.
Systemic neoplasias: 2/50 males in the high dose group presented with malignant lymphoma. These incidences proved to be significant on statistical examination. This finding occasionally occurs in control animals of our strain of rats and is within the range of the historical control data as shown at the end of this section. Furthermore, up to 5.7% malignant lymphomas were observed in other colonies of old control Sprague-Dawley rats. Therefore, it is considered an incidental finding of no toxicological relevance.
Oral cavity: 2/50 males in the high dose group presented with squamous cell carcinoma of the oral cavity versus none in the control group. This incidence was slightly above the historical control values of our strain of rats shown at the end of this section. Oral squamous cell carcinomas may occur consequent to chronic inflammatory changes and foreign bodies in the oral cavity. Since inflammation and/or foreign bodies of the oral cavity were observed in several control and treated animals from this study, the oral squamous cell carcinomas observed in these few animals are considered incidental findings devoid of toxicological relevance.

Additionally, a variety of other non-neoplastic and neoplastic changes such as hemosiderosis and extramedullary hematopoiesis in the spleen, biliary cysts, hepatocellular and cholangiocellular tumors in the liver, cysts, chronic tubular lesion and and chronic nephropathy in the kidneys, foam cells and bronchiolo-alveolar adenoma in the lung, myocardial inflammation with fibrosis, atrophy of the thymus, interstitial edema and tubular atrophy of the testes, inflammatory changes in the prostate gland, hyperplasia, adenoma and carcinoma of the adrenal cortex and medulla, c-cell and follicular hyperplasia and tumors of the thyroid gland, hyperplasia and adenoma of acinar and islet cells of the pancreas, several types of tumors of the skin and subcutaneous tissue, fibroadenoma and adenocarcinoma of the mammary gland, degenerative changes in the eyes and Harderian glands, and schwannoma, hemangioma and hemangiosarcoma in different tissues was found in animals of the oncogenicity group in this study. These and the other changes not mentioned here commonly occur in our colony of rats, and, neither their incidences nor their distribution and morphologic appearance gave any indication of a treatment-related association.

HISTORICAL CONTROL DATA
Historical data can be seen in Tables 12 and 13.
Dose descriptor:
NOAEL
Effect level:
1.14 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: liver necrosis
Dose descriptor:
NOAEL
Effect level:
1.3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: liver necrosis
Critical effects observed:
not specified

Table 6. Mortality Data

Nominal Test Concentrations (mg/kg)

Number of Animals Surviving to Termination

Males

Females

Control (0)

26 (52%)

32 (64%)

10

33 (33%)

35 (70%)

30

31 (62%)

36 (72%)

100

29 (29%)

28 (56%)

300

25 (25%)

30 (60%)

 

Table 7. Cumulative Body Weight Gain (g/animal)

Sex

Week

Nominal Test Concentration (mg/kg)

0

10

30

100

300

g

g

%

g

%

g

%

g

%

Male

12

22.9

225.0

- 2.1

232.1

+ 1.0

233.6

+ 1.6

224.9

- 2.2

50

433.4

431.6

- 0.4

444.6

+ 2.6

435.6

+ 0.5

430.9

- 0.6

130

476.0

468.2

- 1.6

482.1

+ 1.3

493.3

+ 3.6

496.9

+ 4.4

Females

12

98.67

101.5

+ 2.9

103.2

+ 4.6

106.8

+ 8.2

105.9

+ 7.3

50

192.6

191.2

- 0.7

198.0

+ 2.8

201.8

+ 4.8

194.8

+ 1.1

130

264.8

274.0

+ 3.5

264.6

- 0.1

260.9

- 1.5

261.3

- 1.3

 

Table 8. Cumulative Food Consumption (CFC) and Time Weighted Average (TWA) per Week

CFC (g/animal)

TWA (g/week)

Nominal Test Concentrations (mg/kg) (% of control)

0

10

30

100

300

CFC males

16856.0

16962.0 (100.6)

16981.6 (100.7)

16809.7 (99.7)

16722.6 (99.2)

TWA males

163.7

164.7 (100.6)

164.9(100.7)

163.2 (99.7)

162.4 (99.2)

CFC females

11900.0

11573.4 (97.3)

11726.4 (98.5)

11874.5 (99.8)

11488.2 (96.5)

TWA females

115.5

112.4 (97.3)

113.8 (98.5)

115.3 (99.8)

111.5 (96.5)

Table 9. Gross Pathology, Incidence of Findings at Final Sacrifice

Observations

Males

Females

Nominal test concentrations (mg/kg)

0

10

30

100

300

0

10

30

100

300

No. of animals examined

50

50

50

50

50

50

50

50

50

50

Liver: cyst

1

1

4

3

4

7

1

6

8

4

Spleen: large

1

4

3

3

4

3

0

5

1

3

Kidneys: cyst

0

4

3

2

4

2

1

0

1

1

Testes tunica albug: Contents fluid

8

9

10

10

14

-

-

-

-

-

Brain: deformation

2

1

2

2

1

1

1

6

4

4

 

Table 10. Histopathology, Incidence of Findings at Interim Sacrifice

Observations

Males

Females

Nominal test concentrations (mg/kg)

0

10

30

100

300

0

10

30

100

300

No. of animals examined

10

10

10

10

10

10

10

10

10

10

Liver Necrosis:

Minimal

1

0

0

2

1

0

0

0

1

3

Slight

1

1

3

2

2

0

0

0

1

0

Moderate

0

0

0

0

1

0

0

0

0

0

Total

2

1

3

4

4

0

0

0

2

3

Liver Single Cell Necrosis:

Minimal

0

0

0

0

0

0

0

0

0

1

Slight

0

0

0

0

0

0

0

0

0

3

Total

0

0

0

0

0

0

0

0

0

4

Liver Cholangiofibrosis:

Minimal

0

0

0

0

0

3

4

2

4

1

Slight

6

6

6

3

3

2

2

2

3

4

Moderate

0

0

0

1

0

0

0

1

1

3

Total

6

6

6

4

3

5

6

5

8

8

Table 11. Histopathology, Incidence of Findings at Terminal Sacrifice

Observations

Males

Females

Nominal test concentrations (mg/kg)

0

10

30

100

300

0

10

30

100

300

No. of animals examined

50

50

50

50

50

50

50

50

50

50

Treatment Related Increased Incidence

Liver: Fatty change

Minimal

0

0

0

0

0

10

11

11

6

13

Slight

9

12

9

10

8

1

4

0

4

11

Moderate

1

0

2

0

4

1

1

2

2

3

Severe

0

0

1

0

0

0

0

0

1

0

Total

10

12

12

10

12

12

16

13

13

27

Liver: Increased mitotic activity

Minimal

0

0

0

0

0

1

0

1

1

3

Slight

0

0

0

1

0

0

0

0

0

3

Total

0

0

0

1

0

1

0

1

1

6

Differences in Incidences Not Associated with Treatment

Liver: Cholangiofibrosis

35

37

36

33

28

37

41

44

42

45

Liver: Congestion chronic

0

0

0

0

0

0

0

1

0

3

Systemic Neoplasias: Lymphoma malignate

0

0

1

1

2

0

0

0

1

0

Oral Cavity: Carcinoma squamous

0

0

0

0

2

1

0

0

1

0

 

Table 12. Historical Data, Incidence of Malignant Lymphoma (Systemic Neoplasia) in Untreated Male Rats

Date of 1stDose*

No. of Animals with Systemic Neoplasia **

No. of Organs Examined

February 1992

0

50

June 1992

1

50

August 1992

1

50

August 1992

2

50

May 1993

0

50

May 1993

1

50

June 1993

0

50

January 1994

1

60

January 1994

0

60

October 1994

0

50

August 1995

0

50

Total

6 (1.05%)

570

SD

1.36%

 

Range High

2/50 (4.00%)

 

Range Low

0/60 (0.00%)

 

* All studies were performed for 24 months.

** Malignant lymphoma (M-LM-3) in the lymphoreticular tissue (LRT).

 

Table 13. Historical Data, Incidence of Squamous Cell Carinoma, in the Mouth or Nasopharynx of Untreated Male Rats

Date of 1stDose*

No. of Animals with Squamous Carinoma in the Mouth, Oral Cavity (MOU SQ-CE-CC-3)

No. of Animals with Squamous Carinoma in the Nasopharynx, Nasal Cavity (PHA SQ-CE-CC-3)

No. of Organs Examined

February 1992

0

0

50

June 1992

1

0

50

August 1992

0

0

50

August 1992

0

0

50

May 1993

0

0

50

May 1993

0

0

50

June 1993

1

0

50

January 1994

1

0

60

January 1994

0

0

60

October 1994

0

0

50

August 1995

0

0

50

Total

3 (0.53%)

0 (0.00%)

570

SD

0.89%

0.00%

 

Range High

1/50 (2.00%)

0/50 (0.00%)

 

Range Low

0/60 (0.00%)

0/60 (0.00%

 

* All studies were performed for 24 months.

Conclusions:
Under the conditions of the test, the test material was tolerated without occurrence of overt toxicity up to the maximum dose level of 300 mg/kg. All in-life end points (clinical signs, behaviour, body weight development, food consumption, and laboratory data) and mortalities of animals of the treated groups were similar to those of the control group. An overall slightly decreased water consumption was measured for the high dose females only. Organ weights and macroscopic examination at necropsy were without indication of effects related to treatment.
Histopathology revealed the liver as a target organ. Observations at the maximum dose, 300 mg/kg after 12 months of treatment were a slightly increased incidence and/or severity of liver necrosis in males and females, increased incidence of hepatocellular single cell necrosis in females, and increased incidence and/or severity of cholangiofibrosis in females. Observations after 24 months were, overall slightly reduced water consumption in females, increased incidence and slightly increased severity of fatty change in females, and slightly increased incidence and severity of mitotic activity of hepatocytes in females.
Observations at 100 mg/kg after 12 months were, slightly increased incidence and/or severity of liver necrosis in males and females, and increased incidence and/or severity of cholangiofibrosis in females.
There was no evidence for the occurrence of treatment related neoplastic lesions at any dose level in this study.
The findings observed in the liver (necrosis, fatty change, and higher mitotic activity) at 100 and/or 300 ppm, however, clearly indicate these dose levels to have met or exceeded the maximum tolerated dose (MTD) requirement. Therefore the NOEL and NOAEL was determined to be 30 mg/kg in both males and females, corresponded to actual ingested doses of 1.14 mg/kg bw in males and to 1.30 mg/kg bw in females.
Executive summary:

The chronic toxicity of the test material via oral exposure was determined in a 24 month study with both male and female rats, conducted to the standardised guidelines OECD 453, EPA OPP 83-5, EU Method B. 33 and Japanese MAFF.

The test material was administered oral to Sprague-Dawley derived rats at concentrations of 0, 10, 30, 100, and 300 mg/kg in their diet. A total of eighty rats per sex per dose were exposed, consisting of fifty animals per sex for evaluation of carcinogenic potential, and thirty split into satellite groups for the evaluation of haematological parameters, blood chemistry, urine parameters, and the interim sacrifice at month 12. Clinical signs, body weight, food and water consumption, opthalmoscopic effects, and mortality were monitored throughout the study for all animals. Haematological investigations, blood chemistry and urine parameters were examined on weeks 13, 27, 53, 78 and 105. At sacrifices, animals were examined macroscopically and organ weights were recorded. Organs and tissues were collected and prepared for histopathological evaluation. Organs and tissues from all animals in the carcinogenicity group were examined microscopically.

Under the conditions of the test, the test material was tolerated without occurrence of overt toxicity up to the maximum dose level of 300 mg/kg. All In-life end points (clinical signs, behaviour, body weight development, food consumption, and laboratory data) and mortalities of animals of the treated groups were similar to those of the control group. An overall slightly decreased water consumption was measured for the high dose females only. Organ weights and macroscopic examination at necropsy were without indication of effects related to treatment. Histopathology revealed the liver as a target organ. Observations at the maximum dose, 300 mg/kg after 12 months of treatment were a slightly increased incidence and/or severity of liver necrosis in males and females, increased incidence of hepatocellular single cell necrosis in females, and increased incidence and/or severity of cholangiofibrosis in females. Observations after 24 months were, overall slightly reduced water consumption in females, increased incidence and slightly increased severity of fatty change in females, and slightly increased incidence and severity of mitotic activity of hepatocytes in females. Observations at 100 mg/kg after 12 months were, slightly increased incidence and/or severity of liver necrosis in males and females, and increased incidence and/or severity of cholangiofibrosis in females. There was no evidence for the occurrence of treatment related neoplastic lesions at any dose level in this study.

The findings observed in the liver (necrosis, fatty change, and higher mitotic activity) at 100 and/or 300 ppm, however, clearly indicate these dose levels to have met or exceeded the maximum tolerated dose (MTD) requirement. Therefore the NOEL and NOAEL was determined to be 30 mg/kg in both males and females, corresponded to actual ingested doses of 1.14 mg/kg bw/day in males and to 1.30 mg/kg bw/day in females. The study is considered to be reliable, relevant and adequate for risk assessment purposes.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
1.14 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The key study is GLP compliant and of a high quality (Klimisch 1). Five supporting studies were also available, all assaigned Klimisch reliability scores of either 1 or 2. The results of the study were in general agreement with each other. The quality of the database can be said to be high.

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

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to guideline; under GLP conditions
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Tif: RAif (SPF), hybrids of RIII1 x RIII2 (Sprague-Dawley derived)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Recognised animal supplier
- Age at study initiation: Not reported
- Weight at study initiation: males 208.6 - 243.0 g; females 229.5 - 255.8 g
- Fasting period before study: Not reported
- Housing: Housed individually in Macrolon cages type 3 (area: 900 square centimeters) with wire mesh tops and granulated soft wood bedding
- Diet (e.g. ad libitum): certified standard pelleted diet ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 15
- Air changes (per hr): aproximately 15
- Photoperiod (hrs dark / hrs light): 12h light / 12h dark

IN-LIFE DATES: From: October 17, 1995 To: November 22, 1995
Type of coverage:
occlusive
Vehicle:
other: 0.5 % (w/v) carboxymethylcellulose in 0.1% (w/v) aqueous polysorbate 80.
Details on exposure:
TEST SITE
- Area of exposure: Dorsal area (The test article/vehicle suspension was applied to the right side of clipped area (=skin application site). The vehicle only was applied to the left side of clipped area (=skin remote site).
- % coverage: At least 10%
- Type of wrap if used: Gauze patches covered with aluminum wrap, and fastened to the body with adhesive but non-irritating tape.
- Time intervals for shavings or clipplings: 5 days a week for 4 weeks.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Application areas were cleaned with lukewarm water.
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 4 ml/kg body weight
- Concentration (if solution): 0, 10, 100 and 1000 mg/kg

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not reported
- Amount(s) applied (volume or weight with unit): 4 ml/kg body weight
- Concentration (if solution): 0.5% (w/v) carboxymethylcellulose in 0.1% (w/v) aqueous polysorbate 80.

USE OF RESTRAINERS FOR PREVENTING INGESTION: No.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stock solutions of the test substance in acetonitrile with concentrations of 1 000 μg/ml, 205 μg/ml, and 200 μg/ml were prepared as follows: 100 mg, 20.5 mg, and 20 mg portions of test article were weighed into 100-ml volumetric flasks and dissolved in about 70 ml of acetonitrile by means of an ultrasonic bath. Afterwards, the volumetric flasks were filled to volume with HPLC-eluent. Next, various standard solutions were prepared by respective dilution of these stock solutions with HPLC-eluent to yield concentrations in the range from 5 μg/ml to 51.25 μg/ml. These standard solutions were used to calibrate the HPLC.

HPLC conditions:
- Apparatus: Merck L6200A (pump), Merck L4000 (UV-detector), Merck D2000 (integrator), Merck 655A 40 (sampling unit).
- Column: Lichrospher 100 RP-18; 5 μm; 125 x 4.6 mm (o.d.)
- Temperature: room temperature
- Eluent: Acetonitrile/0.02 M H3P04 (60+40/v/v)
- Flow: 1.0 ml/min
- Wavelength: 274 nm
- Injection volume: 10 μl
Duration of treatment / exposure:
6 hours
Frequency of treatment:
5 days a week for 4 weeks
Remarks:
Doses / Concentrations:
0, 10, 100 and 1000 mg/kg bodyweight
Basis:
nominal per unit body weight
No. of animals per sex per dose:
5 animals of each sex were used per dose.
- Group 1: 0 mg/kg
- Group 2: 10 mg/kg
- Group 3: 100 mg/kg
- Group 4: 1000 mg/kg
Control animals:
yes
Details on study design:
- Dose selection rationale: not reported
- Rationale for animal assignment (if not random): random
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily (including weekends)

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Following application 17 hours after removal of patches.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of treatment period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No data
- How many animals: All surviving animals
- Parameters checked: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution, leukocyte count, neutrophils, eosinophils, basophils, lymphocytes, monocytes, large unstained cells, thrombocyte count, prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of treatment period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No data
- How many animals: All surviving animals
- Parameters checked: glucose, urea, creatinine, total bilirubin, total protein, albumin, globulin, A/G ratio, cholestrol, triglycerides, sodium, potassium, calcium, chloride, phosphorous inorganic, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
At necropsy the following weights were recorded from all animals: body (exsanguinated), brain, heart, liver, kidneys, adrenals, thymus, ovaries/testes, spleen.

The following organs and tissues were preserved in neutral buffered 4% formalin:
skin application site, skin remote site, mammary area, spleen mesenteric lymph node, axillary lymph node, sternum with bone marrow, femur with joint skeletal muscle, trachea, lung, heart, aorta, submandibular salivary gland, liver, pancreas, oesophagus, stomach, small intestine, large intestine, kidney (both), urinary bladder, prostate, seminal vesicle, testis (both), epididymis (both), uterus, vagina, ovary (both), pituitary gland, adrenal gland (both), thyroid with parathyroid gland, thymus, peripheral nerve, brain, spinal cord, eye with optic nerve (both), orbital gland (both), extraorbital lacrimal gland (both), Zymbal gland (both), muzzle, tongue, any tissue with gross lesions

HISTOPATHOLOGY: Yes
After the fixation, organ samples listed below were taken, embedded in paraplast, sectioned at 3-5 microns, stained with hematoxylin and eosin, and subjected to a microscopical examination: skin application site, skin remote site, liver, spleen, kidney (both), bone marrow, thymus, thyroid with parathyroid gland.
Statistics:
For each time point and parameter an univariate statistical analysis was performed. Nonparametric methods were applied, to allow for non normal as well as normal data distribution. Each treated group was compared to the control group by Wilcoxon's two-sample test and tested for increasing or decreasing trends from control up to the respective dose group by Jonckheere's test for ordered alternatives.
Clinical signs:
no effects observed
Dermal irritation:
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:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no clinical signs or local irritation attributed to the treatment with test material. Two animals from the 1000 mg/kg dose group, one animal from the 100 mg/kg dose group, one animal from the 10 mg/kg dose group and two control animals developed blisters in the clipped areas of the back. As these findings were noted in treated and non treated animals, they were not attributed to treatment.

All animals survived the treatment period.

BODY WEIGHT AND WEIGHT GAIN
Males treated at 100 and 1000 mg/kg body weight gained less body weight, compared to the control. However, since individual animals, which contributed mainly to this effect, had a lower body weight gain before treatment start (week -1), it was considered to reflect biological variability and was not attributed to the treatment. Throughout the study including the pretest week, the mean body weights of male group 2 were higher than that of the control group. This was also attributed to reflect biological variability.

FOOD CONSUMPTION
During week 2 of treatment increased mean food consumption was measured for females of group 4, which was, however, attributed to biological variability.

FOOD EFFICIENCY
Not examined

WATER CONSUMPTION
Not examined

OPHTHALMOSCOPIC EXAMINATION
Not examined

HAEMATOLOGY
No significant findings.

Higher values were obtained for hemoglobin concentration distribution width (HDW) in male animals of group 4 (1000 mg/kg), however in the absence of other corroborative findings on red blood cell parameters no toxicological relevance was attributed. Other minor deviations which attained a level of statistical significance occurred without any relation to the dose administered andjor the values were within the reference range. Therefore, the following changes were considered not to be treatment-related: In group 2 (10 mg/kg), lower values of red cell volume distribution width (RDW) was noted in males, minimally higher levels of hemoglobin (Hb) and mean corpuscular hemoglobin concentration (MCHC) was observed in females. Females of group 3 (100 mg/kg) had higher white blood cell counts, particularly basophils, lymphocytes and large unstained cells. Males of group 4 (1000 mg/kg) showed minimally lower values of large unstained cells and females of this dose group had slightly lower platelet counts.

CLINICAL CHEMISTRY
In females of group 4 (1000 mg/kg), a minimal increase of plasma potassium concentrations was noted. Since no further alterations in electrolyte concentrations were observed in animals on this study, the relevance of this finding is equivocal. Lower plasma cholesterol concentrations as observed in females of group 4 are within the range of reference values and were therefore considered unrelated to the treatment.

Other minor deviations which attained a level of statistical significance occurred without any relation to the dose administered, and were therefore considered not to be treatment-related: In males of group 2 (10 mg/kg) changes of plasma creatinine concentrations and lower activities of alanine aminotransferase. In group 3 animals (100 mg/kg), decreased albumin to globulin ratios were noted in males, changes of plasma albumin concentrations, higher plasma triglyceride levels and higher activities of alanine aminotransferase and alkaline phosphatase were observed in females.

URINALYSIS
Not examined

NEUROBEHAVIOUR
Not examined

ORGAN WEIGHTS
No significant findings.

Minimal lower values of heart to body weight ratios were calculated for female groups 2, 3, and 4 (10, 100 and 1000 mg/kg body weight). In the absence of any corroborative findings and lack of dose dependency, these deviations were considered of incidental nature.

GROSS PATHOLOGY
One female (0 mg/kg) had a mottled thymus and one male (1000 mg/kg) presented a cyst in one kidney. These lesions were similar to those occurring spontaneously in our colony of rats. Thus no experimental relevance was attributed to these findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
No significant findings.

Changes found at the skin application site occurred in a similar incidence and morphological appearence in controls and treated animals. They were considered to be the result of the application procedure itself and not related to the treatment with the test article. Additionally, a variety of other changes was found in this study. They commonly occur in our colony of rats, and, neither their incidences nor their distribution and morphologic appearance gave any indication of a treatment-related association.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No significant findings.
Critical effects observed:
not specified

Analytical verification of doses:

The mean test article concentrations in the vehicle were 83.2%, 85.0% and 97.7% of the nominal concentrations in dose group 2, 3 and 4, respectively. The test material was found to be homogeneously distributed and to be stable in the vehicle under actual conditions of administration over the period of dosing.

Conclusions:
Under the conditions of this study, the no-observable-effect level (NOEL) for rats of both sexes is 1000 mg/kg body weight.
Executive summary:

The study was performed according the requirements of OECD Guidelines for Testing of Chemicals No. 410 under GLP conditions. The study was performed to investigate the potential toxicity of the test material to male and female Tif: RAif (SPF), hybrids of RIII1 x RIII2 (Sprague-Dawley derived) rats when administered topically 5 days a week for 4 weeks. The dermal route was used as this represents a possible route of exposure in humans. Four groups of 5 male and 5 female rats were dosed with the test material in vehicle (0.5 % (w/v) carboxymethylcellulose in 0.1% (w/v) aqueous polysorbate 80) at 0, 10, 100 or 1000 mg/kg bw/day 5 times a week for 4 weeks. Test material was applied under occlusive dressing for a 6 hour period. Clinical observations, body weight and food consumption were recorded daily. Animals were humanely sacrificed at the end of the study. At termination, blood samples were taken for haematology and blood chemistry investigations. Selected organs were weighed and specified tissues/organs were processed for histopathological examination. Tissues/organs from control and high dose animals were examined by light microscopy, together with all macroscopic abnormalities in any group.

Analytical data confirmed the suitability of the dosing preparations used in the study. There were no adverse treatment-related effects on clinical observations, body weights, food consumption. There were no toxicologically significant effects on haematology, blood chemistry. There were no treatment-related macroscopic or microscopic findings in any organ at examination post mortem. The no effect level (NOEL) in this study was 1000 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study is GLP compliant and of a high quality (Klimisch 1)

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to guideline; under GLP conditions
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Tif: RAif (SPF), hybrids of RIII1 x RIII2 (Sprague-Dawley derived)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Recognised animal supplier
- Age at study initiation: Not reported
- Weight at study initiation: males 208.6 - 243.0 g; females 229.5 - 255.8 g
- Fasting period before study: Not reported
- Housing: Housed individually in Macrolon cages type 3 (area: 900 square centimeters) with wire mesh tops and granulated soft wood bedding
- Diet (e.g. ad libitum): certified standard pelleted diet ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 15
- Air changes (per hr): aproximately 15
- Photoperiod (hrs dark / hrs light): 12h light / 12h dark

IN-LIFE DATES: From: October 17, 1995 To: November 22, 1995
Type of coverage:
occlusive
Vehicle:
other: 0.5 % (w/v) carboxymethylcellulose in 0.1% (w/v) aqueous polysorbate 80.
Details on exposure:
TEST SITE
- Area of exposure: Dorsal area (The test article/vehicle suspension was applied to the right side of clipped area (=skin application site). The vehicle only was applied to the left side of clipped area (=skin remote site).
- % coverage: At least 10%
- Type of wrap if used: Gauze patches covered with aluminum wrap, and fastened to the body with adhesive but non-irritating tape.
- Time intervals for shavings or clipplings: 5 days a week for 4 weeks.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Application areas were cleaned with lukewarm water.
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 4 ml/kg body weight
- Concentration (if solution): 0, 10, 100 and 1000 mg/kg

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not reported
- Amount(s) applied (volume or weight with unit): 4 ml/kg body weight
- Concentration (if solution): 0.5% (w/v) carboxymethylcellulose in 0.1% (w/v) aqueous polysorbate 80.

USE OF RESTRAINERS FOR PREVENTING INGESTION: No.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stock solutions of the test substance in acetonitrile with concentrations of 1 000 μg/ml, 205 μg/ml, and 200 μg/ml were prepared as follows: 100 mg, 20.5 mg, and 20 mg portions of test article were weighed into 100-ml volumetric flasks and dissolved in about 70 ml of acetonitrile by means of an ultrasonic bath. Afterwards, the volumetric flasks were filled to volume with HPLC-eluent. Next, various standard solutions were prepared by respective dilution of these stock solutions with HPLC-eluent to yield concentrations in the range from 5 μg/ml to 51.25 μg/ml. These standard solutions were used to calibrate the HPLC.

HPLC conditions:
- Apparatus: Merck L6200A (pump), Merck L4000 (UV-detector), Merck D2000 (integrator), Merck 655A 40 (sampling unit).
- Column: Lichrospher 100 RP-18; 5 μm; 125 x 4.6 mm (o.d.)
- Temperature: room temperature
- Eluent: Acetonitrile/0.02 M H3P04 (60+40/v/v)
- Flow: 1.0 ml/min
- Wavelength: 274 nm
- Injection volume: 10 μl
Duration of treatment / exposure:
6 hours
Frequency of treatment:
5 days a week for 4 weeks
Remarks:
Doses / Concentrations:
0, 10, 100 and 1000 mg/kg bodyweight
Basis:
nominal per unit body weight
No. of animals per sex per dose:
5 animals of each sex were used per dose.
- Group 1: 0 mg/kg
- Group 2: 10 mg/kg
- Group 3: 100 mg/kg
- Group 4: 1000 mg/kg
Control animals:
yes
Details on study design:
- Dose selection rationale: not reported
- Rationale for animal assignment (if not random): random
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily (including weekends)

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Following application 17 hours after removal of patches.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of treatment period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No data
- How many animals: All surviving animals
- Parameters checked: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution, leukocyte count, neutrophils, eosinophils, basophils, lymphocytes, monocytes, large unstained cells, thrombocyte count, prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of treatment period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No data
- How many animals: All surviving animals
- Parameters checked: glucose, urea, creatinine, total bilirubin, total protein, albumin, globulin, A/G ratio, cholestrol, triglycerides, sodium, potassium, calcium, chloride, phosphorous inorganic, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
At necropsy the following weights were recorded from all animals: body (exsanguinated), brain, heart, liver, kidneys, adrenals, thymus, ovaries/testes, spleen.

The following organs and tissues were preserved in neutral buffered 4% formalin:
skin application site, skin remote site, mammary area, spleen mesenteric lymph node, axillary lymph node, sternum with bone marrow, femur with joint skeletal muscle, trachea, lung, heart, aorta, submandibular salivary gland, liver, pancreas, oesophagus, stomach, small intestine, large intestine, kidney (both), urinary bladder, prostate, seminal vesicle, testis (both), epididymis (both), uterus, vagina, ovary (both), pituitary gland, adrenal gland (both), thyroid with parathyroid gland, thymus, peripheral nerve, brain, spinal cord, eye with optic nerve (both), orbital gland (both), extraorbital lacrimal gland (both), Zymbal gland (both), muzzle, tongue, any tissue with gross lesions

HISTOPATHOLOGY: Yes
After the fixation, organ samples listed below were taken, embedded in paraplast, sectioned at 3-5 microns, stained with hematoxylin and eosin, and subjected to a microscopical examination: skin application site, skin remote site, liver, spleen, kidney (both), bone marrow, thymus, thyroid with parathyroid gland.
Statistics:
For each time point and parameter an univariate statistical analysis was performed. Nonparametric methods were applied, to allow for non normal as well as normal data distribution. Each treated group was compared to the control group by Wilcoxon's two-sample test and tested for increasing or decreasing trends from control up to the respective dose group by Jonckheere's test for ordered alternatives.
Clinical signs:
no effects observed
Dermal irritation:
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:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no clinical signs or local irritation attributed to the treatment with test material. Two animals from the 1000 mg/kg dose group, one animal from the 100 mg/kg dose group, one animal from the 10 mg/kg dose group and two control animals developed blisters in the clipped areas of the back. As these findings were noted in treated and non treated animals, they were not attributed to treatment.

All animals survived the treatment period.

BODY WEIGHT AND WEIGHT GAIN
Males treated at 100 and 1000 mg/kg body weight gained less body weight, compared to the control. However, since individual animals, which contributed mainly to this effect, had a lower body weight gain before treatment start (week -1), it was considered to reflect biological variability and was not attributed to the treatment. Throughout the study including the pretest week, the mean body weights of male group 2 were higher than that of the control group. This was also attributed to reflect biological variability.

FOOD CONSUMPTION
During week 2 of treatment increased mean food consumption was measured for females of group 4, which was, however, attributed to biological variability.

FOOD EFFICIENCY
Not examined

WATER CONSUMPTION
Not examined

OPHTHALMOSCOPIC EXAMINATION
Not examined

HAEMATOLOGY
No significant findings.

Higher values were obtained for hemoglobin concentration distribution width (HDW) in male animals of group 4 (1000 mg/kg), however in the absence of other corroborative findings on red blood cell parameters no toxicological relevance was attributed. Other minor deviations which attained a level of statistical significance occurred without any relation to the dose administered andjor the values were within the reference range. Therefore, the following changes were considered not to be treatment-related: In group 2 (10 mg/kg), lower values of red cell volume distribution width (RDW) was noted in males, minimally higher levels of hemoglobin (Hb) and mean corpuscular hemoglobin concentration (MCHC) was observed in females. Females of group 3 (100 mg/kg) had higher white blood cell counts, particularly basophils, lymphocytes and large unstained cells. Males of group 4 (1000 mg/kg) showed minimally lower values of large unstained cells and females of this dose group had slightly lower platelet counts.

CLINICAL CHEMISTRY
In females of group 4 (1000 mg/kg), a minimal increase of plasma potassium concentrations was noted. Since no further alterations in electrolyte concentrations were observed in animals on this study, the relevance of this finding is equivocal. Lower plasma cholesterol concentrations as observed in females of group 4 are within the range of reference values and were therefore considered unrelated to the treatment.

Other minor deviations which attained a level of statistical significance occurred without any relation to the dose administered, and were therefore considered not to be treatment-related: In males of group 2 (10 mg/kg) changes of plasma creatinine concentrations and lower activities of alanine aminotransferase. In group 3 animals (100 mg/kg), decreased albumin to globulin ratios were noted in males, changes of plasma albumin concentrations, higher plasma triglyceride levels and higher activities of alanine aminotransferase and alkaline phosphatase were observed in females.

URINALYSIS
Not examined

NEUROBEHAVIOUR
Not examined

ORGAN WEIGHTS
No significant findings.

Minimal lower values of heart to body weight ratios were calculated for female groups 2, 3, and 4 (10, 100 and 1000 mg/kg body weight). In the absence of any corroborative findings and lack of dose dependency, these deviations were considered of incidental nature.

GROSS PATHOLOGY
One female (0 mg/kg) had a mottled thymus and one male (1000 mg/kg) presented a cyst in one kidney. These lesions were similar to those occurring spontaneously in our colony of rats. Thus no experimental relevance was attributed to these findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
No significant findings.

Changes found at the skin application site occurred in a similar incidence and morphological appearence in controls and treated animals. They were considered to be the result of the application procedure itself and not related to the treatment with the test article. Additionally, a variety of other changes was found in this study. They commonly occur in our colony of rats, and, neither their incidences nor their distribution and morphologic appearance gave any indication of a treatment-related association.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No significant findings.
Critical effects observed:
not specified

Analytical verification of doses:

The mean test article concentrations in the vehicle were 83.2%, 85.0% and 97.7% of the nominal concentrations in dose group 2, 3 and 4, respectively. The test material was found to be homogeneously distributed and to be stable in the vehicle under actual conditions of administration over the period of dosing.

Conclusions:
Under the conditions of this study, the no-observable-effect level (NOEL) for rats of both sexes is 1000 mg/kg body weight.
Executive summary:

The study was performed according the requirements of OECD Guidelines for Testing of Chemicals No. 410 under GLP conditions. The study was performed to investigate the potential toxicity of the test material to male and female Tif: RAif (SPF), hybrids of RIII1 x RIII2 (Sprague-Dawley derived) rats when administered topically 5 days a week for 4 weeks. The dermal route was used as this represents a possible route of exposure in humans. Four groups of 5 male and 5 female rats were dosed with the test material in vehicle (0.5 % (w/v) carboxymethylcellulose in 0.1% (w/v) aqueous polysorbate 80) at 0, 10, 100 or 1000 mg/kg bw/day 5 times a week for 4 weeks. Test material was applied under occlusive dressing for a 6 hour period. Clinical observations, body weight and food consumption were recorded daily. Animals were humanely sacrificed at the end of the study. At termination, blood samples were taken for haematology and blood chemistry investigations. Selected organs were weighed and specified tissues/organs were processed for histopathological examination. Tissues/organs from control and high dose animals were examined by light microscopy, together with all macroscopic abnormalities in any group.

Analytical data confirmed the suitability of the dosing preparations used in the study. There were no adverse treatment-related effects on clinical observations, body weights, food consumption. There were no toxicologically significant effects on haematology, blood chemistry. There were no treatment-related macroscopic or microscopic findings in any organ at examination post mortem. The no effect level (NOEL) in this study was 1000 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study is GLP compliant and of a high quality (Klimisch 1)

Additional information

Repeat dose - Oral:

OECD 453, Gerspach 1998: The key study was performed according the requirements of OECD Guidelines for Testing of Chemicals No. 453 under GLP conditions. The chronic study was performed to determine repeated dose oral toxicity and carcinogenicity in a 24 month study with both male and female rats. The test material was administered oral to Sprague-Dawley derived rats at concentrations of 0, 10, 30, 100, and 300 mg/kg in their diet. A total of eighty rats per sex per dose were exposed, consisting of fifty animals per sex for evaluation of carcinogenic potential, and thirty split into satellite groups for the evaluation of haematological parameters, blood chemistry, urine parameters, and the interim sacrifice at month 12. Clinical signs, body weight, food and water consumption, opthalmoscopic effects, and mortality were monitored throughout the study for all animals. Haematological investigations, blood chemistry and urine parameters were examined on weeks 13, 27, 53, 78 and 105. At sacrifices, animals were examined macroscopically and organ weights were recorded. Organs and tissues were collected and prepared for histopathological evaluation. Organs and tissues from all animals in the carcinogenicity group were examined microscopically.

Under the conditions of the test, the test material was tolerated without occurrence of overt toxicity up to the maximum dose level of 300 mg/kg. All In-life end points (clinical signs, behaviour, body weight development, food consumption, and laboratory data) and mortalities of animals of the treated groups were similar to those of the control group. An overall slightly decreased water consumption was measured for the high dose females only. Organ weights and macroscopic examination at necropsy were without indication of effects related to treatment. Histopathology revealed the liver as a target organ. Observations at the maximum dose, 300 mg/kg after 12 months of treatment were a slightly increased incidence and/or severity of liver necrosis in males and females, increased incidence of hepatocellular single cell necrosis in females, and increased incidence and/or severity of cholangiofibrosis in females. Observations after 24 months were, overall slightly reduced water consumption in females, increased incidence and slightly increased severity of fatty change in females, and slightly increased incidence and severity of mitotic activity of hepatocytes in females. Observations at 100 mg/kg after 12 months were, slightly increased incidence and/or severity of liver necrosis in males and females, and increased incidence and/or severity of cholangiofibrosis in females. There was no evidence for the occurrence of treatment related neoplastic lesions at any dose level in this study.

The findings observed in the liver (necrosis, fatty change, and higher mitotic activity) at 100 and/or 300 ppm, however, clearly indicate these dose levels to have met or exceeded the maximum tolerated dose (MTD) requirement. Therefore the NOEL and NOAEL was determined to be 30 mg/kg in both males and females, corresponded to actual ingested doses of 1.14 mg/kg bw/day in males and to 1.30 mg/kg bw/day in females. The study is considered to be reliable, relevant and adequate for risk assessment purposes.

Supporting information is available in the form of a 3-month study in rats (Gerspach, 1996a), performed to OECD 408. Under the conditions of the test, treatment with the test material resulted in impaired body weight development of animals treated at 1000 ppm and 4000 ppm. 3 deaths of males occurred at 4000 ppm. At end of treatment period, laboratory investigations revealed effects on the RBC, WBC, liver and kidney parameters. At necropsy, increased organ weights and/or organ to body weight ratios were measured in the 4000 ppm group for liver, spleen and heart (both sexes), and kidneys (males). Histopathological evaluation revealed the liver as target organ of toxicity. Based on mortalities and body weight gain data, it can be concluded that the Maximum Tolerated Dose was met or exceeded at the 1000 ppm dose level, and was clearly exceeded at the 4000 ppm dose level. The NOEL for the test material when offered to rats continuously in their food over a period of 3 months is 100 ppm, corresponding to a mean daily intake of 6.12 mg/kg body weight in males and 7.07 mg/kg body weight in females.

Another supporting 28-day study in rats (Gerspach, 1999), performed to OECD 407 and EU Method B.7 was provided. Under the conditions of the study none of the animals died and no clinical signs were recorded. Effects seen included retarded body weight development, occurrence of anemia consequent to the test material’s inhibitory effect on the heme synthesis, associated with extramedullary hematopoiesis and stimulation of the bone marrow. Down to the low dose level (300 pm) occurrence of porphyrin in the urine was indicated. Additionally, hepato- and/or nephrotoxic effects occurred at 2000 and 10000 ppm. The test material induced a range of effects on the blood system, due to inhibition of heme synthesis, on the liver, and on the kidneys. A NOEL could not be defined as was expected with this study design.

A further 28-day study in rats is available (Bachmann, 1993). The study was not performed to a guideline and was conducted in order to inform on the repeat dose toxicity of the test material over a 28-day period. None of the animals died during the study and no relevant clinical signs were observed. Minor differences in mean bodyweight development in both sexes were not considered related to treatment. A dose-related microcytosis, anisocytosis, hypochromasia and polychromia of red blood cells, resulting in lower hemoglobin and hematocrit values, occurred in males and females dosed at 300 and 1000 mg/kg. Signs of microsytosis and hypochromasia were also seen in males dosed at 100 mg/kg. These alterations are indicative of disturbed hemoglobin synthesis. No treatment-related changes were observed in clinical chemistry parameters.

Macroscopic examination gave no indication of treatment-related gross lesions. Microscopic examination revealed a minimal hypocellularity of the bone marrow in 2/5 males dosed at 300 mg/kg, whereas minimal hypercellularity was found in 4/5 males dosed at 1000 mg/kg. Incidence and severity of splenic extramedullary hematopoesis were slightly increased in males and females dosed at 300 mg/kg and above. The following morphological alterations were noted in hepatocytes: in males, many mitochondria had dilated innermitochondrial cristae, frequently containing helically arranged fibrils. In females, the number of mitochondria with dilated innermitochondrial cristae was only marginal when compared with treated males and fibrils were not observed. All 3 males and 1/3 females investigated showed a moderate amount of glycogenosomes, organelles which contain particulate glycogen.

Under the conditions of the study signs of disturbed hemaglobin synthesis were observed at all treatment levels, although minimally at the low dose of 100 mg/kg bw.

Two studies on dogs are also available as supporting data. The first is a 90 day study (Altmann, 1996) performed to OECD 409, EU B.27 and EPA OPP 82-1. None of the animals died and in–life observations, body weight development and food consumption were not considered to be affected by treatment. Eye and macroscopical examinations revealed no treatment-related effects.

Lower values of hemoglobin concentration and hematocrit, associated with microcytosis, hypochromasia, anisochromia of red blood cells were observed at week 7 and 13 investigations in males dosed at 1000 mg/kg. A tendency to lower cell volume and to hypochromasia of erythrocytes was also noted in females of this group after 13 weeks of treatment. Furthermore, slightly higher platelet counts were recorded for males at 1000 mg/kg at treatment end. Slightly lower plasma cholesterol and triglyceride levels were recorded in males at 1000 mg/kg at weeks 7 and 13, and minimally lower plasma albumin and higher globulin levels resulting in lower albumin to globulin ratios were noted at treatment end in males of this group.

Mean absolute and relative liver weights were increased in females at 1000 mg/kg. An increased incidence and severity of hemosiderosis in the spleen and in the Kupffer cells of the liver was noted histologically in males at 1000 mg/kg.

Under the conditions of the study, administration of the test material via gelatine capsules was well tolerated up to and including the high dose level of 1000 mg/kg. At this dose level, changes on red blood cell parameters and morphology were noted which suggest a disturbance in hemaglobin synthesis. This effect was more pronounced in males than in females. In addition, alterations of liver parameters in the absence of histopathological changes indicate a hepatotrophic effect of the test material. No treatment-related effects were observed at 200 mg/kg and, therefore, this was taken to be the NOEL.

The second is a 12 month study (Altmann, 1999), performed to OECD 452 and EPA OPP 83-1. There were no treatment-related deaths. In-life observations and food consumption were not considered to be affected by treatment. Eye and macroscopical examinations revealed no treatment-related effects and urine analysis revealed no treatment-related effects. A loss in mean overall body weight was observed in males dosed at 1000 mg/kg.

Treatment was associated with effects on red blood cells in males and females dosed at 500 and 1000 mg/kg. These effects included lower hemoglobin concentrations and hematocrit for males and females at 1000 mg/kg, lower mean cell volume and mean corpuscular hemoglobin for males and females dosed at 500 and 1000 mg/kg, indicating a tendency to microcytosis and hypochromasia of red blood cells. Furthermore, a thrombocytosis was recorded for males and females at 1000 mg/kg and for females at 500 mg/kg. Plasma albumin levels, albumin to globulin ratios and cholesterol levels were minimally lower in males treated at 1000 mg/kg throughout the treatment period.

Mean relative liver weight was slightly increased in males at 1000 mg/kg. Treatment-related histopathological findings were observed in the liver of some animals from females dosed at 500 mg/kg and males and females dosed at 1000 mg/kg, and consisted of periportal rarefaction of hepatocytes, most likely due to glycogen deposition.

In conclusion, under the conditions of the study, administration of the test material via gelatine capsules was reasonably well tolerated up to and including the high dose level of 1000 mg/kg. At 1000 mg/kg body weight loss was noted and at dose levels of 500 and 1000 mg/kg changes on red blood cell parameters were noted which suggest a disturbance in hemaglobin synthesis. In addition, alterations of liver parameters and mild histopathological changes indicate a hepatotrophic effect of the test material. No treatment-related effects were observed at 100 mg/kg and, therefore, this was taken to be the NOEL.

The study by Gerspach (1998) was selected as the key study since it was performed on a standard species, for the longest period and gave the lowest NOEL.

 

Repeat dose - Dermal:

OECD 410, Gerspach 1996b: The study was performed according the requirements of OECD Guidelines for Testing of Chemicals No. 410 under GLP conditions. The study was performed to investigate the potential toxicity of the test material to male and female Tif: RAif (SPF), hybrids of RIII1 x RIII2 (Sprague-Dawley derived) rats when administered topically 5 days a week for 4 weeks. The dermal route was used as this represents a possible route of exposure in humans. Four groups of 5 male and 5 female rats were dosed with the test material in vehicle (0.5 % (w/v) carboxymethylcellulose in 0.1% (w/v) aqueous polysorbate 80) at 0, 10, 100 or 1000 mg/kg bw/day 5 times a week for 4 weeks. Test material was applied under occlusive dressing for a 6 hour period. Clinical observations, body weight and food consumption were recorded daily. Animals were humanely sacrificed at the end of the study. At termination, blood samples were taken for haematology and blood chemistry investigations. Selected organs were weighed and specified tissues/organs were processed for histopathological examination. Tissues/organs from control and high dose animals were examined by light microscopy, together with all macroscopic abnormalities in any group.

Analytical data confirmed the suitability of the dosing preparations used in the study. There were no adverse treatment-related effects on clinical observations, body weights, and food consumption. There were no toxicologically significant effects on haematology, blood chemistry. There were no treatment-related macroscopic or microscopic findings in any organ at examination post mortem. The no effect level (NOEL) in this study was 1000 mg/kg bw/day.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only one chronic study was available for this endpoint. This studyhad the lowest NOAEL of any of the repeated dose toxicity studies available. It was also performed on the most typical species for this endpoint, the rat. On these basis, it was selected as the key study.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
One study available.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
One study available. Study did not observe local effects occurring at lower concentrations/area doses than a systemic effect limit dose. Therefore a NOEC (mg/cm2) effect level was not derived.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

Under Directive 67/548/EEC, the substance is classified as R48/R22 ‘Harmful: danger of serious damage to health by prolonged exposure if swallowed’. According to Regulation (EC) no 1272/2008 the substance is classified as a STOT (Specific Target Organ Toxicant) - repeated Exposure Category 2 with Hazard statement: H373: May cause damage to organs through prolonged or repeated exposure and should be accompanied with the signal word 'Warning'.

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