Desmedipham

Carcinogenicity

Description of key information

Studies of dietary carcinogenicity in the rat and mouse are available for desmedipham.

Test method/  species	Result	Assessment	Reference
US EPA 83-5 - Carcinogenicity study in the rat performed at 0, 100, 400 and 1200 ppm	There was no evidence of carcinogenicity in this study.  A NOAEL of 100 ppm (5.4-5.9 mg/kg bw/d) was determined, based on haematological effects and histopathology (spleen, liver, kidneys) at 400 ppm	Supporting study	Everett et al (1991)
Not stated, but comparable to OECD 453 - Carcinogenicity study in the rat performed at 0, 60, 300 and 1500 ppm	There was no evidence of carcinogenicity in this study.  A NOAEL of 60 ppm (3.2 mg/kg bw/d) was determined based on minimal (and non-adverse) MetHb formation and increased spleen weight.	Supporting study	Suter et al (1986)
OECD 451 - Carcinogenicity study in the mouse performed at 0, 400, 1000, 2500 ppm	There was no evidence of carcinogenicity in this study.  A NOAEL of 400 ppm (72 mg/kg bw/d) was determined for females based on liver pathology.  A NOAEL could not be determined for males due to liver pathology at 400 ppm (61 mg/kg bw/d).	Supporting study	Husband et al (1995)
Not stated, but comparable to OECD 451 - Carcinogenicity study in the mouse performed at 0, 30, 150, 750 ppm	There was no evidence of carcinogenicity in this study. A NOAEL of 150 ppm (22-31 mg/kg bw/d) was determined for this study, based on haematological effects.	Supporting study	Suter et al (1986)
Not applicable - Additional pathology	Additional histopathological and statistical evaluation performed for the rat study Suter et al (1986); can be reported with the original study	Supporting study	Jackson and Millar (2000)

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1983-07-11 to 1986-09-15

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

No OECD guideline was mentioned in the study report, however, the study essentially complied with OECD guideline 451 at the time of first assessment. After the study was performed, a new version of the OECD Test Guideline 451 has been adopted 7th September, 2009. Deficiencies in the study were: The study protocol did not included microscopical examination of lacrimal gland, and coagulating gland. Animals were palpated for tissue masses once a week and not daily as recommended in the guideline. It was noted in the previous evaluation that it remained unclear if all grossly visible lesions (e.g. nodules in seminal vesicles and ovaries) were identified in microscopical examination. In addition, it remained unclear, why the total number of some examined organs was much lower than 60 (e.g. in case of mammary glands, thymus, mandibular lymph nodes, mesenteric lymph nodes, thyroids, pituitary gland, ovaries, uterus, testes, epididymides and seminal vesicles). Statistical analysis was insufficient: The tumour data from the animals scheduled for interim kill (K1) were not included in the statistical analysis. Non-neoplastic findings were not analysed by statistical tests. The neoplastic lesions observed in interim kill (K1) were not included in the statistical evaluation (22 neoplastic lesions, 9 of them in the control group). The results of the Peto analysis on tumour data were not presented in the statistical report. Only statistical evaluation on tumours observed in haemolymphoreticular system (lymphomas), lungs, liver, ovaries, Harderian glands, mammary glands and pituitary were described in the pathology report. In these tissues, the Peto test on tumours observed in the animals scheduled for terminal kill (KO) did not yield any positive trend. This study has a lot of deficiencies. Overall the final conclusion that no carcinogenic or organ damaging effect was obvious in this study, is supported by the tabulated data alone without any statistical analysis. A new statistical analysis would only show significances of isolated and irrelevant changes at lower doses which were not dose-related or of even of lower incidences at higher doses. They clearly are due to variability and not indicative of tumorigenic or adverse effects on organs and thus would not change the final conclusion.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

adopted in 7th September, 2009

Deviations:

yes

Remarks:

See "Rationale for reliability incl. deficiencies" for more information

GLP compliance:

yes (incl. QA statement)

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

This test system is recognized by the international regulatory agencies as a suitable rodent model for studies of this type.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 4 weeks
- Weight at study initiation: males: 18 - 25 grams, females: 16 - 22 grams.
- Housing: The animals were housed individually in cages with a mire mesh lids and standard granulated softwood bedding.
- Diet: pelleted standard mouse maintenance diet, ad libitum.
- Water: Tap water was available ad libitum in water bottles.
- Acclimation period: 1 week under test conditions, with pretest veterinary examinations.

DETAILS OF FOOD AND WATER QUALITY: The feed batches mere analyzed for contaminants. Tap water was available ad libitum in water bottles. The water was analyzed for chemical contaminants and for bacteriological contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 55±10
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12. Music mas played during the light period.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSES
The test article mas mixed with microgranulated feed in a Buhler mixer, type DDMA-0.5 and pelleted using a Buhler pelleting machine, Type DFPL. Water was added to the mixtures at a ratio of approximately 1:10 (v:w) to aid pelleting. After pelleting, the pellets were dried for approximately 48 hours and then stored in disposable paper bags. The mixtures mere prepared at least twice monthly.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability, homogeneity and concentration of the test article in the feed preparations were determined prior to study initiation and at monthly intervals. The mean concentrations of desmedipham technical in rodent feed was found to be between 95.2 - 97.6% (± 5.4 - 11.1%) and the homogeneity varied mainly in the range of ±15%, except for a few feed batches were the deviations were greater. The stability of desmedipham in rodent diet at room temperature was confirmed to be at least 21 days.

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

continuously

Post exposure period:

All animals were sacrificed and histological examination was done.

Dose / conc.:

0 ppm

Remarks:

Group 1: Control

Dose / conc.:

30 ppm

Remarks:

Group 2: Achieved test material intakes at the feeding levels above were 4.2 mg/kg bw/day for males and 5.8 mg/kg bw/day for females, respectively

Dose / conc.:

150 ppm

Remarks:

Group 3: Achieved test material intakes at the feeding levels above were 21.7 mg/kg bw/day for males and 30.8 mg/kg bw/day for females, respectively

Dose / conc.:

750 ppm

Remarks:

Group 4: Achieved test material intakes at the feeding levels above were 109.0 mg/kg bw/day for males and 145.0 mg/kg bw/day for females, respectively

No. of animals per sex per dose:

60/sex/group

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The dose levels were selected according to the results of a previous 28-day pilot study.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Observations for mortality were recorded twice daily. Observations for clinical signs and symptoms of toxicity were performed twice daily. In addition to the daily observations, each mouse had a weekly detailed clinical examination which included a palpation for tissue masses. A1 description of any lesion or mass observed at any examination was recorded and the subsequent progress monitored.

HEARING TESTS:
- Ten animals from each sex and group with the lowest identification numbers mere tested for hearing impairment using a tone generator (frequency 10 kHz, volume 80 db, duration of 30 msec, the test was repeated five times with 2-second pauses between each test) . The evidence of Preyer's Reflex constituted a positive reaction. The results mere recorded.
The hearing tests were performed at pretest, and at 6, 12, 18, and 24 months of treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded weekly during months 1 to 3 and twice monthly thereafter.

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 / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

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: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Examinations were performed at pretest, at 6, 12, 18, and 24 months of treatment.
- Dose groups that were examined: ten animals from each sex and group with the lowest identification numbers.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at week 52 from interim sacrifice group (10/sex/group) and from all animals at week 80 and at study termination.
- How many animals: interim sacrifice group (10/sex/group) at week 52 and all animals at week 80 and at study termination.
- Parameters: Additional hematology parameters; red cell morphology, methemoglobin (at week 52) and Heinz body (at week 52 and 104).

EXAMINATION OF TEETH AND MUCOUS MEMBRANES:
- Examinations were performed weekly during the first three months and twice monthly thereafter.

Sacrifice and pathology:

TERMINATION AND POSTMORTEM EXAMINATION
Any moribund mouse was anesthetized by intraperitoneal injection of Pentobarbital, exsanguinated and immediately necropsied to reduce the possibility of tissue autolysis.


GROSS PATHOLOGY: Yes
All mice killed in extremis or found dead were subjected to detailed macroscopic examination; where feasible, a full spectrum of tissue samples was preserved for histopathology .

The same applies to all animals killed at 52 weeks and at termination of the study.
Additional blood was collected at necropsy.
Necropsies were performed by experienced prosectors under the supervision of the RCC pathologist.

HISTOPATHOLOGY: Yes
Necropsy of all mice was performed. Tissue specimens were fixed in 4% formalin and embedded in paraffin. The sections were routinely stained with hematoxylin and eosin. Special stains (Schmoerl's stain, methylene blue, and PAS) were applied to selected tissues.

Sections of the following organs/tissues mere examined microscopically (number of sections per organ):
Adrenal glands, aorta, brain (cerebrum, cerebellum, brain stem), bone (sternum or femur), bone marrow
(sternum or femur), epididymides, esophagus, eyes, gallbladder, Harderian glands, heart, kidneys, large intestine (colon, cecum, rectum), liver, lungs (with mainstem bronchi), lymph nodes (mandibular, mesenteric), mammary glands, ovaries, pancreas, pituitary gland, parathyroid glands (wherever possible), prostate, salivary glands, sciatic nerve, seminal vesicles, skeletal muscle, skin, small intestine (duodenum, jejunum, ileum), spinal cord, spleen, stomach, testes, thymus, thyroid gland, trachea, tongue, urinary bladder, uterus, all gross lesions, and all regional lymph nodes of tissue masses.

Histopathologic examinations of the tissues were undertaken in all animals of all groups, including those which died or were killed in extremis.
In addition, all tissue masses and gross lesions were examined.

Statistics:

Body weights, food consumption, organ weights, and clinical laboratory data were evaluated by statistical analysis (Univariate one-way analysis of variance, Dunnett-test, Steel-test (many-one rank test), Fisher’s exact test). The Peto analysis was applied on the tumour data from the animals scheduled for terminal kill (KO) only. Thus the tumour data from the animals scheduled for interim kill (K1) were not included in the statistical analysis.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

During the course of the study 288 mice died or were killed in moribund state (Table 1). Mortality was slightly increased in mice at 30 (+12%) and 150 ppm (+8%) compared to control groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males, body weights were significantly reduced (p<0.05, p<0.01) from week 25 to week 45 of the treatment period at 750 ppm (Figure 1). In females, body weight was significantly (p<0.05, p<0.01) increased from week 1 to week 23 at 750 ppm (Figure 2). Increased body weight (p<0.05) was observed in females at 30 ppm from week 1 to week 57 of the treatment period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was significantly decreased in both sexes from week 3 to week 32 at 750 ppm.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment related findings were noted in the ophthalmoscopic examinations

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Decreased (p<0.05) hemoglobin and hematocrit values were seen in females at 750 ppm after 52 weeks of treatment. A dose related Heinz body formation was observed in both sexes at 52 and 104 weeks of treatment, statistically significantly at 750 ppm (p<0.05). Methemoglobin formation was significantly increased in males at 750 ppm (p<0.05) after 52 weeks of treatment (increase from 1.5% in controls to 6.5% at 750 ppm). Hemoglobin levels and hematocrit were significantly (p<0.05) reduced in 750 ppm females at week 52. Hematocrit was significantly reduced in 30 ppm females only at 104 weeks. White blood cell count was increased at 750 ppm and MCV was increased after 104 weeks in females.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The weights of adrenal glands, brain, gonads (testes/ovaries), heart, kidneys, liver, spleen, thyroid gland and thymus were recorded of all animals necropsied after 12 (10 males and 10 females/group) and 24 months of treatment (13 - 22 males and 9 - 14 females/group).

At 12 months, the most consistent organ weight changes were observed in females; spleen weights (absolute :15% at 30 ppm, 35% at 150 ppm and 73% at 750 ppm and relative: 35-37% at 30 ppm, 40-43% at 150 ppm and 80%-85 at 750 ppm) were dose dependently increased in treatment groups and significantly increased at 750 ppm. Ovarian weights (absolute and relative to brain weight) were significantly increased at 150 ppm (215%-226%) and absolute and relative to brain weight heart weight increased (17% and 19%, respectively). At 24 months, the only statistically significant organ weight change was a significant increase in absolute testes weights at 150 ppm (22%).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Numerous gross lesions were observed in mice that died during the course of study or were sacrificed in schedule. The type, incidence and severity of these gross lesions did not indicate any toxic or oncogenic effects of the test article.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The total incidence of Kupffer cell proliferation in liver was slightly increased in both sexes at 750 ppm. Hepatocellular hypertrophy was increased in males at 750 ppm. In testes, the incidence of spermatocele was higher in all treated males than in the control group (2 – 4 animals, compared to none in controls). Dacryoadenitis was observed in the harderian gland in males at final necropsy at 14.6%, 20.4%, 22.0% and 29.2 % in control, 30, 150, and 750 ppm, respectively.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In males the incidence of hepatocellular tumours in liver (adenomas/carcinomas) was slightly increased in all treated males. Haemangioma/haemangiosarcomas in liver were only seen in a few males at 30 and 750 ppm.

In mammary glands, the incidence of adenocarcinomas was slightly increased in females at 30 and 150 ppm.

In ovaries, the incidence of cysts (serous/haemorrhagic) was increased in a dose dependent manner. The incidence of Theca/granulosa cell tumours and luteomas in ovaries was increased in females at 150 and 750 ppm (Table 4). The incidence of tubular adenomas in ovaries was higher in all treated females than in the control group.

Appropriate statistical evaluations were performed as stated in the report. They were conducted for neoplastic lesions, for which the incidences appeared increased in the treated groups when compared with the controls. The statistical evaluations testing for a positive trend with respect to dose rate were performed according to PETO et al., 1980. Thus, appropriate statistics were applied on the tumor data from the animals scheduled for terminal kill (KO), however, for the discussed ovarian and uterine tumors discussed before, also for the Theca/granulosa cell tumors, no statistical significance is noted.

Leydig cell tumours in testes were observed in one male at 30 ppm and in three males at 150 ppm, but not in the control or high dose males. There were some indications that all observed macroscopical findings in seminal vesicles were not identified in histopathology. For example, one male at 150 ppm had a nodule (15 mm) in left seminal vesicles, but this nodule was not presented for histological examination according to the individual microscopic findings. Another male at 150 ppm had enlarged seminal vesicles at necropsy, but “Tissue not present for microscopic examination” was stated in the individual microscopic findings. Gland.-cyst hyperplasia in the uterus was increased in the group treated with 150 and 750 ppm desmedipham.

Some rare tumours were found at terminal necropsy. Mesothelioma in body cavities was observed in one male at 750 ppm, and malignant neurinoma was recorded in one male and two females at 150 ppm.

The total number of animals with neoplasms was not affected.

Other effects:

no effects observed

Description (incidence and severity):

No treatment related findings were noted in the hearing test and examinations of teeth and mucous membranes.

Details on results:

Statistical evaluation:
Non-neoplastic findings were not analysed by statistical tests. The neoplastic lesions observed in interim kill (K1) were not included in the statistical evaluation (22 neoplastic lesions, 9 of them in the control group). The results of the Peto analysis on tumour data were not presented in the statistical report. Only statistical evaluation on tumours observed in haemolymphoreticular system (lymphomas), lungs, liver, ovaries, Harderian glands, mammary glands and pituitary were described in the pathology report. In these tissues, the Peto test on tumours observed in the animals scheduled for terminal kill (KO) did not yield any positive trend.

Relevance of carcinogenic effects / potential:

It is obvious that none of the tumor incidences in ovaries and uterus were dose-related. They were compared with published HCD for NMRI mice from 18 oncogenicity NMRI mice studies from July 1981 to August 1988 which covers the time of the study.

In the ovaries, the incidence of Theca/granulosa cell tumors was highest at 150 ppm, but not at the highest dose so that no dose response is seen, a statistical test for a positive trend was negative, furthermore they are within published HCD for NMRI mice.

The incidence of ovarian Sertoli cell tumors was higher in control animals than in the treated groups, so that there is no dose- or treatment relationship. The ovarian luteoma incidences are not clearly dose-dependent, and furthermore within the published HCD, so that they are also not treatment-related. The other tumors occurred isolated without any dose-relationship and thus are due to variation.

The incidences of uterine leiomyomas was only increased in the control and low but not at the high dose group, so that a treatment-relationship can be excluded. The incidence of uterine stromal sarcomas did not show a clear dose-response and further was within HCD so that they are not regarded as treatment-related. The hemangioma incidence at 150 ppm was an isolated finding unrelated to treatment.

Note: HCD for ovarian and uterus tumors from the performing laboratory were not supplied, but published data has been provided by applicant from the same mouse strain and where studies were performed close in time to the study. The relevance and reliability is lower for these HCD due to possible confounding factors and lack of detailed knowledge of the studies. Also, no mean value is provided, only a range.

Dose descriptor:

NOAEL

Effect level:

150 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

Remarks on result:

other: approximately 22 – 31 mg/kg bw/day

See Results' figures and tables in the attached file in "Overall remarks, attachments"

Conclusions:

NOAEL :150 ppm, approximately 22 – 31 mg/kg bw/day, based on hematological and related effects occurring at 750 ppm (decreased hemoglobin and hematocrit values in females at 750 ppm after 52 weeks of treatment. Heinz body formation in both sexes at 52 and 104 weeks, increased methemoglobin in males at 750 ppm. Hemoglobin levels and hematocrit were reduced in 750 ppm females at week 52. Increased spleen hematopoiesis.)

Executive summary:

In this 104-week oncogenicity study, DESMEDIPHAM was administered in the feed to NMRI mice. The study was comprised of four groups. Groups of 60 male and 60 female received desmedipham at dose levels of 0, 30, 150, 750 ppm in the diet for 104 weeks. Achieved test material intakes at the feeding levels above were 0, 4.2, 21.7 and 109.0 mg/kg bw/day for males and 0, 5.8, 30.8 and 145.0 mg/kg bw/day for females, respectively.

Treatment-related hematological and related effects occurred at 750 ppm (decreased hemoglobin and hematocrit values in females at 750 ppm after 52 weeks of treatment. Heinz body formation in both sexes at 52 and 104 weeks, increased methemoglobin in males at 750 ppm. Hemoglobin levels and hematocrit were reduced in 750 ppm females at week 52 in addition to increased spleen hematopoiesis.

In conclusion, NOAEL was found to be 150 ppm, approximately 22 – 31 mg/kg bw/day, based on hematological and related effects occurring at 750 ppm.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Prior to 1994-08-19 to 1995-01-04

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

yes

Remarks:

See Principles of method if other than guideline.

Principles of method if other than guideline:

An OECD guideline was not mentioned in the study report but the study mostly complied with OECD Test Guideline 451 at the time of the first assessment.
After the study was performed, a new version of the OECD Test Guideline 451 has been adopted 7th September, 2009. Deficiencies in the study were:
Food conversion efficiency ratios were calculated for each cage of animals for the first 16 weeks only. According to guideline food efficiency should have been measured weekly for 13 weeks and monthly thereafter.
The study protocol did not included microscopical examination of lacrimal gland and coagulating gland.

GLP compliance:

yes (incl. QA statement)

Species:

mouse

Strain:

CD-1

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

not specified

Details on exposure:

Groups of 50 male and 50 female Crl:CD1(ICR) BR VAF plus mice received desmedipham technical (Purity: 97.5%) at dose levels of 0, 400, 1000 and 2500 ppm in diet for 80 weeks. The test material intakes were 0, 60.8, 153.0 and 402.1 mg/kg bw/day for males and 0, 72.0, 178.1 and 501.1 mg/kg bw/day for females, respectively.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of the study diets indicated that the diets were prepared within 17% of the nominal concentrations. The stability of desmedipham in rodent diet was confirmed to last over 14 days.

Duration of treatment / exposure:

80 weeks

Frequency of treatment:

Continuous.

Dose / conc.:

0 ppm

Dose / conc.:

400 ppm

Dose / conc.:

1 000 ppm

Dose / conc.:

2 500 ppm

No. of animals per sex per dose:

50

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were palpated to detect superficial masses weekly from week 27 onwards.

HAEMATOLOGY: Yes / No / No data
- Time schedule for collection of blood: Blood samples for leucocyte differential counts were collected from 10 surviving males and females in each group at week 52 and at study termination.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10

OTHER: All control and high dose mice and all premature decedents underwent histological examination of a full list of organs, and all remaining animals at 400 and 1000 ppm were subjected to histological examination of kidneys, liver, lungs and all gross lesions and tumours or masses suspected of being tumours.

Statistics:

Mortality, clinical sings and palpable masses, body weight gain, food consumption, hematology, organ weight and histopathological data were evaluated by statistical analysis (Student’s test, Levene’s test, ANOVA, Kruskal-Wallis non-parametric, Shirley’s test; non-parametric). Survival and histopathological lesions were tested by Peto fatal/incidental analysis.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related clinical signs according to the study report. Cranial hair loss was, however, clearly increased in males at 2500 ppm. Neither the incidence nor the time of onset of palpable masses appear to be affected by treatment with desmedipham.

Mortality:

mortality observed, treatment-related

Description (incidence):

A total of 66 males and 53 females were found dead or killed in extremis during the study (Table: Number of animals found dead or killed in extremis.).
The presented survival rate (expressed as % of original group size) showed no difference in mortality between control and the test animals at 1000 ppm. Mortality in animals at 400 ppm was 6% higher than in the control group and the survival rate started to fall earlier (in week 57) than in the control groups. Mortality in animals at 2500 ppm was markedly higher (14 - 20%) than in the control group and especially females at 2500 ppm died or were earlier than females in the control group. There was a statistically significant (p<0.05) trend in mortality at 2500 ppm when the sexes were combined. Desmedipham had a slight adverse effect on longevity at 2500 ppm. There was an increase in the incidence of cardiovascular disease as a factor contributing to death, and as the predominant pathology in animals killed at the termination of the study, in males and females from the 2500 ppm dose group, but this finding was considered not to be a direct toxic effect.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain was significantly reduced at study termination in both males and females at 2500 ppm (16% decrease in males and 26% in females) (Table: Body weight males and Table: Body weight females).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was a treatment related increase (5 - 10%) in overall food consumption at 2500 ppm, whilst food conversion efficiency was decreased by 13% in males and 20% in females at 2500 ppm.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food conversion ratios were calculated for cage animals for the first 16 weeks only.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In hematology treatment related leucocyte differential count changes (p<0.01, neutrophilia and lymphocytopenia) were observed in males at 2500 ppm at the study termination. The observed neutrophil and lymphocyte counts are just within the normal background range for mice of this age.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Brain, kidney and liver (also testes in males) were weighed from 10 surviving animals of each sex at necropsy. The absolute and relative liver weights were significantly (p<0.01) increased in both sexes at 2500 ppm (absolute: 68% in males and 29% in females, relative: 87 % in males and 43 % in females) and in males receiving 1000 ppm (15%). The relative liver weights of males receiving 2500 ppm were increased almost by 50%, those of females receiving 2500 ppm by 43% , and males receiving 1000 ppm by 23%. An increase (14 - 20%; p<0.001) in relative kidney weights was observed in all treated males, and in 2500 ppm females (15%; p<0.05).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic observations showing a treatment related increase in incidence were seen in the liver, spleen, heart and thoracic cavity. Abnormal colour, the presence of nodules or masses, abnormal shape, abnormal areas, accentuation of lobular pattern or enlargement were observed in liver, spleen and heart of both sexes mainly at 2500 ppm. Macroscopic changes in the liver were more frequent in males than in females. Accumulation of fluid in the thoracic cavity was seen in 3 and 11 males at 1000 and 2500 ppm, respectively, but not in controls or in males at 400 ppm. This effect was less clear in females; fluid in the thoracic cavity was seen in 2, 2 and 5 females at 0, 400 and 2500 ppm, but not in females at 1000 ppm.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Only liver, lungs, kidneys and ovaries were microscopically examined from all test animals.
Significantly increased incidences of chronic hepatocyte necrosis in the liver were seen in males and females at 1000 and 2500 ppm (Table: Mortality and histopathology). Regenerative hepatocyte hyperplasia, associated with chronic hepatocyte necrosis, was also observed in males at 1000 and 2500 ppm. There was a treatment and dose related reduction in the incidences of focal chronic inflammation and hepatocyte vacuolation in both sexes.
Alveolar macrophage accumulation in lungs was increased in treated males.
In kidneys, increased number of cystic tubules and tubule dilatation was observed in both sexes. One male at 1000 ppm had renal carcinoma.
In heart, the incidence of auricular trombosis and myocardial fibrosis was higher in males at 2500 ppm than in the control group (Table: Histopathological findings in organs and tissues which were not systematically examined from all test animals). Also ventricular trombosis was seen in males at 1000 and 2500 ppm, but not in the control group. In males and females, myocardial fibrosis in heart was increased at 2500 ppm. The increased incidences of histopathological findings in the heart were not statistically significant, but it is noted in the test report that there was an increase in cardiovascular disease as factor contributing to the death of animals which died or were killed in extremis during the study.
Extramedullary hematopoiesis in spleen was increased in both sexes at 2500 ppm.
Increased reactive hyperplasia in submandibular and mesenteric lymph nodes was observed in both sexes at 2500 ppm.
There was a reduction in the incidence of adrenal cortical cortical atrophy in males and of spindle cell hyperplasia in the adrenal cortex of males and females from the 2500 ppm dose group compared with controls (See Table: Cortical atrophy and spindle cell hyperplasia).
According to the study pathologist, this reduction in the incidence of common age-related changes in high dose group animals was probably the result of the reduced survival rate in these animals and was therefore not significant.
Total number of benign tumors seemed to be increased at 2500 ppm but no statistical analysis were performed (Table: Tumour incidence).

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In males, a slight treatment related increase in the incidence of benign hepatocellular tumours was observed. In some males these benign tumours appeared to have developed in close association with the treatment-related chronic hepatocyte necrosis and regenerative hepatocyte hyperplasia. In females, the incidence of hepatocellular adenomas and the overall incidence of hepatocellular tumours was significantly increased at 2500 ppm. For the sexes combined, both the the pairwise difference and the trend were statistically significant (p<0.05).
Benign pulmonary adenomas were increased in a dose related manner in treated females. Pulmonary adenocarcinomas showed no treatment relationship in either sex. The overall incidence of pulmonary tumours was increased in treated females, but the increase was not statistically significant.

Key result

Dose descriptor:

NOAEL

Effect level:

< 400 ppm

Based on:

test mat.

Sex:

male

Remarks on result:

other: < 61mg/kg bw/ day

Key result

Dose descriptor:

NOAEL

Effect level:

< 400 ppm

Based on:

test mat.

Sex:

female

Remarks on result:

other: < 72 mg/kw bw/ day

Key result

Dose descriptor:

LOAEL

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

LOAEL

Effect level:

< 400 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

not specified

Lowest effective dose / conc.:

400 ppm

System:

hepatobiliary

Organ:

liver

Table: Liver results

Organ	Doses (ppm)
	Males				Females
	0	400	1000	2500	0	400	1000	2500
Liver
No. animals examined	50	49	49	50	50	49	48	48
Chronic hepatocyte necrosis	1	6	39***	48***	1		35***	42***
Regenerative hyperplasia			1	4
Carcinoma	8	4	5	6				1
Adenoma	11	14	15	19				3**
Adenoma (%)	22	28	30	38				6

* p ≤ 0.05; ** p ≤ 0.01; ***p≤ 0.001

Empty field: organs examined, but no findings

Table: Overview of HCD.

HCD Liver adenoma (%)	Study duration	Reference
Males
0 – 19.23	18 months	Charles River, 1995
4.08 – 37.5	24 months	Charles River, 1995
Females
0 – 2.00	18 months	Charles River, 1995
0 – 11.27	24 months	Charles River, 1995

 

Conclusions:

The study is acceptable. Increased incidences of chronic hepatocyte necrosis of the liver was observed in both sexes at 1000 and 2500 ppm. Regenerative hyperplasia in the liver, associated with the strikingly increased hepatocyte necrosis, was also observed in males at 1000 and 2500 ppm. Total hepatocellular tumors were significantly increased in females at 2500 ppm, but an increase in males at the high dose was also evident. For the combined incidences of total hepatocellular tumors in both sexes, both pairwise difference and the trend were statistically significant. The changes observed in the liver may be seen as typical effects following chronic toxic injury, accompanied by reparative hepatocyte hyperplasia which can progress to the development of hepatocellular adenomas.

Accumulation of fluid in the thoracic cavity was seen at 1000 and 2500 ppm, and alveolar macrophages were increased in males. The incidences of auricular trombosis and myocardial fibrosis of the heart were increased in 2500 ppm males, and a few cases of ventricular thrombosis were found in 1000 and 2500 ppm males.

Extramedullary hematopoiesis in the spleen was noted in both sexes at 2500 ppm. This histopathological finding is consistent with similar findings noted in subchronic studies in mice, and probably caused by the hematological (red blood cell) effects observed in these subchronic studies which included more complete hematology analyses.

Hepatotoxicity was the primary toxicological effect caused by administration of desmedipham. The histopathological changes observed in the spleen and in the heart may be seen as secondary effects to the marked hepatotoxicity at the high dose. No evidence of primary oncogenic effects were observed. The NOAEL in females is 400 ppm, corresponding to 72 mg/kg bw/day, based on hepatotoxic effects. In males, a slight increase in chronic hepatocyte necrosis was also seen at 400 ppm. The NOAEL for hepatotoxicity in males is therefore considered to be less than 400 ppm (61 mg/kg bw/day).

NOAEL females : 400 ppm (72 mg/kg bw/day) based on hepatotoxic effects (increased incidences of chronic hepatocyte necrosis).
NOAEL males : < 400 ppm (61 mg/kg bw/day) based on hepatotoxicity with slight increase in chronic hepatocyte necrosis also seen at 400 ppm and increased relative kidney weight seen at all doses.

Executive summary:

Groups of 50 male and 50 female Crl:CD1(ICR) BR VAF plus mice received desmedipham technical (Purity: 97.5%) at dose levels of 0, 400, 1000 and 2500 ppm in diet for 80 weeks. The test material intakes were 0, 60.8, 153.0 and 402.1 mg/kg bw/day for males and 0, 72.0, 178.1 and 501.1mg/kg bw/day for females, respectively. Analysis of the study diets indicated that the diets were prepared within ±17% of the nominal concentrations. The stability of desmedipham in rodent diet was confirmed to last over 14 days. All animals were palpated to detect superficial masses weekly from week 27 onwards. Blood samples for leucocyte differential counts were collected from 10 surviving males and females in each group at week 52 and at study termination. Leucocyte differential counts were performed on smears of control and 2500 ppm dose animals on both occasions, and on smears of 400 and 1000 ppm dose animals after 80 weeks. All control and high dose mice and all premature decedents underwent histological examination of a full list of organs, and all remaining animals at 400 and 1000 ppm were subjected to histological examination of kidneys, liver, lungs and all gross lesions and tumours or masses suspected of being tumours.

Increased incidences of chronic hepatocyte necrosis of the liver was observed in both sexes at 1000 and 2500 ppm. Regenerative hyperplasia in the liver, associated with the strikingly increased hepatocyte necrosis, was also observed in males at 1000 and 2500 ppm. Total hepatocellular tumors were significantly increased in females at 2500 ppm, but an increase in males at the high dose was also evident. For the combined incidences of total hepatocellular tumors in both sexes, both pairwise difference and the trend were statistically significant. The changes observed in the liver may be seen as typical effects following chronic toxic injury, accompanied by reparative hepatocyte hyperplasia which can progress to the development of hepatocellular adenomas.

Accumulation of fluid in the thoracic cavity was seen at 1000 and 2500 ppm, and alveolar macrophages were increased in males. The incidences of auricular trombosis and myocardial fibrosis of the heart were increased in 2500 ppm males, and a few cases of ventricular thrombosis were found in 1000 and 2500 ppm males.

Extramedullary hematopoiesis in the spleen was noted in both sexes at 2500 ppm. This histopathological finding is consistent with similar findings noted in subchronic studies in mice, and probably caused by the hematological (red blood cell) effects observed in these subchronic studies which included more complete hematology analyses.

Hepatotoxicity was the primary toxicological effect caused by administration of desmedipham. The histopathological changes observed in the spleen and in the heart may be seen as secondary effects to the marked hepatotoxicity at the high dose. No evidence of primary oncogenic effects were observed. The NOAEL in females is 400 ppm, corresponding to 72 mg/kg bw/day, based on hepatotoxic effects. In males, a slight increase in chronic hepatocyte necrosis was also seen at 400 ppm. The NOAEL for hepatotoxicity in males is therefore considered to be less than 400 ppm (61 mg/kg bw/day).

NOAEL females : 400 ppm (72 mg/kg bw/day) based on hepatotoxic effects (increased incidences of chronic hepatocyte necrosis).
NOAEL males : < 400 ppm (61 mg/kg bw/day) based on hepatotoxicity with slight increase in chronic hepatocyte necrosis also seen at 400 ppm and increased relative kidney weight seen at all doses.

Reference 3

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1989-04-12 to 1992-07-08

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EPA OPP 83-5 (Combined Chronic Toxicity / Carcinogenicity)

Deviations:

yes

Remarks:

See Principles of method if other than guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

See Principles of method if other than guideline.

Principles of method if other than guideline:

According to study report the guideline used was US EPA Pesticide Assessment Guidelines Subdivision F, Series 83-5 corresponding to OECD Test Guideline 453. After the study was performed, a new version of the OECD Test Guideline 453 has been adopted 7th September, 2009. Deficiencies in the study were: The study protocol did not include microscopical examination of seminal vesicles, vagina, submandibular lymph node, tongue and nasal cavity, lacrimal gland, Harderian gland, cervix, coagulating gland and gall bladder.
According to the OECD guideline, haematology, biochemistry and urine examinations should be carried out on all study animals (10 male and 10 female animals per group) at 3, 6, and 12 months, as well as at study termination (if longer than 12 months). In the study blood samples were taken after approximately 79 and 104 weeks in 10 males and 10 females.
Haematology: Prothrombin time, and activated partial thromboplastin time was not investigated. Animals were checked “at least” once a week for tumor development instead of once a day. Biochemistry: animals not fasted over night. Organ weight: Thyroid was not weighed. Ophthalmoscopy was not carried out before dosing.

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Dose / conc.:

0 ppm

Dose / conc.:

100 ppm

Dose / conc.:

400 ppm

Dose / conc.:

1 200 ppm

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Dose range finding study.

Observations and examinations performed and frequency:

Ophthalmoscopy was carried out on all animals in control and high dose groups during week 104 of the study. Samples for hematology, clinical chemistry (extra determinations; RBC, plasma, brain cholinesterase) and urinanalysis were taken from 10 males and 10 females of each group after approximately 79 and 104 weeks of treatment. Differential blood cell counts were performed on all surviving animals after approximately 53, 78 and 102 weeks of dosing. Organ weights were determined from the same 10 males and 10 females used for laboratory investigations on week 104. All control and high dose rats and all premature decedents underwent histological examination including all recommended organs with the exception of nasal cavity, seminal vesicles, submandibular lymph nodes, tongue and vagina.

Statistics:

Hematology, clinical chemistry, organ weight and body weight data were evaluated by statistical analysis (F-max test, parametric ANOVA, Student’s t-Test, Fisher’s F-protected LSD, Kruskal-Wallis ANOVA). Mortality data were presented graphically using Kaplan- Meier survival curves and pairwise comparisons made using Wilcoxon rank sum test modified for censored survival data. Comparison of histology lesion incidences was made using Fisher’s Exact Probability test.

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The red blood cell count was significantly decreased in males at 1200 ppm at 104 weeks (Table: Biochemistry). In clinical chemistry parameters, the albumin/globulin-ratio was significantly reduced in females at 400 (10% ) and 1200 ppm (12 %) during week 79 and total bilirubin was increased in females at 400 (50%) and 1200 ppm (71 %) during week 79. Lymphocytes were increased in both sexes at 1200 ppm. Reticulocytes had not been counted. There were no notable intergroup differences in differential blood cell counts in samples taken from all surviving animals of the main study. In week 79 MHC was significantly increased in high dose females (4%) but not in other treated groups.

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The incidence of Kupffer cell/macrophage pigmentation in liver was significntly increased in males at 1200 ppm (Table: Histopathological findings in organs examined from all test animals). Further investigation in a representative sample of these animals showed the pigment to stain positively for haemosiderin. In liver, the incidence of bile duct hyperplasia was slightly higher in males at 1200 ppm (40%) than in the control group (30%), as were the incidences of hepatocellular adenoma and carcinomas in the liver.
In lungs, alveolar macrophages were significantly increased in both sexes at 1200 ppm and at 400 ppm in males (Table: Histopathological findings in organs examined from all test animals). This compound related effect was considered to correlate with the macroscopic finding of pale foci in the lungs of both sexes at 1200 ppm. Inflammations and interstitial pneumonitis in lungs were predominantly seen in males at 400 and 1200 ppm.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Infiltration by mesothelioma cells in lungs and in aorta and mesothelioma in thorax (in pleura) was observed in one male at 100 ppm. This tumour is rarely seen in rats. In females a decreased incidence of enlarged adrenals was observed: 17, 8, 10 and 6 out of 50 was seen in control, 100 ppm, 400 ppm and 1200 ppm, respectively. The incidence of tubular pigmentation in kidneys was significantly increased in males at 1200 ppm. Further investigation in a representative sample of these animals showed the pigment to stain positively for haemosiderin. Tubular adenoma in kidneys was observed in one male at 1200 ppm. The incidence of urothelial hyperplasia was significantly increased in 1200 ppm females.
In spleen, the incidence of hemosiderin deposition was significantly increased in both sexes at 1200 ppm (Table: Histopathological findings in organs not systematically examined from all animals). In females, a few cases of follicular adenoma of thyroids was observed at 400 and 1200 ppm, compared to no recorded cases in the control group. In thymus, the incidence of epithelial hyperplasia was slightly, but not significantly, increased in females at 1200 ppm. The incidence of cystic ducts was also slightly higher in females at 1200 ppm. Interstitial-cell adenoma in testes was slightly, but not significantly, increased at 1200 ppm. Overall incidence of tumors did not seem to be affected in a dose dependent way but no statistical analysis were performed (Table: Tumours).

Key result

Dose descriptor:

LOAEL

Effect level:

400 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

Key result

Dose descriptor:

NOAEL

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Key result

Critical effects observed:

not specified

Lowest effective dose / conc.:

100 ppm

Organ:

kidney

liver

spleen

Conclusions:

Hematological effects were seen mainly at 1200 ppm, as decreased red blood cell counts in males and increased bilirubin in females. Increased bilirubin was also observed in 400 ppm females. Pigmentation of the liver (Kupffer cells), spleen and liver was observed at 1200 ppm, mostly in both sexes. The observed pigmentation was at least in the case of the spleen, but probably also in liver and kidney, caused by deposition of hemosiderin, and related with effects of hemolytic anemia observed at 1200 ppm. Urothelial hyperplasia of the kidneys in females was increased at 1200 ppm. Increased alveolar macrophages were observed at 400 and 1200 ppm in males and 1200 in females. The systemic NOAEL was 100 ppm, equivalent to 5.4 and 6.9 mg/kg bw/day in males and females, respectively.
NOAEL : 100 ppm, equivalent to 5.4 and 6.9 mg/kg bw/day in males and females, respectively based on hematological effects; decreased RBC parameters, histopathological effects in spleen, liver and kidneys (pigmentation), urothelial hyperplasia in kidneys.

Executive summary:

Groups of 50 male and 50 female Sprague-Dawley rats were dosed daily for 104 weeks with Desmedipham via the diet at dose levels of 0, 100, 400 or 1200 ppm. The study was conducted in the same room as a concurrent 52 week toxicity study on Desmedipham and was performed according to US EPA Pesticide Assessment Guidelines, Subdivision F, Series 83-5. This report contains the results of the 104 week carcinogenicity animals only.

Ophthalmoscopy was carried out on all animals in the Control and High dose groups during Week 104 of the study. After approximately 77 and 103 weeks a nominal 10 males and 10 females were subjected to investigations of haematology, clinical chemistry and urinalysis parameters. After approximately 53, 77 and 102 weeks of dosing differential blood counts were performed on all surviving animals.

At the end of 104 weeks treatment, all surviving carcinogenicity animals were killed and subjected to necropsy. Selected organs were weighed from the same animals used for laboratory investigations. All Control and High dose rats, as well as all premature decedents, underwent histological examination of a full list of organs. All other animals in the Low and Intermediate dose groups underwent a histological evaluation of kidneys, liver and lungs.

Dosing Sprague-Dawley rats for 104 weeks orally via the diet with Desmedipham showed a slight, equivocal reduction in body weight gain in females and slight, equivocal reductions in RBC parameters in males and females at 1200 ppm.

Increased haemosiderin deposition was noted in the liver and kidney of males and in the spleen of males and females at 1200 ppm. The incidence of this finding was also higher in the spleen of females at 400 ppm.

A higher incidence of foci of increased alveolar macrophages was noted in the lungs of males at 400 and 1200 ppm. and females at 1200 ppm. This finding was considered to correlate with the macroscopic finding of pale foci in the lungs, which were higher in incidence in High dose males and females at 1200 ppm.

Mild changes in clinical chemistry parameters were seen in females at 400 and 1200 ppm.

Males and females receiving 100 ppm showed no effects considered to be of toxicological significance. There was no evidence of carcinogenic effect at any dose level. The no-effect level is therefore considered to be 100 ppm.

Reference 4

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1983-04-27 to 1986-09-25

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

assessment report

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

See Principles of method if other than guideline.

Principles of method if other than guideline:

An OECD guideline was not specified in the study report but the study was in compliance with OECD guideline 453 at the time of the previous evaluation with the above mentioned deficiencies. After the study was performed, a new version of the OECD Test Guideline 453 has been adopted 7th September, 2009. Deficiencies in the study were: Deficiencies in histopathological analyses and statistical analyses of the histopathological data, has been dealt with in the following position papers. Animals were palpated once weekly and not once a day. Food conversion efficiency or water consumption were not recorded. Organ weights that were not recorded : epididymides and uterus. The study protocol did not include microscopical examination of vagina, lacrimal gland, cervix, coagulating gland and gall bladder.

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Groups of 70 male and 70 female Wistar KFM-Han, SPF quality outbred rats received desmedipham technical (Purity: 97.8 - 98.2%) at dose levels of 0, 60, 300 and 1500 ppm in the diet for 106 weeks.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

The calculated nominal dosage levels were 0, 3.18, 15.71 and 79.90 mg/kg bw/day for males and 0, 3.86, 19.84 and 100.46 mg/kg bw/day for females, respectively. The mean concentrations of desmedipham technical in rodent feed were between 94.6 - 96.5%, and the homogeneity was ±15%. The stability of desmedipham in rodent diet was confirmed to be at least 21 days.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

106 weeks

Dose / conc.:

0 ppm

Dose / conc.:

60 ppm

Dose / conc.:

300 ppm

Dose / conc.:

1 500 ppm

No. of animals per sex per dose:

There were 20 animals/sex/group in the chronic toxicity study and 50 animals/sex/group in the oncogenicity study.

Control animals:

yes, concurrent no treatment

Details on study design:

- Toxicokinetic data: not mentioned.
- Dose selection rationale: based on range finding study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly.

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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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 AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples for hematology and clinical biochemistry and urine samples were collected from all animals of each sex and group after 3, 6, 12, 18 and 24 months.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples for hematology and clinical biochemistry and urine samples were collected from all animals of each sex and group after 3, 6, 12, 18 and 24 months.
- Animals fasted: No data
- How many animals: All.

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

OTHER: Analyses of red cell morphology, methemoglobin levels and Heinz body determination were included in hematological parameters, and thyroid hormones (T3, T4) were measured after 12 and 24 months of treatment. 10 rats per sex and group were tested for hearing impairment and ophthalmoscopic changes.
Histopathological examination of a full list of organs was performed on all control and high dose rats including premature decedents. The animals at 60 and 300 ppm were subjected to histological examination of the liver, lungs, kidneys, spleen, thyroid gland and all macroscopic abnormalities. Organ weights of adrenal glands, brain, gonads (testes/ovaries), heart, kidneys, liver, spleen, thyroid glands were recorded for all animals at scheduled necropsy

Sacrifice and pathology:

10 rats per sex and group were killed after 52 weeks (interim sacrifice) and the rest were killed after a treatment period of at least 2 years.

Statistics:

Body weights, food consumption, organ weights and clinical laboratory data were evaluated by statistical analysis (Univariate one-way analysis of variance, Dunnett-test, Steel-test, Fisher’s exact test). Spontaneous mortality was analysed by Fisher’s exact test for 2x2 tables. Histopathological findings were not statistically analysed. In the statistical report, only significant results remaining after adjustment for multiple testing were presented in hematology, clinical biochemistry, urinanalysis and organ weights and organ weight ratios.

Mortality:

mortality observed, treatment-related

Description (incidence):

During the course of the oncogenicity study, 145 rats (77 males, 68 females) died or were killed in a moribund state (Table: Mortality during the 2 year chronic toxicity / oncogenicity study with desmedipham technical). There was no difference in the total mortality of the animals between groups. However, there was a difference in survival rate between males at 1500 ppm and the control group in the oncogenicity study. It seemed that males at 1500 ppm died somewhat earlier than the control males, 20% of the males were dead by week 80 at 1500 ppm, and by week 95 in the control group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weight gain was slightly but significantly reduced in chronic toxicity and oncogenicity group animals at 1500 ppm. At study termination the mean body weights were 4% lower in males and 13% in females at 1500 ppm, compared to controls, in animals of the oncogenicity study part. In the chronic study from week 57 to week 93 body weight was significantly decreased in males (up to approximately 15%).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the oncogenicity study, food consumption was reduced at some time in both sexes at 60, 300 and 1500 ppm during the treatment period. Significantly reduced food consumption was sometimes observed in all treated males between week 1 and week 70 and in all treated females between week 19 and week 70. In the chronic toxicity study, significantly reduced food consumption was seen in animals at 300 and 1500 ppm between weeks 1 and 84. Overall, the mean food consumption of the rats was generally comparable between the groups.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Significant decreases were observed in erythrocyte count in males at 60 ppm and 1500 ppm, and in females at 1500 ppm. Hemoglobin concentrations were decreased significantly at 60 ppm and 1500 ppm in males and at 300 and 1500 ppm in females. Hematocrit values were decreased in males at 300 ppm, and in females at 1500 ppm (Table: Changes in red blood cells after 24 months). Mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV) was significantly decreased in 60 and 300 ppm females, but increased at 1500 ppm. Methemoglobin was significantly increased at 300 and 1500 ppm in both sexes, with a maximum of 4.6% in males at the high dose. Methemoglobin was increased at all doses in males at 3 months and in females at 6 and 12 months. Reticulocytes and Heinz bodies were increased in both sexes at 1500 ppm. Polychromatophilia, anicytosis, poikilosytosis and increased numbers of nucleated erythrocytes were noted in both sexes at 1500 ppm after 3 and/or 6 months.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Total bilirubin was increased at most intervals in both sexes at 1500 ppm, the difference was significant in males at 12 months, and in females at 12 and 24 months. Direct bilirubin was significantly increased only in males at 1500 ppm after 3 months.
In males, alpha-1-globulin (rel/abs) was significantly reduced in males at 1500 ppm after 3 and 6 months and in all treated males after 12 months, but not at study termination after 24 months. At 6 months, alpha- 2-globulin (abs) was significantly reduced in males at 300 and 1500 ppm. Albumin to globulin ratio (A/G) was significantly increased in males at 1500 ppm after 3, 6 and 24 months. In females, alpha-1-globulin (rel/abs) was significantly reduced at 300 and 1500 ppm after 3,12 and 18 months. In all treated females, alpha-2-globulin (abs) was significantly reduced at 18 months. The A/G ratio was significantly reduced in females at 1500 ppm after 18 months. Significantly decreased blood levels of T4 were observed in 1500 ppm males and 300 and 1500 ppm females at 12 months, and all treated female groups at 24 months. T3 was decreased in females at 12 months at 300 and 1500 ppm. A summary of the main changes in clinical biochemistry is given in Table: Clinical biochemistry male to Table: Clinical biochemistry females.
The blood level of phosphorus (unreduced phosphomolybdate complex without deproteinization) was reduced significantly in males at 300 and 1500 ppm after 6 months and after 24 months in males at 1500 ppm. In females, phosphorus was reduced in all treated groups at 6 months, but not after that.

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weights were recorded on all animals at scheduled necropsy. Organ weights of animals found dead or killed prematurely were not recorded and the number of animals whose organs were weighed varied in groups. At 12 months, absolute spleen weights were significantly increased in males at 1500 ppm, and relative (to body weight) spleen weights were increased in both sexes at 1500 ppm. At 24 months, absolute and relative spleen weights were significantly increased in 1500 ppm males and 60 and 1500 ppm females. Absolute and relative (to brain) liver weights in females were significantly increased at 300 ppm. Liver weights relative to body weight were significantly increased (10 – 15%) in females at 60, 300 and 1500 ppm. Summary of main changes in organ weights are summarized in Table: Absolute organ weights at 12 months; Table: Organ to body weight ratios at 12 months; Table: Organ to brain weight ratios at 12 months; Table: Absolute organ weights at 24 months; Table: Organ to body weight ratios at 24 months; Table: Organ to brain weight ratios at 24 months. Liver weight relative to body weight was increased in females (10-15%). Hemosiderosis of the liver was increased in 1500 ppm males.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In thyroid glands, follicular hyperplasia was increased in 300 and 1500 ppm males and 1500 ppm females (Thyroid hormones in blood and histopathological findings in thyroid glands). C-cell hyperplasia was increased in males at 300 and 1500 ppm. In parathyroid glands, the incidence of hyperplasia was increased in males at 1500 ppm.
In liver (Histopathological changes in the liver and in the spleen), the incidence of Kupffer cell proliferation was higher in all treated groups than in the control group. Lymphoid cell infiltration in the liver was high in all males and slightly increased in treated males. In females, lymphoid cell infiltration was increased at 1500 ppm (these effects were not seen during the re-evaluation , Jackson et al 2000). The incidence of hematopoiesis in the liver was increased in females at 1500 ppm (please see Jackson et al, 2000 below).
Increased extramedullary hematopoiesis and hemosiderosis in the spleen was observed in all animals. Marked hematopoiesis was increased in both sexes at 1500 ppm, compared to controls.
In lungs, perivascular cuffing (lymphoid cell infiltration) and macrophage accumulation was increased in all treated males and in 1500 ppm females (Histopathological findings in lungs and kidneys). Accumulation of macrophages and pulmonal inflammations were generally increased in rats at 1500 ppm. In kidneys, tubular dilatation was increased mainly in high dose males, and tubular pigmentation was increased in females at 1500 ppm (Histopathological findings in lungs and kidneys) (not observed in re-evaluation). Lymphoid cell infiltration in kidneys was increased in all treated females and the incidence was higher in all treated males than in the control group (not observed in re-evaluation). One male at 1500 ppm had adenoma in kidneys.
The incidence of medullary cysts in thymus was increased in both sexes at 1500 ppm (Histopathological findings in the hemolymphoreticular system) (not observed in re-evaluation). Thymus, one of the main organs in haemolymphoreticular system was poorly examined in this chronic/oncogenicity study. For example, in males at 1500 ppm 20% of thymuses (48/60) at terminal necropsy were not examined histologically. The code “tissue not present/not sufficiently cut” after thymus was recorded several times in individual animal data without any additional information.
In the liver, lungs (perivascular cuffing) and kidneys, lymphoid cell infiltration was higher in treated rats, specially in males. In males, increased lymphoid cell infiltration was also observed in trachea, stomach, and urinary bladder, predominantly at 1500 ppm. In females, increased lymphoid cell infiltration was also observed in pancreas, salivary glands and Harderian glands. Inflammations in the above mentioned organs were predominantly observed in rats at 1500 ppm. For example trachitis in trachea was observed in treated rats, but not in the control groups.
Increased hematopoiesis in the adrenal cortex was observed in 2/60 males at 1500 ppm (3.3%) and in 2/21 females at 60 and in 2/17 females at 300 ppm, but not in the control group (Histopathological findings in the endocrine system and mammary glands) (please see Jackson et al, 2000). The incidence of cellular alteration in adrenal cortex was higher in both sexes at 1500 ppm than in the control group. Cellular alteration in adrenal medulla was not described in more detail in the study report.
The incidence of acinar atrophy in pancreas was higher in both sexes, especially in females at 1500 ppm than in the control group (Histopathological findings in the endocrine system and mammary glands).
Aspermia in epididymides and alveolar atrophy of seminal vesicles was slightly increased in males at 1500 ppm. One male at 60 ppm had carcinoma in seminal vesicles (Histopathological findings in the male genital tract).

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Follicular adenoma/carcinoma was seen in a few females at 60, 300 and 1500 ppm, but not in the control group. Haemangioma was observed in one male at 300 and 2 at 1500 ppm and haemangiosarcoma in one male at 60 and 1500 ppm (not seen after re-evaluation), but not in the control groups or in any of the females.
Malignant lymphomas/lymphoma cells in haemolymphoreticular system were occasionally observed in the spleen, thymus, lymph nodes, liver, kidneys, bone and gastrointestinal and genital tract. According to the used classification, malignant lymphomas were counted as single neoplasias even when they were widely spread throughout the body (multicentric tumors). In males, the total incidences of malignant lymphomas were 1% and 3% in the control and high dose groups (Histopathological findings in the hemolymphoreticular system). In females, the total number of lymphomas was highest (6/21) in females at 60 ppm. The major part of the examined females at 60 ppm had died prematurely (17/21). Only few lymphomas were found after histopat re-evaluation.
According to the individual animal data, lymphoma combination, lymphoma with leukemic cells or lymphoma/leukemia in haemolymphoreticular system was observed in one male at 60 ppm, in one male at 1500 ppm and in one female at 60 ppm, but not in the control group. Leukemic cells were observed in lungs, adrenal cortex, liver, kidneys or heart. Other tumours, lymphangiomas, haemangiomas and haemangiosarcomas in haemolymphoreticular system were noted mainly in mesentery lymph nodes and spleen.
In the first evaluation, islet cell hyperplasia was observed in one male both at 60 and 300 ppm. The incidence of islet cell adenoma in pancreas was higher in males at 1500 ppm (5.0%) than in the control group (1.8%).
In interim kill after 52 weeks, adenomas in the pituitary were predominantly seen in males (3/10) at 1500 ppm and less in the control males (1/10). At study termination after 104 week, the incidence of pituitary adenomas in animals at interim + terminal was higher in males at 1500 ppm ( 40%) than in the control group (32.8%). Almost all females found dead or killed in moribund at 60 and 300 ppm had pituitary adenoma.
It can be seen that the pituitary tumor incidences at termination do not follow a clear dose- response relationship, no dose-related incidence increase was seen in females and in males only a slight increase in the incidence at 1500 ppm was seen. In the other long-term rat studies no effect on pituitary tumor incidences were seen. The total incidences for this benign tumor are 32.8 % in controls and 40 % in the highest dose group. HCD from the same rat strain (Wistar Hannover) and the same laboratory which conducted this study are available.
The incidence of pituitary adenoma in the high dose males is within the range of HCD.
Adenomas and adenocarcinomas in mammary glands were only observed in treated females. Fibroadenomas were noted in 5/58, 9/27, 14/28 and 5/60 at 0, 60, 300 and 1500 ppm, respectively. Thus the number of fibroadenomas in mammary gland was markedly higher in females at 60 and 300 ppm than in the control or high dose groups, although all animals were not evaluated in those groups. However after the re-evaluation of the material, it was concluded that the tumours were within historical control (see Jackson et al, 2000 for full evaluation).
In ovaries, cysts were increased in females at 1500 ppm. Stromal hyperplasia in ovaries was slightly increased in females at 1500 ppm (see Histopathological changes in the female genital tract). Result of the re-examination (data from Jackson et al (2000) study) can be seen in the table below for neoplasic findings in ovaries and uterus. The re-evaluation resulted in adjusted values but did not change the overall conclusion (see also Jackson et al, 2000 in the Table: Histopathological findings in the female genital tract (updated according to Jackson et al, 2000).
In uterus, squamous hyperplasia was higher in females at 1500 ppm than in the control group. Squamous carcinoma was noted in one female at 1500 ppm and a few animals with schwannomas were observed in all treated female groups, but not in the control group. Invasive tumours in adrenal medulla were observed in one female at 60 and 300 ppm, only. In males, pheochromocytoma in adrenal medulla was mainly observed in males at 60, 300 ppm and 1500 ppm, respectively. There seemed to be increased incidence of number of animals with neoplasm and benign tumors in males at 1500 ppm (see table Total tumor incidence). No statistical analysis were performed on these data.

Relevance of carcinogenic effects / potential:

There was no evidence of carcinogenicity in this study.

Key result

Dose descriptor:

NOAEL

Effect level:

60 ppm

Based on:

test mat.

Sex:

male/female

Key result

Dose descriptor:

LOAEL

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

histopathology: neoplastic

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Key result

Critical effects observed:

not specified

Lowest effective dose / conc.:

60 ppm

System:

cardiovascular

Organ:

blood

Table: Mortality during the 2 year chronic toxicity / oncogenicity study with desmedipham technical

End-point		Males ( ppm) 0           60		300	1500	Females (ppm) 0           60          300			1500
Mortality
Total number of animals		70	70	70	70	70	70	70	70
Chronic toxicity		20	20	20	20	20	20	20	20
Oncogenicity		50	50	50	50	50	50	50	50
Interim sacrifice at week 52		10	10	10	10	10	10	10	10
Number of dead animals
Interim sacrifice group		1/10	0/10	0/10	1/10	2/10	0/10	0/10	1/10
Chronic toxicity		2/10	4/10	3/10	3/10	2/10	5/10	0/10	5/10
Oncogenicity		13/50	16/50	14/50	19/50	17/50	12/50	15/50	8/50
(%)		(26%)	(32%)	( 28%)	(38%)	(34%)	(24%)	(30%)	(16%)
Total                Mortality	in	15/60	20/60	17/60	22/60	19/60	17/60	15/60	13/60
chronic/oncogenicity study (%)		(25%)	(33%)	(28%)	(37%)	(32%)	(28%)	(25%)	(22%)

Table 151 Histopathological findings in lungs and kidneys

End-point	Males ( ppm) 0              60		300	1500	Females (ppm) 0               60		300	1500
Lungs Macrophage accumulation interim	0/10	0/10	1/10	3/10	1/10	1/10	0/10	1/10

 

terminal	11/60	12/60	17/60	24/60	13/60	11/60	12/60	28/60
total (%)	(16%)	(17%)	(26%)	(38%)	(20%)	(17%)	(17%)	(41%)
Perivascular cuffing            interim	1/10	0/10	0/10	3/10	0/10	1/10	1/10	0/10
terminal	9/60	14/60	16/60	14/60	9/60	7/60	11/60	18/60
total (%)	(14%)	(20%)	(23%)	(24%)	(13%)	(11%)	(17%)	(26%)
Pulmonal inflammation^1)    interim	1/10	1/10	1/10	1/10	0/10	0/10	0/10	0/10
terminal	8/60	9/60	5/60	19/60	11/60	7/60	9/60	15/60
total (%)	(13%)	(14%)	(8%)	(28%)	(16%)	(10%)	(13%)	(21%)
Kidney
Tubular pigmentation         interim	0/10	0/10	0/10	1/10	0/10	0/10	1/10	3/10
terminal	1/60	0/60	0/60	2/60	15/60	19/60	10/60	30/60
total (%)					(21%)	(27%)	(16%)	(47%)
Tubular dilatation               interim	2/10	3/10	7/10	2/10	1/10	3/10	0/10	2/10
terminal	7/60	11/60	8/60	17/60	30/60	27/60	31/60	22/60
total (%)	(13%)	(20%)	(21%)	(27%)
Lymphoid cell infiltration, interim	3/10	10/10	6/10	9/10	4/10	2/10	2/10	4/10
terminal	7/60	15/60	10/60	19/60	39/60	31/60	35/60	35/60
total (%)	(14%)	(35%)	(23%)	(40%)	(61%)	(47%)	(53%)	(56%)
Adenoma				1

1) Bronchitis/pneumonitis/bronchopneumonia/interst.pneumonia/pleuritis Empty place = organ examined, no findings

Table: Updated histopathological findings in lungs and kidneys ( see Jackson et al, 2000)

Endpoint	Dose (ppm)
	Males				Females
	0	60	300	1500	0	60	300	1500
Lungs
Focus(i)           of          alveolar macrophages	12	9	7	18	5	3	3	12
Interim	0/10	0/10	1/10	3/10	2/10	1/10	0/10	1/10
Terminal	17/60	12/60	10/60	30/60	16/60	13/60	14/60	29/60
Total (%)	17/70	12/70	11/70	33/70	18/70	14/70	14/70	30/70
Perivascular cuffing	0/70	0/70	0/70	0/70	0/70	0/70	0/70	0/70
Pulmonal inflammation (incl. acute           bronchopneumonia, pneumonitis)
Interim	1/10	1/10	0/10	1/10	0/10	1/10	0/10	0/10
Terminal	0/60	1/60	1/60	0/60	11/60	5/60	7/60	11/60
Kidney
Tubular pigmentation	0/70	0/70	0/70	0/70	0/70	0/70	0/70	0/70
Tubular dilatation	0/70	0/70	0/70	0/70	0/70	0/70	0/70	0/70
Lymphoid cell infiltration	0/70	0/70	0/70	0/70	0/70	0/70	0/70	0/70
Inflammatory  cells  in  renal pelvis
Interim	2/10	0/10	0/10	0/10	0/10	0/10	0/10	0/10
Terminal	4/60	3/60	8/60	6/60	0/60	0/60	0/60	0/60
Adenoma (b)	1/70	0/70	0/70	0/70	0/70	0/70	0/70	0/70

(b) = benign, (m) = malignant

Table 152 Histopathological findings in the hemolymphoreticular system

End-point	Males ( ppm) 0            60		300	1500	Females (ppm) 0             60		300	1500
Thymus
Total number of examined	57/70	16/70	12/70	54/70	55/70	17/70	14/70	53/70
Medullary cysts                   interim	0/8	-	-	1/6	3/8	-	0/1	3/8
terminal	4/49	2/16	1/12	8/48	15/47	6/17	7/13	25/45
total (%)	4/57	2/16	1/12	9/54	18/55	6/17	7/14	28/53
	(7%)			(17%)	(33%)			(53%)
Epithelial hyperplasia          terminal	1/49							3/45
Thymoma	1/49			2/48
	(2%)			(4%)
Bone marrow Myeloid hyperplasia           terminal	0/59	1 /21	5/17	2/60	2/58	1/16	2/15	2/59
Mesenteric lymph node
Hemosiderosis                   terminal	0/58	0/21	0/16	1/59	0/58	0/18	0/13	0/60
Haemangioma	4/58	2/21	0/16	1/59	1/58	1/18	0/13	0/60
Haemangiosarcoma	2/58	1/21	1/16	0/59	1/58	0/18	1/13	1/60
Lymphangioma		1/21		1/59				1/60
Mandibular lymph node
Hematopoiesis                   terminal	0/60	0/18	0/15	1/59	0/59	0/15	0/14	0/59
Hemosiderosis                   terminal	0/60	0/18	0/15	1/59	0/59	0/15	0/14	0/59
Lymphoid hyperplasia         terminal	26/60	10/18	8/15	21/59	20/59	4/15	8/14	20/59

 

Haemolymphoreticular system
Malignant Lymphoma     interim	0/10	-	-	1/10	0/10	-	0/1	0/10
terminal	1/60	1 /21	0/17	1/60	1/60	6/21	1/16	1/60
total (%)	(1%)			(3%)	(1%)	6/21	1/17	(1%)
Malignant histiocytoma		2/21

Empty place = organ examined, no findings

Table: Updated histopathological findings in the hemolymphoreticular system after re-evaluation (Jackson et al, 2000)

Endpoint	Dose (ppm)
	Males				Females
	0	60	300	1500	0	60	300	1500
Thymus
Medullary cysts	0/70	0/70	0/70	0/70	0/70	0/70	0/70	0/70
Epithelial hyperplasia	0/70	0/70	0/70	0/70	0/70	0/70	0/70	0/70
Thymoma (b)	1/70	1/70	1/70	1/70	0/70	5/70	3/70	1/70
Thymoma (m)	1/70	0/70	0/70	0/70	0/70	0/70	0/70	0/70
Bone marrow
Myeloid hyperplasia	0/70	0/70	0/70	0/70	0/70	0/70	0/70	0/70
Lymph node, mesenteric
Sinusoidal  erythrocytes  and erythrophagocytosis	1/70	0/70	0/70	0/70	0/70	2/70	2/70	0/70
Haemangioma (b)
Interim	0/10	0/10	1/10	0/10	0/10	0/10	0/10	0/10
Terminal	6/60	4/60	3/60	3/60	1/60	3/60	0/60	2/60
Haemangiosarcoma (m)	1/70	1/70	1/70	0/70	1/70	1/70	1/70	0/70
Mandibular        node        (no findings)
Haemolymphoreticular system
Malignant lymphoma (m)	0/70	1/70	0/70	1/70	0/70	1/70	0/70	0/70
Histiocytic sarcoma (m)	0/70	2/70	0/70	0/70	0/70	0/70	0/70	0/70

(b) = benign, (m) = malignant

Table 153 Histopathological findings in the endocrine system and mammary glands

End-point	Males ( ppm) 0            60          300			1500	Females (ppm) 0             60		300	1500
Adrenal cortex
Cellular alteration              interim	3/10	-	-	1/10	4/10	-	0/1	2/10
terminal	29/60	14/24	11/18	41/60	23/60	8/21	7/17	42/60
total (%)	(45%)			(60%)	(38%)			(63%)
Hematopoiesis				2/60	0/60	2/21	2/17	0/60
Invasive tumour					0/60	1/21	1/17	0/60
Adrenal medulla
Pheochromocytoma (%)	2/60	4/25	3/19	3/60	1/58	2/20	0/16	0/60
	(3%)			(5%)
Pancreas
Acinar atrophy                 interim	0/10	0/10	0/10	1/10	1/10	0/10	0/10	1/10
terminal	4/57	1/20	1/17	6/60	1/59	0/17	1/15	9/60
total (%)	(6%)			(10%)	(1%)			(14%)
Islet cell hyperplasia         terminal	0/57	1/ 20	1/17	0/60	0/59	0/17	0/15	0/59
Islet-cell adenoma (%)	1/57	0/20	1/17	3/60
	(2%)			(5%)
Pituitary
Adenoma                        interim	1/10	-	-	4/10	1/10	-	0/1	0/10
terminal	21/58	15/27	20/24	27/59	42/59	33/37	36/39	41/60
total (%)	(32%)			(45%)
Mammary gland
Alveolar hyperplasia	0/60	0/20	2/17	6/60	17/58	7/27	9/28	23/60
Adenoma                      terminal					0/58	2/27	3/28	1/60
Adenocarcinoma         terminal						2/27	3/28	1/60
total						4/27	6/28	2/60
Fibroadenoma            terminal					5/58	9/27	14/28	5/60
(%)					(9%)	(33%)	(50%)	(8%)

Empty place = organ examined, no findings

Table: Updated histopathological findings in the endocrine system and mammary glands after re-evaluation (Jackson et al, 2000)

Endpoint	Dose (ppm)
	Males				Females
	0	60	300	1500	0	60	300	1500
Adrenal cortex
Focal cortical vacuolation
Interim	1/10	2/10	4/10	1/10	1/10	1/10	1/10	0/10
Terminal	31/60	34/60	28/60	36/60	12/60	10/60	14/60	13/60
Total	32/70	36/70	32/70	37/70	13/70	11/70	15/70	13/70
Focal hyperplasia	1/70	2/70	4/70	0/70	1/70	4/70	1/70	2/70
Cortical adenoma (b)	1/70	0/70	0/70	1/70	2/70	0/70	0/70	0/70
Cortical adenocarcinma (m)	0/70	0/70	1/70	0/70	0/70	0/70	1/70	1/70
Adrenal medulla
Phaeochromocytoma (b) (%)	2/70 (2.8)	3/70 (4.3)	5/70 (7.1)	2/70 (2.8)	0/70 (0)	1/70 (1.4)	1/70 (1.4)	0/70 (0)
Phaeochromocytoma (m) (%)	0/70	0/70	0/70	0/70	0/70	1/70	0/70	0/70

 

	(0)	(0)	(0)	(0)	(0)	(1.4)	(0)	(0)
Pancreas
Acinar atrophy
Interim	0/10	0/10	0/10	2/10	2/10	2/10	0/10	1/10
Terminal	6/60	3/60	4/60	10/60	1/60	1/60	4/60	7/60
Total (%)	6/70 (8.6)	3/70 (4.3)	4/70 (5.7)	12/70 (17.1)	3/70 (4.3)	3/70 (4.3)	4/70 (5.7)	8/70 (11.4)
Islet cell hyperplasia, terminal	1/70	4/70	2/70	2/70	0/70	2/70	1/70	0/70
Islet cell adenoma (b)	2/70	2/70	4/70	2/70	0/70	2/70	1/70	0/70
Islet cell adenocarcinoma (m)	1(70) (1.4)	0/70 (0)	0/70 (0)	0/70 (0)	0/70 (0)	0/70 (0)	0/70 (0)	0/70 (0)
Pituitary
Adenoma
Interim	1/10	2/10	1/10	3/10	1/10	1/10	1/10	0/10
Terminal	22/60	18/60	32/60	25/60	38/60	39/60	39/60	37/60
Total (%)	23/70 (32.8)	20/70 (28.6)	33/70 (47.1)	28/70 (40)	39/70 (55.7)	40/70 (57.1)	40/70 (57.1)	37/70 (52.8)
Mammary gland
Diffuse     and     focal     lobular hyperplasia					5/70	2/70	3/70	3/70
Focal atypical hyperplasia					4/70	4/70	8/70	5/70
Fibroadenoma terminal					6/70 (8.6)	12/70 (17.1)	16/70 (22.8)	2/70 (2.8)
Adenocarcinoma terminal					0/70 (0)	1/70 (1.4)	5/70 (7.1)	3/70 (4.3)

(b) = benign, (m) = malignant

Mammary gland findings more detailed table from Jackson et al 2000:

LESION	Dose level (ppm)
	0		60		300		1500
	D	T	D	T	D	T	D	T
Number of animals	19	41	17	43	15	45	13	47
Benign mammary tumours – absent	15	37	14	34	10	34	13	45
One fibroadenoma	2	4	1	9	5	9	0	2
Two fibroadenomas	0	0	2	0	0	1	0	0
Three fibroadenomas	0	0	0	0	0	1	0	0
Tumour bearing animals (%)	6/58 (10%)		12/60 (20%)		16/60 (27%)		2/60 (3%)
Malignant  mammary  tumours  – absent	17	41	16	43	13	42	12	45
One adenocarcinoma	0	0	1	0	2	3	1	2
Tumour bearing animals (%)	0/60 (0%)		1/60 (2%)		5/60 (8%)		3/60 (5%)

T:terminal kill, D: decedents

Table 154 Histopathological changes in the female genital tract

End-point		Dose level ( ppm) 0                 60		300	1500 ppm
Ovaries/Number of animal examined
Cysts	interim	3/10	0/1	0/2	2/10
terminal		9/60	6/26	2/19	17/60
total (%)		(15%)			(28%)
Stromal hyperplasia	terminal	1/60	1/26	2/19	3/60
Theca-Granulosa cell tumour	terminal	2/60	1/26	2/19	4/60
total (%)		(3%)			(7%)
Leiomyoma	terminal				1/60
Uterus
Squamous hyperplasia	interim	0/10	0/1	0/2	2/10
	terminal	3/60	1/26	0/19	6/60
	total (%)	(5%)			(10%)
Squamous carcinoma	terminal	0/60	0/26	0/19	1/60
Fibroma	interim	0/10	0/1	1/2	0/10
	terminal	0/60	1/26	0/19	0/60
	total	0/70	1/27	1/21	0/70
Stromal sarcoma	terminal		1/26
Schwannoma	terminal		1/26	2/19	2/60
Adenoma	terminal	2/60
Adenocarcinoma	terminal	1/60	1/26	0/19	3/60

Empty place = organ examined, no findings

Table: Histopathological findings in the female genital tract (updated according to Jackson et al, 2000)

Endpoint	Dose (ppm)
	Females
	0	60	300	1500
Ovaries
Follicular cysts
Interim	3/10	2/10	1/10	1/10
Terminal	8/60	9/60	13/60	20/60
Total	11/70	11/70	14/70	21/70
Tubulostromal hyperplasia	0/70	3/70	3/70	0/70
Granulosa/thecal cell tumor terminal	1/70 (1.4)	2/70 (2.8)	1/70 (1.4)	1/70 (1.4)
Leiomyoma	0/70 (0)	0/70 (0)	1/70 (1.4)	1/70 (1.4)
Uterus
Focal squamous hyperplasia
Interim	0/10	1/10	0/10	0/10
Terminal	1/60	0/60	0/60	3/60
Total	1/70	1/70	0/70	3/70
Squamous cell carcinoma	0/70	0/70	0/70	1/70
Fibroma	0/70	0/70	0/70	0/70
Endometrial stromal sarcoma	0/70	1/70	0/70	0/70
Schwannoma	0/70 (0)	1/70 (1.4)	1/70 (1.4)	1 /70 (1.4)
Endometrial adenoma (b)	3/70	0/70	1/70	0/70
Endometrial carcinoma (m)	2/70	1/70	0/70	3/70

(b) = benign, (m) = malignant

Table 155 Histopathological findings in the male genital tract

 

End-point		Dose level ( ppm) 0                 60		300	1500 ppm
Testes
Calcification	terminal	2/60	2/28	4/22	4/59
Leydig cell hyperplasia	terminal	2/60	0/28	0/22	3/59
Leydig cell tumour	terminal	1/60	2/28	0/22	1/59
Hemangiosarcoma					1/59
Epididymides
Aspermia	interim	1/10	-	-	0/10
	terminal	6/60	6/24	5/17	10/59
	total (%)	(10%)			(17%)
Seminal vesicles

 

 

 

Alveolar atrophy	interim	0/10	-	-	0/10
	terminal	7/59	8/20	7/17	13/59
	total (%)	(11.9%)			(22%)
Carcinoma			1/20

Empty place; organ examined, no findings

 

Table: Histopathological findings in the male genital tract (updated with result of re-analysis from Jackson et al, 2000)

Endpoint	Dose (ppm)
	Males
	0	60	300	1500
Testes
Calcification terminal	0/70	0/70	0/70	0/70
Focal          interstitial           cell hyperplasia
Interim	0/10	0/10	0/10	0/10
Terminal	4/60	2/60	5/60	6/60
Total	4/70	2/70	5/70	6/70
Leydig cell tumor (b) terminal	1/70	2/70	0/70	1/70
Haemangiosarcoma	0/70	0/70	0/70	1/70
Epididymides
Uni-/bilateral          reduced or absent spermatozoa
Interim	2/10	0/10	2/10	0/10
Terminal	12/60	18/60	16/60	22/60
Total (%)	14/70 (20)	18/70 (25.7)	18/70 (25.7)	22/70 (31.4)
Seminal vesicles
Alveolar atrophy	0/70	0/70	0/70	0/70
Adenocarcinoma	0/70	1/70	0/70	0/70

(b) = benign, (m) = malignant

 

Conclusions:

Based on the available data, long-term effects of treatment with desmedipham were mainly seen as changes in hematology and findings related to these changes. Increased levels of methemoglobin were observed at 300 and 1500 ppm in both sexes, and reticulocytes in blood and Heinz bodies were clearly increased at 1500 ppm in both sexes. The highest increase in methemoglobin was observed in males treated with 1500 ppm desmedipham; 4.6% of red blood cells contained methemoglobin. The complete picture of hematological effects during the whole course the study is unclear; slight and mostly transient, but sometimes statistically significant, changes in red blood cell parameters indicating hemolytic anemia were observed. The NOAEL for hematological effects is 60 ppm, based on significant increases in methemoglobin levels at the two highest dose levels in both sexes.

Spleen weights were increased at 1500 ppm in both sexes, and histopathology showed increased hemosiderosis and erythropoiesis in the spleen at the high dose. Liver weights w ere increased at all doses in females, and increased hematopoiesis in the liver was noted in females at 1500 ppm.
Increased hemosiderosis of the liver was observed in males at 1500 ppm. Increased tubular pigmentation was noted at 1500 ppm in females, and tubular dilatation was observed in males, mainly at 1500 ppm. Most of the toxic effects seen in spleen, liver and kidneys seem to be related with the observed hematological effects of desmedipham.

Decreased levels of T4 were observed mainly in females after 24 months. The total incidence of follicular hyperplasia in thyroid glands (interrim and terminal sacrifice) was increased in both males and females, mainly at 1500 ppm. C-cell hyperplasia of the thyroid glands was also increased in males at 1500 ppm.

Infiltration of inflammatory cells was observed at increased incidences in the liver, lungs and especially in kidneys. Pulmonal inflammation was slightly increased in both sexes at the high dose.


Histopathological examination of mammary glands indicated an increased incidence of fibroadenomas at 60 and 300 ppm, based on a limited number of examined animals (9/27 at 60 ppm and 14/28 at 300 ppm).

Because of deficiencies in histopathological analyses and statistical analyses of the histopathological data, only a temporary NOAEL, based on hematological and related effects, may be set at 60 ppm (3 – 4 mg/kg bw/day). Full histopathological analysis of all isolated tissues at all dose levels, accompanied by relevant statistical analysis of the data is required.

The conclusion was corrected afterwards and can be seen below.

NOAEL : 60 ppm / 3.2 mg/kg bw/day, based on increased mineral deposition and associated urothelial hyperplasia in the kidneys and hemosiderin deposition, increased methemoglobin and extramedullary haemopoiesis in the spleen observed at mid or high dose levels.

Executive summary:

In this 2-year chronic toxicity/ oncogenicity study, DESMEDIPHAM TECHNICAL was administered in the feed to Wistar rats. The study was comprised of four groups, each containing 70 male and 70 female rats.

NOAEL : 60 ppm / 3.2 mg/kg bw/day, based on increased mineral deposition and associated urothelial hyperplasia in the kidneys and hemosiderin deposition, increased methemoglobin and extramedullary haemopoiesis in the spleen observed at mid or high dose levels.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

153 mg/kg bw/day

Study duration:

chronic

Species:

mouse

Quality of whole database:

GLP- and guideline-compliant studies are available for the rat and mouse.

System:

hepatobiliary

Organ:

liver

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

The data were considered by RAC (2019), and their evaluation is summarised below.

Rat carcinogenicity studies: (1991) and (1986)

The top dose levels in these studies were comparable, 1200 ppm and 1500 ppm, respectively.  The main toxic effect was haematotoxicity (Hb reduced by ~10%, increased MetHb and Heinz bodies). The maximum tolerated dose in 90-day studies was around 4000 ppm (body weight reduction by ~20 %, Hb reduction by ~15%.  The top dose selection in these two carcinogenicity studies was therefore considered acceptable.  In study M-146980-01 only premature decedents were examined histopathologically at the low- and mid-dose levels.  The following findings and incidences were considered for their relevance to classification:

Thyroid follicular cell adenoma in females [0/50-0/25-2/30-1/50] with no increased in males
Leydig cell adenoma in males [1/50-1/21-0/28-4/48] with no associated increase in hyperplasia
Pituitary adenoma in males [33%-29%-47%-40%; HCD 20-54% (mean 38%)]
Mammary gland fibroadenoma in females [9%-17%-23%-3%; HCD 6-44% (mean 26%)]
Mammary gland adenocarcinoma in females [0%-1%-7%-4%; HCD 0-10% (mean 3%)]
Uterine squamous cell carcinoma in females [0%-0%-0%-1%]

Taking into account the lack of statistical significance at the top doses, the lack of a clear dose-response relationship in most cases and the incidences remaining within a relevant HCD range, where available, RAC considered it questionable whether any of these increases is treatment-related.  In addition, each of the increases is found in only one and not in the other of the two rat carcinogenicity studies using similar top dose levels.  Therefore, RAC did not consider any of these findings relevant for classification.

Mouse carcinogenicity study, 1990

Significant hepatotoxicity was seen in this study at the top dose level of 2500 ppm (moderate to marked hepatocyte necrosis in almost all animals, a 1.9-fold increase in liver weight in males), indicating that the MTD had been reached and possibly exceeded.  Increases in the incidence of hepatocellular adenomas at the top dose level, although not statistically significant on pairwise comparison, were considered by RAC likely to be treatment-related in view of the marked hepatotoxicity.  There was also an increased incidence of pulmonary adenomas in top dose females [3, 5, 6, 9 of 49-50 animals at 0, 400, 1000 and 2500 ppm respectively.  The increase is not statistically significant on pairwise comparison and was not accompanied by non-neoplastic findings.  There was no increase in pulmonary adenomas in males [13, 10, 11, 10].

Mouse carcinogenicity study, 1986

The top dose level of 750 ppm in this study caused haematotoxicity (methaemoglobinaemia, Heinz body formation, increased spleen weight) and in males also bodyweight reduction by ~10 %.  A dose of 1600 ppm increased MetHb levels to 14% and splenic weights by 60% in male NMRI mice after a 28-day administration.  In view of the haematotoxicity, the top dose level in the carcinogenicity study was considered  by RAC to be sufficiently high.  The following findings and incidences were considered for their relevance to classification:

Ovarian tubular adenoma (females) [2%-13%-15%-8%]
Ovarian theca/granulosa cell tumour (females) [8%-9%-23%-14%]
Leydig cell adenoma (males) [0%-2%-6%-0%]

Combined hepatocellular tumours (adenoma/carcinoma) in males [7%-17%-13%-10%]

Taking into account the lack of statistical significance at the top dose level and the lack of a clear dose-response relationship, RAC considered it  questionable whether any of these increases is treatment-related.  In addition, no increase in ovarian or testicular tumours was reported at 2500 ppm in the other mouse carcinogenicity study. Therefore, RAC did not consider any of these findings relevant for classification.

Justification for classification or non-classification

Carcinogenicity studies in the rat and mouse are available for desmedipham.  The same carcinogenicity studies for desmedipham were also considered by RAC (2019).  RAC concluded that, out of the neoplastic findings in the available studies, there was sufficient indication of a treatment-related effect only for the hepatocellular tumours in the mouse.  However, taking into account the lack of statistical significance on pairwise comparison, the excessive liver toxicity at the tumorigenic dose (marked chronic hepatocyte necrosis and liver enlargement), lack of increase in hepatocellular carcinomas in males and lack of genotoxicity, RAC concluded that no classification for carcinogenicity is justified.

Consequently, on the basis of the available data and the recent RAC conclusion, no classification for carcinogenicity is required for desmedipham.