Desmedipham

Endpoint summary

Description of key information

Studies of repeated dose oral toxicity in the rat, mouse and dog of duration up to 2 years are available for desmedipham.  The key study is identified as the 90-day rat study, as this identifies the lowest NOAEL (equivalent to 2.6-2.7 mg/kg bw/d).

Test method/  species	Result	Assessment	Reference
Non-guideline  - 28-day dietary mouse study	The NOAEL for this study is reported to be 100 ppm (22-26 mg/kg bw/d) based on haematological effects).	Supporting study	Suter et al (1984)
Non-guideline  - Non-guideline, 5-week dietary RF study performed with desmedipham (2000, 4000 ppm)	Investigations in this study were very limited and are not sufficient to derive a NOAEL.  Notably there was no assessment of haematological parameters or histopathology	This is a non-guideline and non-GLP range-finding study, which included very limited parameters.	Mulhern and Perry (1989)
Non-guideline  - 28-day dietary RF study in the dog	Group size was very small (1/sex) and investigations were limited (no histopathology).  A NOAEL of 200 ppm (7 mg/kg bw/d) was determined for this study, based on haematological effects.	This is a non-guideline range-finding study, which included very limited parameters.	Bathe et al (1984)
Non-guideline  - 6-week dietary RF study in the dog	Group size was (3 males) and investigations were limited (no histopathology).  A NOAEL of 15 ppm (~0.5 mg/kg bw/d) was determined for this study, in the absence of any effects in any dose group.	This is a non-guideline range-finding study, which included very limited parameters.	Harling et al (1985)
US EPA 82-1 - 90-day dietary study in the rat	A NOAEL was not identified for this study due to haematological effects at the lowest tested concentration of 300 ppm (~24 mg/kg bw/d).	Supporting study	Suter et al (1984)
US EPA 82-1 - 90-day dietary study in the rat	A NOAEL of 30 ppm (2.6-2.7 mg/kg bw/d) was determined for this study, based on haematological effects at 60 ppm.	Key study	Suter et al (1985)
Non-guideline  - 90-day dietary study in the rat	A NOAEL of 160 ppm (10.6-12.3 mg/kg bw/d) was determined for this study, based on haematological effects at 800 ppm and higher.	Supporting study	Elliott et al (1987)
Non-guideline  - 90-day dietary RF study in the mouse	A NOAEL of 750 ppm (134-148 mg/kg bw/d) was determined for this study, based on haematological effects at 1300 ppm and higher.	Supporting study	Hill and Taupin (1992)
Non-guideline  - 90-day dietary study in the dog	A NOAEL of 150 ppm (5.24 mg/kg bw/d) can be determined for this study, based on marginal haematological effects at the highest dose level.	Supporting study	Hounsell and Martin (1986)
Non-guideline  - 90-day dietary study in the dog	A NOAEL of 100 ppm (4.1 mg/kg bw/d) can be determined for this study, based on thyroid effects (increased weight, follicular hypertrophy) in females.	Supporting study	Oshodi and MacNaughtan (1991)
Non-guideline  - 80-day dietary mechanistic study in the dog, designed to identify a NOAEL for methaemoglobin formation	A NOAEL was not identified for this study, due to splenic congestion (considered to be an effect secondary to MetHb formation) at the lowest dose level of 300 ppm.	Supporting study	Allen et al (1991)
Non-guideline  - 12-month dietary toxicity study in the dog (considered to be chronic under REACH); broadly comparable to OECD 408	A NOAEL of 300 ppm (9.7-10.4 mg/kg bw/d) was agreed for this study, based on non-adverse findings (increased iron deposition in the liver, bone marrow and haematological parameters) at this dose level.	Supporting study	Bathe (1985)
None stated - 52-week oral (dietary) chronic toxicity study in the rat performed at 0, 100, 400 and 1200 ppm	A NOAEL of 100 ppm (6.5-8.0 mg/kg bw/d) was determined for this study, based on clinical chemistry changes at 400 ppm.  Haematological effects at 400 ppm were seen but were not considered to be adverse.	Supporting study	Everett et al (1991)
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 (report and amendment)	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 ast 400 ppm (61 mg/kg bw/d).	Supporting study	Husband et al (1990) in IUCLID title; (add in data source)
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.	Not required - reassessment of study findings	Not specified (2000)

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1989-04-12 to 1991-11-01

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Deviations:

yes

Remarks:

See Principles of method if other than guideline.

Principles of method if other than guideline:

No OECD guideline was mentioned in the study report, however, the study essentially complied with OECD guideline 452 at the time of first assessment. After the study was performed, a new version of the OECD Test Guideline 452 has been adopted 7th September, 2009.

Deviations:
Following endpoints are mentioned in the new guideline and were not investigated in the study:
Not fasted at time of blood sample.
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 26 and 52 weeks in 10 males and 10 females.
Haematology: Prothrombin time, methemoglobin and activated partial thromboplastin time was not investigated.
Biochemistry: animals not fasted over night.
Organ weight: Thyroid was not weighed.
Not microscopically examined: lacrimal gland, Harderian gland, cervix, coagulating gland and gall bladder, seminal vesicles, vagina, submandibular lymph node, tongue and nasal cavity. Test for neuro toxicity not conducted, however, neuro toxicity tests are triggered.
Ophthalmoscopy was not carried out before dosing.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Groups of 20 male and 20 female Sprague-Dawley rats received desmedipham technical (Purity: 98%) at dose levels of 0, 100, 400 and 1200 ppm in the diet for 52 weeks.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On week 25, there was evidence of desmedipham in the control diet (8.5 ppm), but in repeat analysis of the diet prepared for week 25 showed no contamination of the control diet. Analyses of formulated diets were generally within acceptable limits (±10%) for accuracy of preparation and homogeneity. The calculated values for achieved test material intakes were 6.5, 25.2, 75.0 mg/kg bw/day for males and 8.0, 31.7 and 97.1 mg/kg bw/day for females, respectively.
On week 25, there was evidence of desmedipham in the control diet (8.5 ppm), but in repeat analysis of the diet prepared for week 25 showed no contamination of the control diet.

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: Based on range finding study.

Observations and examinations performed and frequency:

Samples for hematology, clinical chemistry (extra determinations; plasma, RBC and brain cholinesterase) and urinanalysis were taken from all animals at 25/26 and 52 weeks of treament. Histological examinations were carried out on a full list of organs (except seminal vesicles, vagina, submandibular lymph node, tongue and nasal cavity, as required by current guidelines) from all control, high dose animals and all premature decedents.. In addition kidneys, liver and lungs of all test animals at 100 and 400 ppm were examined.

Statistics:

Haematology, clinical chemistry, organ weight and body weight data were statistically analysed for homogeneity of variance using the Fmax test. If the group variances appeared homogeneous a parametric ANOVA was used and pairwise comparisons made via Student's t-test using Fisher's F-protected LSD. If the variances were heterogeneous log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a non-parametric test such as a Kruskal-Wallis ANOVA was used. Organ weights were also analysed conditional on body weight (i.e. analysis of covariance). Histological data were analysed using Fisher's Exact Probability test.

Clinical signs:

no effects observed

Mortality:

mortality observed, treatment-related

Description (incidence):

There were five premature deaths during the study; one control male and female, one male at 400 ppm and one female at 100 and 1200 ppm. The control animals and the female at 1200 ppm had malignant lymphoma in lymphoreticular/hematopoietic tissues and these animals died between weeks 31 to 43. One female at 100 ppm died on week 25 and was autolysed. The male at 400 ppm had glioma (B) in the brain and died on week 50.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weight was statistically significantly (8%) reduced in females at 1200 ppm from week 5 until week 28. In females at 100 ppm, food consumption (11%) and body weight gain (17%) were increased, the body weight gain was significantly increased on week 52 only. The body weight gain (expressed as % of control, weeks 0-52) was 99%, 96% and 94% in males and 117%, 106% and 95% in females at 100, 400 and 1200 ppm, respectively.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematological data revealed reductions in red blood cell parameters. Hemoglobin and hematocrit were decreased in males at all dose levels after 26 weeks, and red blood cells were decreased at 1200 ppm. No statistically significant effects on red blood cell parameters were recorded in males at terminal sacrifice on week 52. In females, hemoglobin was decreased at all dose levels, and hematocrit and red blood cells were decreased at 400 and 1200 ppm at week 26. At week 52 in females, decreases were noted in Ht and Hb at 1200 ppm and and in RBC at 400 and 1200 ppm. Total bilirubin and creatinine were increased in males at 400 and 1200 ppm and total bilirubin also in females at 1200 ppm. Albumin and albumin/globulin-ratio was decreased in females at 1200 ppm after 26 weeks. In males albumin and albumin/globulin-ratio was decreased at 1200 ppm 26 and 52 weeks and at 400ppm after 26 weeks. Alkaline phosphatase was reduced in males at 1200 ppm after 52 weeks.

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Kidney weights were reduced in males at 1200 ppm. Thymus weight (absolute) was significantly higher in males at 1200 ppm compared with control males. Spleen weight was increased in females at 1200 ppm.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Hemosiderin deposition (Kupffer cell pigmentation) in the liver was increased in males at 1200 ppm. In kidneys, tubular pigmentation (mainly in the cytoplasm of the cortical tubules) was increased in males at 1200 ppm. In lungs, the total incidences of foci of increased alveolar macrophages (graded minimal or mild) were increased in a dose dependent manner in both males and females. Pigment deposition in the spleen was increased in severity in both sexes at 1200 ppm. Histological examinations were not carried out on the spleen of all test animals at 100 and 400 ppm.
Thymic atrophy (minimal to moderate) was significantly reduced in 1200 ppm males (1/20) compared to control males (10/20).

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

In the study report, there was no overall summary table on the incidence of externally palpable masses and tumour incidence in the control and treated groups. It remained unclear, if all masses and macroscopical findings observed at necropsy were examined. For example, one male at 100 ppm had a mass on the left epididymis, and one male at 1200 ppm had a mass on the right testes, according to necropsy findings presented in the individual data. In the presentation of histological findings, these organs were listed and examined, but the masses observed at necropsy were not mentioned or identified. Reproductive organs were not systematically examined. Seminal vesicles and vagina were excluded from histopathology in the study protocol, and testes, epididymides, ovaries and uterus were examined only from some animals at 100 and 400 ppm. The available data, keeping in mind the limitations mentioned above, gave no indications of neoplastic effects.

Key result

Dose descriptor:

LOAEL

Effect level:

400 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

Key result

Dose descriptor:

NOAEL

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 ppm

System:

other: Erythrocyte (Met-Hb formation)

Organ:

blood

Conclusions:

Dosing Sprague-Dawley rats for 52 weeks with Desmedipham showed reductions in parameters in both sexes at 100, 400 and 1200 ppm and increased haemosiderin deposition in the liver (males), kidney (males) and spleen (males and females) at 1200 ppm. Males at 1200 ppm showed an equivocal increase in thymus weight and decreased incidence of thymic atrophy. Effects on various clinical chemistry parameters were also seen, in males at 400 ppm and in males and females at 1200 ppm.
NOAEL= 100 ppm (6.5 mg/kg bw/day in males and 8.0 mg/kg bw/day in females) based on the increased values for bilirubin and creatinine and on the decreased A/G ratio after 26 weeks.

Executive summary:

Groups of 20 male and 20 female Sprague-Dawley rats were dosed with Desmedipham via the diet at concentrations of 0, 100, 400 or 1200 ppm. Ophthalmoscopy was carried out on all animals in the Control and High dose groups before treatment commenced and during Week 51 of the study. After approximately 25 and 51 weeks a nominal 10 males and 10 females were subjected to investigation of haematology, clinical chemistry and urinalysis parameters. On completion of 52 weeks of dosing all surviving animals were killed and necropsied and selected organs weighed. Premature decedents were also necropsied. Histological examination was carried out on a full list of organs from all Control and High dose animals and all premature decedents. In addition, histological examination was carried out on the kidneys, liver and lungs of all other animals.
The results can be summarised as follows:
Mortality: There were five premature deaths, one Control male, one Intermediate dose male, one Control female, one Low dose female and one High dose female. There was no evidence to suggest that any of these deaths were due to administration of Desmedipham.
Clinical Signs:There were no clinical signs that were thought to be attributable to administration of Desmedipham. There were no notable intergroup differences in the incidence of externally palpable masses in either sex.
Body Weight: There were no notable intergroup differences in either sex.
Food Consumption: There were no notable intergroup differences in food consumption.
Water Consumption: There were no visual intergroup differences in either sex.
Analyses of Formulated Diets: Analyses of formulated diets were generally within acceptable limits (±10%) for accuracy of concentration and homogeneity.
Ophthalmoscopy: There were no notable intergroup differences.
Haematology: Haemoglobin was decreased in all male groups receiving Desmedipham at Weeks 26 and 52, all female groups receiving Desmedipham at Week 26 and in High dose females at Week 52. RBC was decreased in High dose males at Weeks 26 and 52 and in Intermediate and High dose females at Weeks 26 and 52. HCT was decreased in all male groups receiving Desmedipham at Weeks 26 and 52, Intermediate and High dose females at Week 26 and High dose females at Week 52.
Clinical Chemistry: Total bilirubin was increased in Intermediate and High dose males at Weeks 26 and 52 and in High dose females at Weeks 26 and 52. Creatinine was increased in Intermediate and High dose males at Weeks 26 and 52. Albumin was decreased in High dose males at Weeks 26 and 52 and High dose females at Week 26. AG-R was decreased in Intermediate and High dose males at Week 26, High dose males at Week 52 and High dose females at Week 26. Alkaline phosphatase was reduced in High dose males at Week 52.
Urinalysis: There were no notable intergroup differences.
Organ Weights: Kidney weight was reduced in High dose males. Spleen weight was increased in High dose females. Thymus weight was equivocally increased in High dose males.
Necropsy Findings: There were no notable intergroup differences.
Histological Findings: There was an increased incidence of Kupffer cell pigmentation in the liver of High dose males. Tubular pigmentation in the kidney was increased in High dose males. Pigment deposition in the spleen was increased in severity in High dose males and females. There was a decrease in incidence of thymic atrophy in the High dose males.

NOAEL= 100 ppm (6.5 mg/kg bw/day in males and 8.0 mg/kg bw/day in females) based on the increased values for bilirubin and creatinine and on the decreased A/G ratio after 26 weeks.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1990-12-03 to 1991-09-06

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Deviations:

yes

Remarks:

See Principles of method if other than guideline .

Principles of method if other than guideline:

Deviations: Histopathology was not always adequate. For example, hypercellularity in bone marrow was observed in females at 1500 ppm, but this tissue was not systematically examined from lower dose groups.

In the study report it was not specified which guideline was used, however, Guideline 409 has been used to assess if the study complies with the data requirements because the study was conducted on dogs and not rodents. Conclusion has not been changed. After the study was performed, a new version of the OECD Test Guideline has been adopted: OECD Test Guideline 409 adopted 21st September 1998. The study complies with the current guideline except for some issues regarding histopathology : as already mentioned in the previous evaluation, hypercellularity in bone marrow was observed in females at 1500 ppm, but this tissue was not systematically examined from lower dose groups. Epididymides were not weighed and accessory sex organs were not examined histopathologically.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

Groups of 4 male and 4 female beagle dogs received desmedipham technical (Purity: 98%) at dose levels of 0, 100, 500 and 1500 ppm in diet for 90 days. Achieved test material intakes were not reported (in Tier II, 100 ppm was reported to be equivalent to 3.7 and 4.3 mg/kg bw/day in males and females).

Route of administration:

oral: feed

Details on oral exposure:

The animals were dosed by inclusion of the test compound in the diet for 7 days/week for 13 consecutive weeks up to 24 h before sacrifice.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were obtained from each test formulation prepared for Weeks 1, 9 and 11 and analysed for concentration and homogeneity at IRI. Sampling dates deviated from those stated in the protocol which were Weeks 1, 6 and 13 respectively, this was due to work load. A further sample was obtained during Week 2 to assess the stability of the test material in the diet. Analyses for stability, concentration and homogeneity were undertaken at IRI under a separate protocol and contract (!RI Project No. 350942). Additional samples were also obtained from each test diet formulation prepared during the study and retained frozen.
The mean concentrations of desmedipham in feed samples were 97.8 - 95.9% of the nominal dose levels and stability was within ± 5% for all dose levels.

Duration of treatment / exposure:

90 days

Dose / conc.:

0 ppm

Dose / conc.:

100 ppm

Dose / conc.:

500 ppm

Dose / conc.:

1 500 ppm

No. of animals per sex per dose:

4

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The above dose levels were agreed with the Sponsor following evaluation of the available toxicity data and taking into account other factors such as the anticipated possible human exposure.
- Fasting period before blood sampling for clinical biochemistry: Over night.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Not specified

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Not specified

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: Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

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: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: once pretrial and again during weeks 6 and 12 of the study.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: all dogs

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once pretrial and again during weeks 6 and 12 of the study.
- Animals fasted: Not specified
- How many animals: all dogs

PLASMA/SERUM HORMONES/LIPIDS: Not specified

URINALYSIS: Yes
- Time schedule for collection of urine: once pretrial and again during weeks 6 and 12 of the study.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified

NEUROBEHAVIOURAL EXAMINATION: Not specified

IMMUNOLOGY: Not specified

OTHER: A full histopathology (except spinal cord) was carried out on organs/tissues of all control and high dose animals. The extended histological evaluation included liver and thyroids of all test animals at 100 and 500 ppm and spleen of the females at 100 and 500 ppm.

Statistics:

Hematology, clinical chemistry and organ weight data were evaluated by statistical analysis (F-max-test, ANOVA, Student’s t-test, Kruskall-Wallis test).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematological data revealed an increase in reticulocytes (94%) together with a decrease in hemoglobin (15%) and MCHC (4%) in females at 1500 ppm during week 12. Reticulocytes were also decreased in females at 100 ppm during week 12. A decrease in hemoglobin levels at 1500 ppm, and an increase in platelet counts was observed at 500 and 1500 ppm males at week 6 and a decrese in platelets were observed in week 6 in females. No effects on hematology was observed in males at week 12.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The albumin/globulin ratio (AG-R) was reduced in females at 1500 ppm during weeks 6 and 12. Blood cholesterol levels were increased at 1500 ppm in males at week 6 and in females at week 12.

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Thyroid weights were increased for animals in 500 and 1500 ppm females, achieving statistical significance for 1500 ppm females after absolute and covariance analyses (38%) and 14 % at 500 ppm (covariate). In males the thyroid weight also seem to increase (16% in high dose). Absolute adrenal weights were increased among 1500 ppm males (26%), and absolute pituitary weights were increased in males at 500 ppm (29%). A statistically non-significant increase in absolute and adjusted weight were observed in spleen (59 % abs, adj at 1500 ppm) and liver (abs, adj 14%, at 1500 ppm ) in females and in liver in males (13% abs, 11% adj. at 1500 ppm).

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Increased hemosiderin deposition (Kupffer cells/pigmented macrophages) in the liver was observed in both sexes at 1500 ppm (Table 76). Extramedullary hematopoiesis in the spleen was observed in two (2/4) females at 1500 ppm, but not in males. Hypercellularity (predominantly normoblasts) in bone marrow was noted in three (3/4) females at 1500 ppm; the bone marrow of dogs at 500 and 100 ppm were not examined microscopically. Follicular epithelial hypertrophy (minimal/ mild) in thyroids was significantly increased in males at 1500 ppm and in females at 500 and 1500 ppm. The follicular epithelial hypertrophy was characterised by an increase in cytoplasm, sometimes slightly vacuolated with the nucleus located basally. The cells often appeared columnar rather than cuboidal. Epithelial hyperplasia was not evident. In lungs, focal pleural/subpleural fibrosis was observed in 2/4 females at 1500 ppm, but not in the control groups or in males at 1500 ppm. Lungs from lower dose groups were not microscopically examined.

Histopathological findings: neoplastic:

no effects observed

Details on results:

No treatment related effects were observed in clinical signs, opthalmoscopical investigations, body weight, food consumption and in urinanalysis. No test animal deaths occurred during the study.

Key result

Dose descriptor:

NOAEL

Effect level:

4.1 other: mg/kg bw/d

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Effect level:

18.7 other: mg/kg bw/d

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

4.1 mg/kg bw/day (actual dose received)

Organ:

thyroid gland

Conclusions:

NOAEL = 100 ppm corresponding to 4.1 mg/kg bw /day in females and 500 ppm in males corresponding to 18.7 mg/kg bw/day based on increased incidence of thyroidal follicular epithelial hypertrophy and increased thyroid weight.

Executive summary:

This study was undertaken to investigate the oral toxicity of Desmedipham in Beagle dogs. Three groups of 4 male and 4 female Beagle dogs were dosed once daily via the diet on 7 days per week for 13 consecutive weeks at dose levels of 100, 500 and 1500 ppm. A fourth similarly constituted group received normal diet and acted as Controls.

The animals were observed daily for any signs of ill health or reaction to treatment. Food consumption was measured daily whilst body weight was monitored weekly. Ophthalmoscopic examinations and laboratory investigations of haematology, clinical chemistry and urinalysis were preformed pretrial and during Weeks 6 and 12 of treatment. Blood samples were collected for pharmacokinetic studies on Day 1 and on one day during Week 13 of the study.

On completion of the 13 week treatment period all the animals were killed and subjected to a detailed necropsy, organ weight analyses and histological evaluation (Control and High dose groups only).

Treatment with Desmedipham at dose levels of 500 and 1500 ppm for 13 weeks was associated with a reduction in AG-R (at 1500 ppm), a tendency towards an increase in cholesterol and an increase in thyroid weight. Follicular epithelial hypertrophy was observed in the thyroids of the majority of treated animals. In the female animals, dosed at 1500 ppm, mild red blood cell damage was indicated by the haematology findings and the histopathological findings of increased haemosiderin in the liver, extramedullary haemopoiesis in the spleen and hypercellular bone marrow.

NOAEL = 100 ppm corresponding to 4.1 mg/kg bw /d in females and 500 ppm in males corresponding to 18.7 mg/kg bw/d based on increased incidence of thyroidal follicular epithelial hypertrophy and increased thyroid weight.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Remarks:

a range-finding study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1991-06-21 to 1992-04-10

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

The study was designed as a dose range finding study and therefore apparently not conducted in full compliance with current OECD Guideline 408 (Rodent) and 87/302/EEC B test requirements. Histopathology was insufficient and inadequate. Only kidneys, liver and spleen from control and high dose animal and some gross abnormalities were preserved for histopathology and microscopically examined. Observed gross lesions e.g. in lungs, heart, ovaries and uterus were not microscopically identified. Parameters of clinical biochemistry were not determined. The study was carried out according to the principles of GLP/OECD.

Protocol
Groups of 10 male and 10 female mice (Crl:CD-1(IRC) BR strain, VAF plus) were administered desmedipham at nominal concentrations of 0, 750, 1300, 2300 or 4000 ppm in diet for 13 weeks.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

mouse

Strain:

ICR

Remarks:

Crl:CD-1(ICR) BR strain, VAF plus

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 26-28 days.
- Weight at study initiation: males: 25-31g and the females: 20-24g.
- Housing: in groups of 5, by sex, in grid bottomed stainless steel cages.
- Diet: All animals were offered a powdered diet (SQC Rat and Mouse Maintenance Diet, No. 1).
- Water: Mains water was provided in polypropylene bottles, both were freely available throughout the study.
- Acclimation period: 13 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 19-27°C.
- Humidity: 39-68%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Details on route of administration:

Formulated diets were freely available up to the day of necropsy.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): For each group a weighed amount of test article were mixed with the required amount of diet for at least 15 minutes in a double cone rotary mixer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean recovery of desmedipham in feed samples were between 92.6 - 98.8% of the nominal dose levels. Homogeneity was within ± 14% and the stability for at least 8 days for the concentrations of 50 and 5000 ppm. Correspondingly, the mean calculated test material intakes were 0, 134, 209, 402 and 714 mg/kg bw/day for males and 0, 148, 237, 484 and 1008 mg/kg bw/day for females. The mean recovery of desmedipham in feed samples were between 92.6 - 98.8% of the nominal dose levels.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

continuously

Dose / conc.:

0 ppm

Remarks:

Control

Dose / conc.:

750 ppm

Remarks:

Correspondingly, the mean calculated test material intakes were 134 mg/kg bw/day for males and 148 mg/kg bw/day for females.

Dose / conc.:

1 300 ppm

Remarks:

Correspondingly, the mean calculated test material intakes were 209 mg/kg bw/day for males and 237 mg/kg bw/day for females.

Dose / conc.:

2 300 ppm

Remarks:

Correspondingly, the mean calculated test material intakes were 402 mg/kg bw/day for males and 484 mg/kg bw/day for females.

Dose / conc.:

4 000 ppm

Remarks:

Correspondingly, the mean calculated test material intakes were 714 mg/kg bw/day for males and 1008 mg/kg bw/day for females.

No. of animals per sex per dose:

10/sex/group

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during week 13 of treatment.
- How many animals: All survived animals

Sacrifice and pathology:

HISTOPATHOLOGY: Yes,
Histopathological examinations were only carried out on kidneys, liver and spleen of the control and high dose animals and on enlarged lymph nodes. Remaining tissues were discarded.

Statistics:

Homogeneity was within ± 14% and the stability for at least 8 days for the concentrations of 50 and 5000 ppm. Samples for hematology were taken from all animals during week 13. Histopathological examinations were only carried out on kidneys, liver and spleen of the control and high dose animals and on enlarged lymph nodes. Remaining tissues were discarded. Body weight, hematological and organ weight data were statistically analysed (analysis of variance, t-test, Kruskal-Wallis test, Wilcoxon rank sum test).

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two females dosed at 4000 ppm were found dead during week 1, without previous changes in animal condition and behaviour or showing any significant macroscopic findings at necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight gain was reduced by 38% in females and by 21% in males at 4000 ppm and by 14% in males at 2300 ppm compared with the control group. However, the terminal body weights at 4000 ppm were reduced by only 10% in the females and 7% in the males.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption was somewhat variable, with some cages of mice scattering food excessively. Although intergroup differences in total food intake of up to 122 were noted, there was no evidence of a dose relationship.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Dose related decreases in red blood cell count, hemoglobin and hematocrit with associated increased MCH and MCHC- values was observed in females. Only red blood cell count was statistically significantly (p<0.05) reduced in the females at 750 ppm. Hematological effects in the males were seen only at 4000 ppm. A significant increase in reticulocytes was seen only in females at 4000 ppm.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute liver (23%) and spleen weights (36%) were significantly increased in males at 4000 ppm. In females, absolute spleen weights were increased at 1300 and 2300 ppm (36%). Relative spleen weights (49%) were increased at 4000 ppm and relative liver weights were increased at 2300 (14%) and 4000 ppm (33%) in males. In females, relative spleen weights were increased at 1300 (41%), 2300 (46%)and 4000 ppm (37%), relative liver weights at 2300 (15%) and 4000 ppm (29%), and relative kidney weights at 4000 ppm (17%).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Apparently treatment-related macroscopic post-mortem findings included enlargement and/or swelling of the spleen together with discolouration; enlargement and/or swelling of the kidneys and enlargement of lymph nodes. However, incidences of these observations did not show a strict relationship to dose.
The raised incidences of splenic enlargement in males given 4000 ppm and females given 1300 or 2300 (but not 4000) ppm were notable in that they are supported by increased absolute spleen weight.
The apparently increased incidence of kidney swelling in all treated male groups was not supported at all by altered kidney weight (absolute or relative) in males. This is therefore considered to be of doubtful significance. Equally, the increased incidence of enlarged lymph nodes, without microscopic changes in lymph nodes, is also considered to be of dubious toxicological significance.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Tissues (except some lymph nodes) were not microscopically examined from mice at 750, 1300 and 2300 ppm. Only kidneys, liver and spleen of control and high dose animal and gross abnormalities were preserved for histopathology. Remaining tissues were discarded. Microscopical examination was carried out on kidneys, liver and spleen of control and high dose animals. Enlarged lymph nodes, when observed, were microscopically examined.

Histopathological changes were noted in the liver (chronic inflammation, single hepatocyte necrosis and minimal hemosiderin deposits), kidneys (chronic inflammation, minimal focal cortical scarring, minimal hemosiderin deposits, occasional basophilic tubules/males) and spleen (increased hemosiderin deposition) mostly in both sexes at 4000 ppm.

Dose descriptor:

NOAEL

Effect level:

750 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

organ weights and organ / body weight ratios

Remarks on result:

other: corresponding to 134 mg/kg bw /day (males) and 148 mg/kg bw/day (females)

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

Conclusions:

No NOEL could be set for hematological effects in females because of reduced RBC counts at 750 ppm, although the decrease was slight (less than 4% lower than in controls). The NOAEL for hematological effects in males was 2300 ppm. Organ weight changes were observed at 1300 ppm in females and 2300 ppm in males. The NOAEL of the study is 750 ppm (134 - 148 mg/kg bw/day), based on hematological changes evident mainly at 1300 ppm and higher dose levels in females, and increased organ weights (liver, spleen, kidneys) in both sexes.

Executive summary:

One hundred mice of the Crl:CD-l(ICR) BR strain (VAF plus) were divided into 5 groups each of 10 males and 10 females. Four groups received Desmedipham in the diet at nominal concentrations of 750, 1300, 2300 or 4000 parts per million. The fifth group received untreated diet and acted as a control.
Animals were observed daily, body-weights and food consumption were recorded weekly. A blood sample for haematology was obtained from all surviving animals during week 13 of treatment.
After 13 weeks of treatment all surviving animals were killed by carbon dioxide asphyxiation and subjected to a detailed necropsy during which the weights of 3 organs were recorded and a limited range of tissues was preserved. The liver, lymph nodes, spleen and kidneys of all control and high dose animals were examined microscopically; as were tissues, from other dose groups, showing macroscopic abnormalities.
Analysis of formulated diets before treatment started confirmed homogeneity and stability under the conditions of use in this study. Diets prepared for weeks one and 13 were found to have actual concentrations acceptably close to nominal concentrations (± 7.4%).
Two females dosed at 4000 ppm were found dead during week one. There were no previous changes in animal condition or behaviour or significant macroscopic findings at necropsy. The cause of death could not be established histologically.
No further mortalities occured.
There were no treatment related changes in animals condition or behaviour.
Following weight losses in weeks one and 12, females dosed at 4000ppm showed a moderate (38%), reduction in total weight gain relative to controls. Males given 2300 or 4000 ppm showed lesser (14 and 21%) reductions in total weight gain respectively.
There was no evidence of an adverse effect on food intake.
Females dosed at 2300 ppm and above and males dosed at 4000ppm showed minimal to slight reductions in red blood cell count, with associated alterations in other red blood cell parameters. Although minor, these differences were statistically significant.
At necropsy enlargement of the spleen, kidneys and lymph nodes was noted in treated groups, although without a strict dose relationship. Only the splenic enlargement in females dosed at 1300 or 2300 ppm and in males given 4000 ppm was backed by organ weight increases.
A dose related increase in liver weight was found in all treated groups. The increases over control values were statistically significant at 2300 and 4000 ppm. At 4000 ppm mean relative liver weights were about a third higher than those of controls.
Absolute and relative spleen weights were statistically significantly greater than control values for males dosed at 4000 ppm and females dosed at 1300 or 2300 ppm. Females dosed at 4000 ppm had a significantly raised mean relative (but not absolute), spleen weight value.
Definite treatment related microscopic changes were noted for the liver (hepatocyte necrosis, haemosiderin deposits), kidney (minimal focal cortical scarring, basophilic tubules and minimal haemosiderin deposits) and spleen (increased haemosiderin deposition) in animals given 4000 ppm.
Because of the above findings it was concluded that 4000 ppm lay above the MTD and would not be suitable as a high dose level for a carcinogenicity study.
The no observed effect level was concluded to be 750 ppm.

Reference 4

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1986-08-20 to 1987-08-26

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

A specific OECD Guideline is not mentioned in the study report, however, the study complied with OECD guideline 408 at the time of the last assessment: Mammary glands and seminal vesicles were excluded from the histopathological examination already in the study protocol without any explanation. After the study was performed, a new version of the OECD Test Guideline 408 has been adopted 21st September, 1998. Besides the deficiencies mentioned in the DAR, the main study fulfils these data requirements except that no examination of reactivity to stimuli examinations was performed as mentioned in the current OECD Test Guideline 408. Thymus from all animals should also have been weighed and accessory sex organs should have been examined histopatologically according to current guideline.

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

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

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

yes

Remarks:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

A caesarean-derived strain of Sprague-Dawley origin

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 74 to 86 g (males) or 71 to 84 g (females).
- Housing: The rats were placed at random in suspended cages with wire mesh floors, according to sex, so that each cage contained, as far as possible, 5 rats of the same sex.
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 18.5 to 21.5°C.
- Humidity: Relative humidity was not controlled but was recorded daily at a mean of 57.5% RH.
- Photoperiod (hrs dark / hrs light): 12/12.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Remarks:

via diet

Details on oral exposure:

DOSE AND DIET PREPARATION
- Mixing appropriate amounts with (Type of food): A pre-mix was prepared each week by grinding the test substance directly into Labsure Laboratory Animal Diet No. 2 and mixing in an inflated polythene bag for a minimum period of 3 minutes. The required concentrations were then prepared by direct dilution of the pre-mix with further quantities of untreated diet; homogeneity being achieved by mixing in a double cone blender for a minimum period of 7 minutes.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to commencement of the study the proposed diet mixing procedures were checked by chemical analysis of trial diets to confirm that the proposed procedures produced homogenous diet, that the accuracy of mixing was acceptable and that the concentration of test substance in the diet remained unchanged between preparation and administration.

Samples of diets prepared in the first week and in Weeks 6 and 13 were also analysed to check the accuracy of preparation.

The analysed mean concentrations of desmedipham in feed samples were within ±7.0% of the nominal concentrations. Desmedipham remained constant in the rodent diet for up to 14 days during storage under animal room conditions.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Continuous

Dose / conc.:

0 ppm

Remarks:

Control

Dose / conc.:

160 ppm

Remarks:

Achieved test material intakes were 10.6 mg/kg bw/day for males and 12.3 mg/kg bw/day for females, respectively.

Dose / conc.:

800 ppm

Remarks:

Achieved test material intakes were 54 mg/kg bw/day for males and 60 mg/kg bw/day for females, respectively.

Dose / conc.:

4 000 ppm

Remarks:

Achieved test material intakes were 275 mg/kg bw/day for males and 339 mg/kg bw/day for females, respectively.

No. of animals per sex per dose:

10/sex/group

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The dosage levels selected for this study were based on the results of a four-week preliminary toxicity study in rats by dietary administration of Desmedipham.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily.

BODY WEIGHT: Yes
- Time schedule for examinations: at the time of allocation of animals to groups, on the day of commencement of treatment (Week 0), and once a week thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined on weekly basis. Diet consumption calculated as g food/rat/week.

FOOD EFFICIENCY:
- Food conversion ratios were calculated, where appropriate, from bodyweight and food consumption data as weight of food consumed per unit gain in bodyweight.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment commenced and during Week 13.
- Dose groups that were examined: Control and high dosage level groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Samples for hematology (extra determinations; cell morphology, cholinesterase levels) were taken from all animals during week 13.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Samples for clinical biochemistry were taken from all animals during week 13.

Sacrifice and pathology:

Lungs, liver, kidneys, spleen, thyroid glands and any macroscopically abnormal tissue were microscopically examined from all rats. The full list of organs with some exceptions was microscopically examined from all control and high dose rats after a treatment period of 13 weeks. However seminal vesicles and mammary glands were not processed for histopathological study and thus not examined.

Statistics:

Food and water consumption, body weight, organ weights and clinical laboratory data were evaluated by statistical analyses (Bartlett’s test, one-way ANOVA, Kruskal-Wallis analysis of ranks, Student’s test, Williams’s test).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment related pallor was observed periodically throughout the study in both sexes and hair loss in 3/20 females from days 79/81 onwards at 4000 ppm.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male rat in the control group died during the blood sampling procedure on Day 86.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights and body weight gains at 4000 ppm decreased by 23% and 50% in females, respectively, and by 14% and 23% in males, respectively. Body weights were also slightly reduced (about 11%) in females at 160 and 800 ppm.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was significantly lower in both sexes at 4000 ppm and water consumption was higher in females at 4000 ppm, compared with controls.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematological observations included dose related changes in red and white cell parameters and increased platelet counts in both sexes at 4000 ppm. Red cell parameters (RBC, Hb, Ht) were significantly reduced, whereas mean corpuscular volume (MCV), white cell parameters (WBC total, neutrophils, lymphocytes) and platelet counts were increased significantly in both sexes at 4000 ppm. Significantly higher neutrophil counts were also observed in males at 800 ppm. Total WBC counts for male rats at 800 ppm were higher than those of the control group, but not statistically significantly.

Similar significant, treatment related changes in red blood cell parameters (RBC, Hb, Ht, MCV, MCHC) and platelet counts were observed in females at 800 ppm. Individual platelet counts for majority of animals at 4000 ppm and for several animals at 160 and 800 ppm were upper limit of the expected range for this parameter. Morphological changes were observed in the erythrocytes as polychromatophilia and anisocytosis in the blood films of both sexes at 800 and 4000 ppm

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related changes in a number of clinical biochemistry parameters were apparent in both sexes at 4000 ppm and in males at 800 ppm. Statistically significantly lower glucose levels than those of the controls were recorded in both sexes at 4000 ppm. Globulin levels and total protein levels were significantly higher in the males at 800 and 4000 ppm and significantly higher globulin levels were detected in females at 4000 ppm than in controls. Statistically significantly higher levels were noted at 4000 ppm for alanine aminotransferase in both sexes, aspartate aminotransferase in males and alkaline phosphatase in the females.

Significantly higher cholesterol levels were recorded in the females at 4000 ppm, the apparent shift in cholesterol levels was observed for the males at 4000 ppm. Significantly lower brain cholinesterase levels were recorded in the males at 800 (20%) and 4000 ppm (21%) than those of the controls (see Table 7) and significantly higher plasma cholinesterase levels in the males at 4000 ppm. Sodium was decreased in males in all dose groups.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The relative (to body weight ratios) spleen weights for both sexes were statistically significantly increased at 4000 ppm. Similarly, statistically significantly higher relative liver weights were recorded for males at 4000 ppm (11%). The relative (to body weight ratios) thyroid weights in males (44%) and absolute thyroid weights in females were statistically significantly increased at 4000 ppm (27%). Weights of pituitary gland were significantly lower in females at 4000 ppm than in the control group (15%). Brain weight of females at 4000 ppm were not changed.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Trachea, thymus, aorta, pancreas, epididymides, parathyroids, adrenals, pituitary, salivary glands, oesophagus, duodenum, jejunum, ileum, caecum, colon, rectum, sciatic nerve, sternum, ovaries and cervix were not microscopically examined from rats at 160 and 800 ppm. Exceptional microscopical findings in these tissues were not observed in rats at 4000 ppm. Mammary glands and seminal vesicles were not examined.

Treatment related changes were observed in the spleen (enlargement, hemosiderosis, congestion), kidneys (pigmentation) and thyroid glands (follicular cell hypertrophy).

Congestion of the spleen was noted in males already at 160 ppm. Splenic congestion is an early, minor change and may be regarded, in the abscence of other treatment-related observations at the low dose, as being of little or no toxicological importance. Brown pigment in cortical tubules of the kidneys was noted in some males (7/10) and in one female (1/10) at 4000 ppm, possibly representing lipofuscin deposition according to the study investigators.

Dose descriptor:

NOAEL

Effect level:

160 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

histopathology: non-neoplastic

Remarks on result:

other: corresponding to 10.6 mg/kg bw /day

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

Conclusions:

Mammary glands and seminal vesicles were excluded from the histopathological examination already in the study protocol without any explanation. Otherwise the study was acceptable.
Based on available data, NOAEL: 160 ppm corresponding to 10.6 mg/kg bw /day based on congestion of the spleen, changes in red cell parameters in females and morphological changes in red cell in both sexes at 800 ppm. In addition, enlargement and hemosiderosis was observed in spleen and minimal to moderate follicular cell hypertrophy in thyroids in both sexes at 800 ppm.

Executive summary:

Desmedipham was administered to rats for thirteen consecutive weeks by admixture with the diet at levels of 160, 800 or 4000 ppm. Control animals received normal untreated diet.

There were no treament related deaths. Treatment related pallor was observed periodically throughout the study in both sexes and hair loss in 3/20 females from days 79/81 onwards at 4000 ppm. Body weights and body weight gains at 4000 ppm decreased by 23% and 50% in females, respectively, and by 14% and 23% in males, respectively. Body weights were also slightly reduced (about 11%) in females at 160 and 800 ppm. Food consumption was significantly lower in both sexes at 4000 ppm and water consumption was higher in females at 4000 ppm, compared with controls.

In the histopathological examination, congestion of the spleen was noted in males already at 160 ppm. Mammary glands and seminal vesicles were excluded from the histopathological examination already in the study protocol without any explanation.

Hematological treatment-related effects were observed such as: Red cell parameters (RBC, Hb, Ht) were significantly reduced, whereas mean corpuscular volume (MCV), white cell parameters (WBC total, neutrophils, lymphocytes) and platelet counts were increased significantly in both sexes at 4000 ppm, specifically in females at 800 ppm. Significantly higher neutrophil counts were also observed in males at 800 ppm. Total WBC counts for male rats at 800 ppm were higher than those of the control group, but not statistically significantly.

In conclusion, NOAEL: 160 ppm corresponding to 10.6 mg/kg bw /day based on congestion of the spleen, changes in red cell parameters in females and morphological changes in red cell in both sexes at 800 ppm. In addition, enlargement and hemosiderosis was observed in spleen and minimal to moderate follicular cell hypertrophy in thyroids in both sexes at 800 ppm.

Reference 5

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1985-07-08 to 1987-10-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Deviations:

yes

Remarks:

See Principles of method if other than guideline.

Principles of method if other than guideline:

In the study report it was not specified which guideline was used, however, Guideline 409 has been used to assess if the study complies with the data requirements because the study was conducted on dogs and not rodents. After the study was performed, a new version of the OECD Test Guideline has been adopted: OECD Test Guideline 409 adopted 21st September 1998. The study complies with the current guideline except that epididymides, uterus and thymus in all animals were not weighed.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Route of administration:

oral: feed

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

90 days.

Dose / conc.:

0 ppm

Dose / conc.:

1 ppm

Dose / conc.:

5 ppm

Dose / conc.:

150 ppm

No. of animals per sex per dose:

1

Control animals:

yes, concurrent no treatment

Details on study design:

Groups of 4 male and 4 female beagle dogs received desmedipham technical (Purity: 98.3%,) at dose levels of 0, 1, 5, 150 ppm in their diet for 90 days. The overall mean group intakes were 0.035, 0.17 or 4.97, mg/kg/d, and 0.035, 0.19 or 5.50 mg/kg/d in males and females respectively.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Not specified

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Not specified

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: Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

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: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected before the start of the treatment period and after that on days 15, 29, 43, 57, 71 and 85
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: Not specified.
- Parameters examined: extra determinations; Heinz bodies and methemoglobin.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected before the start of the treatment period and after that on days 15, 29, 43, 57, 71 and 85
- Animals fasted: Not specified
- How many animals: Not specified.
- Parameters examined: extra determination; thyroid hormones.

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: Blood samples were collected before the start of the treatment period and after that on days 15, 29, 43, 57, 71 and 85
- Animals fasted: Not specified
- How many animals: Not specified.

URINALYSIS: Not specified

NEUROBEHAVIOURAL EXAMINATION: Not specified

IMMUNOLOGY: Not specified

OTHER: A full histopathology was carried out on all test animals.

Statistics:

Food and water consumption, body weight, organ weights and clinical laboratory data were evaluated by statistical analysis (Bartlett’s Test, One-way analysis of variance, t-test, Kruskal-Wallis analysis of ranks).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Methemoglobin levels were increased slightly but significantly in males on days 43, 57, 71 and 85 and in females on day 85 at the 150 ppm dose level. Erythrocytes with Heinz bodies were not increased, but increased numbers of reticulocytes were observed on days 71 and 85 in the blood of males treated at 5 ppm. White blood cells were significantly increased in males at 1 ppm on days 57, 71 and 85. White blood cells and neutrofils were significantly increased on day 85 in females only at 5 ppm.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Thyroxine levels were significantly lower in females on days 58 and 86 at the 150 ppm dose level compared to controls. In males, thyroxine and the free T4 index was higher on day 30 at 150 ppm. The T4-binding capacity was significantly lower on day 58 at 5 and 150 ppm dose levels (thyroxine and free T4 indices were slightly higher at 5 and 150 ppm).

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Adrenal relative weight was statistically significantly reduced by 18% in the high dose compared with control in male dogs. In females, absolute and relative ovary weight seemed to be reduced in the two top doses (41% rel) and approximately 43% (abs). The reduction was not statistically significant but such a large effect may be biologically significant. Absolute thyroid/parathyroid in males was larger in high dose (not statistically significant) but the dogs also weighed a bit more, this was not observed in females.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Mononuclear cell infiltrations in the parotid salivary glands were increased in treated females at 150 ppm (3/4 animals). The distribution of mononuclear cell infiltrations gave a significant positive trend. Hemorrhagic cystitis in the urinary bladder was seen in 3/4 females in all treated groups, but not in the control group. Although none of these occured in the control group the distribution was such that the incidence and severity of this finding was about the same throughout all treated groups, and both trend test and pair-wise comparisons with the controls were not significant. Pituitary cysts were found in females (3/4) at 150 ppm, but not in the control group.

Histopathological findings: neoplastic:

no effects observed

Details on results:

No treatment related effects were observed in mortality, clinical signs, ophthalmological and electrocardiography examinations, body weight gain and food consumption.

Key result

Dose descriptor:

NOAEL

Effect level:

> 150 ppm

Based on:

test mat.

Sex:

not specified

Basis for effect level:

haematology

Dose descriptor:

LOAEL

Effect level:

5.24 other: mg/kg bw/d

Based on:

test mat.

Sex:

not specified

Dose descriptor:

NOAEL

Effect level:

5.24 other: mg/kg bw/d

Based on:

test mat.

Sex:

male/female

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

150 ppm

System:

other: Target system / organ toxicity Other: Erythrocyte (Met-Hb formation)

Organ:

blood

The analysed mean concentrations of desmedipham in feed samples were between 87.9 -104.8% of the nominal concentrations. The test material was stable at 5 and 150 ppm but slightly unstable at 1 ppm; a reduction of up to 1% per day of the nominal concentration was observed.

 

Conclusions:

Hematological findings, mainly slight increases in methemoglobin levels, were observed at 150 ppm, almost exclusively in males. The only other red blood cell parameter change accompanying this finding in males was a slight increase in reticulocytes at 5 ppm on days 71 and 85. No decreases in RBC counts, Heinz bodies or hemoglobin levels were observed. Transient changes in white blood cells and levels of thyroxine in blood were also observed. No histopathological changes in the thyroid were reported. A significant positive trend for an increase in mononuclear cell infiltrations in the parotid salivary glands in females was noted; the toxicological significance of this finding is unclear. Slight or moderate hemorrhagic cystitis of the urinary bladder was observed in females at all doses of desmedipham. The NOEL for methemoglobinemia in males is 5 ppm (0.18 mg/kg bw/d) based on transitional increases in methemoglobin levels at 150 ppm. As no consistent clinical signs of hemolytic anemia were observed, the NOAEL of the study is 150 ppm (5.24 mg/kg bw/d).
It should be noted that there is a reduction of adrenal weight in males and decreased Thyroxine at high dose both without histopat effects, which were not regarded as adverse.
NOAEL: >150 ppm (higher than 5.24 mg/kg bw/d) based on only transitional increases in methemoglobin levels at 150 ppm but with no changes in RBC counts, Heinz bodies or hemoglobin levels

Executive summary:

Groups of 4 male and 4 female beagle dogs were fed diets containing 0, 1, 5 and 150 ppm of technical (production) desmedipham for at least 90 days. Observations were made daily of clinical signs and weekly of bodyweights and food consumption. A clinical examination was carried out pre-test and during week 13. Ophthalmology and electrocardiography were carried out pre-test and in week 13. Laboratory investigations of clinical chemistry, haematology and urinalysis were carried out pre-test and up to six times during the treatment period. Detailed necropsies were carried out at the end of the treatment period on all dogs. The weights of selected organs were recorded and tissues taken for histopathological examination. A microscopic examination was carried out on all tissues taken.
The group mean (and individual range) of test material intake over weekly intervals achieved by administering dietary levels of 1, 5 and 150 ppm desmedipham was calculated to be 0.035 (0.022 - 0.049), 0.17 (0.14 - 0.21) or 4.97 (4.01 - 6.09), mg/kg/d, and 0.035 (0.020 - 0.046), 0.19 (0.14 - 0.23) or 5.50 (4.31 - 6.72) mg/kg/d in males and females respectively. The overall group mean intake for both sexes was therefore 0.035, 0.18 or 5.24 mg/kg/d. Increased methaemoglobin levels were detected in male and female dogs at 150 ppm. No other treatment related effects were detected.

NOAEL: 150 ppm (5.24 mg/kg bw/d) based on only transitional increases in methemoglobin levels at 150 ppm but with no changes in RBC counts, Heinz bodies or hemoglobin levels

Reference 6

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1989-08-15 to 1991-03-11

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

The aim of the experiment was to establish a dietary NOAEL for methemoglobin formation and to study the time-course of methemoglobin formation in dog at repeated administration. The study was therefore not conducted according to OECD or other test guidelines. The study was performed according to the principles of GLP/OECD.

Principles of method if other than guideline:

Dose escalation study to investigate a NOAEL for MetHb production

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Route of administration:

oral: feed

Dose / conc.:

0 ppm

Remarks:

group 1 days 1-80

Dose / conc.:

150 ppm

Remarks:

group 2 days 1-21

Dose / conc.:

200 ppm

Remarks:

group 2 days 22-42

Dose / conc.:

500 ppm

Remarks:

group 2 days 43-80

Dose / conc.:

75 ppm

Remarks:

group 3 days 1-21

Dose / conc.:

300 ppm

Remarks:

group 3 days 22-40

Dose / conc.:

0 ppm

Remarks:

group 3 days 41-42

Dose / conc.:

1 500 ppm

Remarks:

group 3 days 43-80

Details on study design:

Three groups of 2 male and 2 female beagle dogs received desmedipham technical (Purity: 97.6%) at increasing dose levels of 0, 150/200/500 and 75/300/0/1500 ppm daily in the diet over a total of 80 days.
Group 1 was given untreated diet through the study and group 2 was given 150 ppm (days 1- 21), then 200 ppm (days 22-42) and then 500 ppm (days 43-80). Group 3 was given 75 ppm (days 1- 21), then 300 ppm (days 22-40), then 0 ppm (days 41-42) and then 1500 ppm (days 43-80).
Achieved daily intakes of desmedipham were to males and females 2.5 (75 ppm) 5.1 and 4.3 (150 ppm), 6.5 and 5.3 (200 ppm) 9.7 and 11.1 (300 ppm) 15.5 and 15.7 (500 ppm), 45.0 and 49.2 mg/kg/body weight/day (1500 ppm), respectively. The mean concentrations of desmedipham technical in feed were between 96.7 - 116.7% of the nominal concentrations and the variation in homogeneity was ±4%. The stability of desmedipham in diet was at least 21 days.
Blood samples for hematology were collected from all animals on every weekday during the study. The following hematological parameters were determined; RBC, Hb, Ht, MCV, MCH, MCHC, platelets, reticulocytes, normoblast, Heinz bodies, methemoglobin and red cell morphology. Necropsy of all dogs was performed at the study termination and samples of spleen, liver and bone marrow (sternum, and femur) and bone marrow smears were collected from all animals at necropsy.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The only clinical sign associated with the administration of desmedipham technical was the appearance of grey urine, seen occasionally in dogs (3/4) at 150/200/500 ppm, and frequently in males and almost every day in females at 75/300/0/1500 ppm. Severity was not stated.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematological parameters other than methemoglobin formation were unaffected by treatment. An increase in levels of methemoglobin was noted in both sexes at 1500 ppm and in females at 500 ppm. Histopathology revealed possible treatment-related congestion of the spleen in one dog at 150/200/500 ppm and in two dogs at 75/300/0/1500 ppm. There was no effect on myelograms or on decalcified bone marrow samples. There was only one male at 75/300/0/1500 ppm, in which a marginal increase in the relative number of total erythroid cells was noted as a possible treatment-related effect.
There was considerable day-to-day variation in methemoglobin values in untreated dogs, but that the values remained generally in the same range for at least 80 days. Considerable between-dog variation is evident and it may be noted that pretest values are necessarily not representative of “normal” values in any animal.

Key result

Dose descriptor:

NOAEL

Effect level:

9.7 - 11.1 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

other:

Remarks on result:

not determinable because of methodological limitations

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 ppm

System:

other: Erythrocyte (Met-Hb formation)

Organ:

blood

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

A NOAEL was not identified for this study, due to splenic congestion (considered to be an effect secondary to MetHb formation) at the lowest dose level of 300 ppm.

Executive summary:

In this methemoglobin production study, DESMEDIPHAM TECHNICAL was administered orally, by feed admixture, to pure-bred Beagle dogs. There were three groups, each comprising two male and two female dogs. The dosing regimen, the dietary concentrations administered and average test article intakes achieved are given as in Table 1.

It is concluded from the findings that the administration of DESMEDIPHAM TECHNICAL induced methemoglobinemia in both sexes at 1500 ppm and in females at 500 ppm and that 300 ppm may be regarded as the no effect level for this parameter. There was no evidence to suggest that DESMEDIPHAM TECHNICAL may have produced any toxic effect upon the bone marrow.

A NOAEL was not identified for this study, due to splenic congestion (considered to be an effect secondary to MetHb formation) at the lowest dose level of 300 ppm.

Reference 7

Endpoint:

chronic toxicity: oral

Remarks:

104-week oncogenicity study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1983-07-11 to 1986-09-15

Reason / purpose for cross-reference:

reference to same study

Reference 8

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1983-03-14 to 1985-03-22

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

See principles of method if other than guideline.

Principles of method if other than guideline:

In the study report it was not specified which OECD guideline was used, however, Guideline 452 has been used to assess if the study complies with the data requirements because the study was a one year study conducted on dogs.
After the study was performed, a new version of the OECD Test Guideline has been adopted: OECD Test Guideline 452 adopted7th September 2009. The study complies with the current guideline except for some issues regarding histopathology : Epididymides were not weighed as usual. Seminal vesicle and coagulating- and lacrimal gland not histopathologically examined. As was mentioned in the previous evaluation, it remained unclear whether all tissues were systematically examined and all observed findings e.g. in mammary glands, parathyroid glands and thymus were reported.

GLP compliance:

yes (incl. QA statement)

Remarks:

See principles of method if other than guideline.

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Route of administration:

oral: feed

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean analytical concentrations of desmedipham in feed samples were between 95.5 - 97.4% of the target dose levels, the homogeneity was within ±13.3% for all dose levels, and desmedipham technical was stable in dog feed for 21 days.

Dose / conc.:

0 ppm

Remarks:

Group 1

Dose / conc.:

300 ppm

Remarks:

Group 2

Dose / conc.:

1 500 ppm

Remarks:

Group 3

Dose / conc.:

7 500 ppm

Remarks:

Group 4 day 1-28

Dose / conc.:

5 000 ppm

Remarks:

Group 4 day 29 - end

Details on study design:

Groups of 6 male and 6 female beagle dogs received desmedipham technical (Purity: 97.8%) at dose levels of 0, 300, 1500 and 7500/5000 ppm in diet for 12 months. The test material intakes were 0, 9.7, 52.5 and 167.7 mg/kg bw/day for males and 0, 10.4, 57.4 and 200.7 mg/kg bw/day for females, respectively. After the first 28 days, the high dose level 7500 ppm was reduced to 5000 ppm because of weight loss and marked toxicity.

Observations and examinations performed and frequency:

Samples for hematology, clinical chemistry and urinanalysis were taken from all animals at pretest and at 13, 27 and 52 weeks of treament. Additional examinations of hematological parameters (Heinz-bodies, Met- Hb), bone marrow smears and of tri-iodothyronine (T3) and thyroxine (T4) were performed. Two dogs per sex and group were killed after 13 week (the interim kill group K1) and four dogs per sex and group after 52 weeks (terminal sacrifice group KO). Histopathology was carried out on a full list of organs from all test animals.

Statistics:

Hematology, clinical chemistry and organ weight data were evaluated by statistical analysis (one-way ANOVA, Student’s t-test, Williams’ test, Fisher’s test).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Animals treated at 5000 and 1500 ppm showed higher incidence of vomiting, blood stained and mucoidal faeces together with intermittent dark coloration of feces. Eight animals at 5000 ppm and one animal in each of the 1500 and 300 ppm groups showed occasional signs of decreased activity, tremors, ataxia, spasms and episodes of lateral or ventral recumbency which persisted for 2 - 20 minutes.

Mortality:

mortality observed, treatment-related

Description (incidence):

Three dogs (2 males, 1 female) in 5000 ppm dose groups died or had to be killed during the course of the study. All deaths were considered to be treatment related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean food consumption of the group 4 males was reduced during the first 6 months and in the group 4 females throughout the study. In females at 5000 ppm, body weight gain and food consumption were significantly reduced throughout the study. In females, the body weights were significantly reduced at 5000 ppm throughout the treatment period and at 1500 ppm during the first 5 months. There were no significant differences between body weights of males in control and treatment groups. In males, body weights and food consumption returned to the level of controls after the first 6 months.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean food consumption of the group 4 males was reduced during the first 6 months and in the group 4 females throughout the study. In females at 5000 ppm, body weight gain and food consumption were significantly reduced throughout the study. In females, the body weights were significantly reduced at 5000 ppm throughout the treatment period and at 1500 ppm during the first 5 months. There were no significant differences between body weights of males in control and treatment groups. In males, body weights and food consumption returned to the level of controls after the first 6 months.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment related increases in Heinz bodies and methemoglobin levels were noted in all treated animals, statistically significant in males at 1500 and 5000 ppm and in females at 5000 ppm. Red blood cells, hematocrit, hemoglobin and mean corpuscular hemoglobin concentrations were decreased at 5000 ppm (up to 46%, up to 31 %, up to 16% and up to 40 %, respectively), whereas mean corpuscular hemoglobin , platelets and reticulocytes, nucleated erythrocytes and total leucocytes were increased. Morphological changes in erythrocytes (polychromatophilia, Howell-Jolly bodies) as well as increased erythrocyte precursors in the bone marrow were also observed at 5000 ppm.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Several clinical chemistry parameters such as total bilirubin, cholesterol, alkaline phosphatase, lactic dehydrogenase and albumin/globulin ratio were affected in dogs at 1500 and 5000 ppm. Total bilirubin levels were increased in both sexes at 1500 ppm. Total cholesterol levels and lactate dehydrogenase activities in both sexes and alkaline phosphatase activity in females were increased at 5000 ppm (and non-significant in males at 5000 ppm). Urea levels were decreased in males at 5000 ppm. The albumin/globulin ratios were decreased in dogs at 1500 and 5000 ppm. Serum tri-iodothyronine (T3) was decreased in all three treated groups of females, and in males at 300 and 1500 ppm, but not 5000 ppm. Thyroxine (T4) levels were decreased in females at 1500 and 5000 ppm. Data are summarized in Table 78.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased kidney and liver weights (absolute and relative) were noted in both sexes at 5000 ppm. The absolute and relative thyroid weights were increased in females at 1500 and 5000 ppm, and in males at 5000 ppm. The relative weight of thymus (to body and brain ratios) were reduced in females at 1500 and 5000 ppm after 13 weeks; at study termination only absolute thymus weights were decreased in 5000 ppm females. Increased relative weight of adrenals of females (to body (15%) and brain ratio (13%)) was observed. Absolute (33%) and relative (to body (60%) and brain ratio (48%)) weight of ovaries were increased at 1500 ppm, however, not statistically significantly. Testes seemed to be decreased in the top dose (48% abs, 45% rel to bw, 47% brain ratio). Pancreas weights seemed to be increased in males in the top dose. In females spleen weights were increased at 7500/5000 ppm and in males at 1500 and 7500/5000 ppm. Due to the very low number of animals (2/gender/group), the numbers are very uncertain and it is difficult to draw a firm conclusion.

After 12 months, weights of adrenals (absolute, relative to brain weight) increased in males at 1500 and 5000 ppm and liver (rel to bw) were significantly increased in males and females at 1500 and 5000 ppm. Absolute increase in liver weight was 23 % and 66% in males at 1500 and 5000 ppm . Testes weight relative to brain weight was statistically significantly increased at 1500 ppm. In females at 5000 ppm, the relative weight of adrenals (to body ratios) was also increased. After 52 weeks, the absolute brain weights were reduced in females at 300 ppm and 5000 ppm, but not at 1500 ppm. Pancreas weight was increased in both males and females at 5000 ppm. Spleen weight relative to brain weight ratio was also significantly increased at 5000 ppm in males.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The main pathology findings in the deceased dogs were anemia and lesions secondary to anemia (erythropoiesis in spleen, increased erythropoiesis in the bone marrow, bone marrow atrophy, and iron deposition in Kupffer cells of the liver). Moderate myocardial necrosis was noted in one prematurely killed/deceased dog and moderate multifocal broncopneumonia and chronic gastric ulcer in another dog.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Increased erythropoiesis in the bone marrow was observed at interim and at terminal sacrifice in all examined animals at 1500 and 5000 ppm (Table 81). At low dose in 2/4 dogs after 13 weeks and in ¼ after 12 months. Bone marrow atrophy was observed in two (one male and one female) 5000 ppm group dogs killed after 121 days and 12 months. Slight to moderate deposits of iron in the liver were noted in 3/4 females at 300 ppm and almost in all dogs at 1500 (slight to moderate) and 5000 ppm (slight to marked) after 12 months. After 13 weeks iron deposits in the liver was noted in 0/4 in control, ¼ low-dose (minimal), ¾ in mid-dose (minimal to slight) and 4/4 dogs in high-dose (slight to moderate). Extramedullar erythropoiesis in the spleen was found in all 5000 ppm animals and in 1 mid-dose dog both after 13 weeks and 12 months. After 13 weeks, the erythropoises was slight in the mid-dose and marked to severe in high-dose dogs. Slightly increased extramedullar hematopoiesis in the liver was observed at 5000 ppm (3/8 animals). Increased intrahepatic cholestasis was noted in 5000 ppm animals after 13 and 52 weeks. Increased hemosiderin deposition in Kupffer cells of the liver was noted in 0/4, 1/4, 1/3 and 4/4 dogs after 13 weeks and 0/8, 3/8, 7/8 and 8/8 dogs after 52 weeks of treatment at 0, 300, 1500 and 5000 ppm, respectively. Multifocal extramedullary hematopoiesis in abdominal adipose tissue was observed in one prematurely killed 5000 ppm group dog. Thyroid follicular cell hyperplasia was observed in 0/4, 0/4, 4/4 and 4/4 dogs after 13 weeks and in 0/8, 0/8, 4/8 and 8/8 dogs after 52 weeks of treatment at 0, 300, 1500 and 5000 ppm, respectively. At study termination, follicular hyperplasia in thyroids was noted only in the 1500 ppm female group (4/4 dogs). Increased vacuolation of cortical cells in adrenal glands, particularly in zona fasciculata was noted in one (1/4) male at both dose levels of 300 and 1500 ppm and in all (4/4) males at 5000 ppm after 52 weeks. Cortical hyperplasia in adrenal glands was noted in 3/8 dogs (2 males, 1 female) at 5000 ppm after 52 weeks.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

LOAEL

Effect level:

1 500 ppm

Based on:

test mat.

Sex:

male/female

Key result

Dose descriptor:

NOAEL

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Effect level:

9.7 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 ppm

System:

other: Target system / organ toxicity Other: Erythrocyte (Met-Hb formation)

Organ:

blood

other:

Conclusions:

A NOAEL of 300 ppm (9.7-10.4 mg/kg bw/d) was agreed for this study, based on non-adverse findings (increased iron deposition in the liver, bone marrow and haematological parameters) at this dose level.

Executive summary:

In this oral toxicity study, deposits of iron in the liver were noted in 3/4 females at 300 ppm and almost in all dogs at 1500 and 5000 ppm and increased erythropoiesis in the bone marrow was also observed at lowest dose. At the low dose the following effects were observed: After 13 weeks, 1 male and 1 female (50% of animals) had increased erythropoiesis in bone marrow. Increased erythropoiesis in bone marrow was observed in 25% of female animals (1/4 females) after 52 weeks.

DESMEDIPHAM TECHNICAL was administered in the diet for one year to pure-bred Beagle dogs. The study was comprised of four groups, each containing six male and six female dogs. The following nominal dose levels of DESMEDIPHAM TECHNICAL were administered:

Group 1 0 ppm Group 2 300 ppm Group 3 1500 ppm Group 4 7500 ppm (days 1-28) 5000 ppm (day 29-end)

Two dogs of each group and sex were killed after 13 weeks of treatment

MORTALITY

The following group 4 animals died or were killed in moribund condition:

No. 46 female (killed) - day 121 of treatment

No. 19 male (killed) - day 189 of treatment

No. 45 female (died) - day 358 of treatment

All deaths were considered to be treatment related. These animals showed lower food consumption during the study until death when compared with the other treated and control dogs and maintained their initial body weights during this time period. Further, these animals shamed decreased activity, particularly during the days just prior to death. Dog no. 19 displayed pale mucous membranes, decreased body temperature and increased heart rate just prior to its sacrifice in moribund condition.

SYMPTOMS

A higher frequency of feces containing traces of mucus and/or blood as well as a higher frequency of spontaneous vomiting were noted in the groups 3 and A animals. Dark discolored feces were noted sporadically in the animals of these groups as well as in two dogs of group 2. The urine of the dogs of groups 3 and 4 appeared normal at urination. After a short time, a dark discoloration of the urine on the kennel floor became evident. One group 2 and one group 3 animal, as well as eight animals of group 4 showed occasionally symptoms like decreased activity or isolated cases of tremor, ataxia, spasms and episodes of lateral or ventral recumbency persisting for about 2 to 20 minutes. Ruffled fur was observed in one group 3 male and three group 4 females occasionally during the second, half of the study. Alopecia was also noted in two of these females during this time period.

OPHTHALMOSCOPIC EXAMINATIONS

No treatment-related changes were seen.

FOOD CONSUMPTION

The mean food consumption of the group 4 males was reduced during the first 6 months and in the group 4 females throughout the study. The reduced food consumption was most marked during the first 4 weeks of treatment. After the dose level of group 4 (week 5- from 7500 to 5000 ppm) was reduced, the mean food consumption increased rapidly, but the food consumption of the group 4 females was decreased when compared with the group 2 and. 3 animals. Further, the groups 2 and 3 females showed slightly lower food consumption than the controls during the entire treatment period.

BODY WEIGHTS

The group 4 animals lost weight during weeks 1-4. At week 5: the dose level of this group was reduced from 7500 to 5000 ppm and the mean body weight gain of the group 4 males became similar to the control group. However, despite the dose level reduction; the mean body weight gain of the group 4 females decreased from week 8 onwards. The mean body weight gain of the group 3 females was decreased during the first 5 months of treatment when compared with that of the control group.

CLINICAL LABORATORY INVESTIGATIONS

Hematological findings indicated toxic hemolytic anemia. This was mainly characterized by a dose-dependent increase in Heinz bodies and methemoglobin formation in the dogs of groups 3 and 4. A trend for increased methemoglobin formation urns also observed, in the group 2 dogs. Other characteristic findings of toxic hemolytic anemia observed in the group 4 dogs during treatment were:

- a decreased erythrocyte count, hemoglobin concentration and hematocrit value,

- increased mean corpuscular volume and decreased, mean corpuscular hemoglobin concentration,

- increased platelet count for the group 4 males,

- increased reticulocyte count,

- increased number of nucleated erythrocytes,

- increased total leukocyte count.

Morphological changes in the erythrocytes of the group 4 dogs were indicated by increased polychromatophilia, an isocytosis, poikilocytosis, target cells and Howell-Jolly bodies.

Biochemical findings indicated:

- a decreased urea level for the group 4 males.,

- an increased total bilirubin level for the group 3 dogs,

- an increased total cholesterol level for group 4 dogs,

- increased lactate dehydrogenase activity for group 4 dogs,

- increased alkaline phosphatase activity for the group 4 females,

- decreased T3 level for groups ?., 3 and. 4 dogs and

- decreased T4 level for the groups 3 and 4 female dogs.

Other changes were observed in the plasma protein fractions of the electrophoretic pattern, indicated by the presence of a pre-albumin fraction, decreased, albumin fraction, increased beta and gamma globulins, and decreased albumin to globulin ratios for the group 3 and/or group 4 dogs. The changes indicated are supported by the morphological findings observed in this study. All other differences in the results of the hematological, biochemical and urinalysis data were considered to be incidental and reflections of normal biological variation.

BONE MARROW EXAMINATION

A test article- and dose-related expansion of the erythrocytes and precursors population, due to an increased erythropoietic activity, was observed in both sexes of groups 3 and 4, principally caused by individual dogs with marked increased values. The cell population showed predominantly normoblasts. Marked to strong deposits of iron were noted in almost all animals of group 3 and 4, and in two females of group 2 <no. 33, 34). Parallel to these findings, the leukopoiesis was relatively decreased. The thrombopoietic system and the other cells examined showed no relevant test article-related quantitative or qualitative - differences between the control and the treated animals. The erythroblastic hyperplasia observed in this study may be a reactive response of the bone marrow to compensate for the increased destruction rate of the red cells.

PATHOLOGY

Organ Weights

Increased absolute and relative thyroid weights were noted in groups 3 and 4 after 3 and 12 months of treatment. After 3 months of treatment, the absolute and relative spleen weights were increased, as well as the liver weights of the group 4 dogs after 12 months of treatment.

Gross and Histopathologic Findings:

Three group 4 dogs died or had to be killed during the course of the study. The main pathologic findings in these dogs were anemia and lesions secondary to anemia, emaciation in two of the dogs, myocardial necrosis in one, and bronchopneumonia and gastric ulcer in another. Dark red bone marrow was observed in one group 3 and one group 4 dog killed after 13 weeks. After 13 weeks and after 12 months, dose-dependent increases in erythropoiesis in the bone marrow was observed in all treated groups. Beginning bone marrow atrophy was seen in two dogs killed at the end of the 12-month treatment period.

After 13 weeks, extramedullary erythropoiesis was seen in the spleen of one group 3 dog and all group 4 dogs, and also in the abdominal adipose tissue of one group 4 dog. After 12 months, extraneously erythropoiesis was seen in the spleen of one group 3 dog and all group 4 dogs, also in the liver of one group 3 dog and three group A dogs. Intrahepatic cholestasis was noted in all group 4 dogs killed after 13 weeks and in six group 4 dogs killed after 12 months. Dose-dependent hemosiderin deposition in the Kupffer cells was noted in one group 2 dog, three group 3 and all group 4 dogs killed after 13 weeks as well as in three group 2, seven group 3 and all group 4 dogs killed after 12 months. Dose-dependent follicular hyperplasia was noted in the thyroid gland of all group 3 and group 4 dogs killed after 13 weeks and in four group 3 and all group 4 dogs killed after 12 months. The type, incidence, and severity of these treatment-related findings were similar in the dogs killed after 13 weeks and in those killed after 12 months. The other lesions noted in this study are not considered to be treatment related. They are spontaneous findings commonly encountered in dogs of this strain and age.

A NOAEL of 300 ppm (9.7-10.4 mg/kg bw/d) was agreed for this study, based on non-adverse findings (increased iron deposition in the liver, bone marrow and hematological parameters) at this dose level.

Reference 9

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1983-06-01 to 1984-11-07

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

According to the Statement of Compliance; this study was conducted according to the principles of GLP with the possible exceptions of minor items, none which could be considered to affect the validity of the data or the interpretation of the results in the report. After the study was performed, a new version of the OECD Test Guideline 407 has been adopted 3rd October, 2008. The main study fulfils these data requirements except that no examination of reactivity to stimuli examinations was performed as mentioned in the current OECD Test Guideline 407. Epididymides and prostate from all animals should also have been weighed according to current guideline. Furthermore, it is now standard to perform histopathological examination of at least the control and the high dose group of the following tissues, which were not performed in this study: spinal cord, eye, stomach, small and large intestines, thyroid, trachea and lungs, epididymides, prostate + seminal vesicles with coagulating glands, vagina, urinary bladder, lymph nodes, peripheral nerve (sciatic or tibial), skeletal muscle, accessory sex organs.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

one exception; blood samples were taken from non-fasted animals at the study termination.

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

yes

Remarks:

one exception; blood samples were taken from non-fasted animals at the study termination.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

Standard laboratory species/strain with background data available

Sex:

male/female

Details on test animals or test system and environmental conditions:

SPF-bred mice (NMRI KFM-Han., outbred) were used as the test system. Forty male and 40 female mice were obtained. At delivery, all mice were 4 weeks of age. The mice were given health examinations by a veterinarian and were assigned to groups using a random algorithm. They were individually identified by cage number and by toe clip to denote the treatment group. All mice were acclimated to test conditions for 7 days prior to treatment on day 1. The body weight range, recorded at pretest week 1 was 20 - 24 g for males and 16 - 20 g for females. The experiment was conducted under Optimal Hygienic Conditions. Each animal was housed individually in a Makrolon type-1 cage with wire mesh top and sterilized standard granulated softwood bedding . The animal room was air-conditioned, with 15 air changes per hour, the room temperature was 22 +/- 2 degrees centigrade. The relative humidity was 55 +/- 10 percent. The light/dark cycle consisted of 12 hours of artificial fluorescent light each day. Music was played for at least 8 hours of each light period. Each mouse was provided with repelleted standard Kliba no. 343 rat/mouse maintenance diet ad libitum. The feed batch 78/83 was analyzed . Tap water was available in water bottles ad libitum. The water quality was in compliance with the requirements of the Schweizerisches Lebensmittelbuch. The water was analyzed for chemical contaminants and bacteriological contaminants.

Route of administration:

oral: feed

Vehicle:

water

Details on oral exposure:

Each mouse of group 2, 3 and 4 received feed containing the test article for the duration of the study. The animals of group 1 received similar feed without the test article. The feed was weighed and then presented for a feeding period of 7 days. After each 7-day feeding period, the remaining feed was weighed and then discarded; the results were used to calculate the mean daily food consumption.
Water was added to the feed preparations at a 1:10 volume/ weight ratio to ensure pelleting. The preparations were repelleted with a Buehler pelleting machine. The pellets were dried and stored at room temperature in disposable containers (paper bags) .

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability, concentration and homogeneity of DESMEDIPHAM TECHNICAL in the feed preparations was determined in the analytical laboratories of RCC.

Duration of treatment / exposure:

28 days

Frequency of treatment:

ad libitum in feed.

Dose / conc.:

0 ppm

Dose / conc.:

100 ppm

Dose / conc.:

400 ppm

Dose / conc.:

1 600 ppm

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control:

None.

Observations and examinations performed and frequency:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at 4 weeks of treatment.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- How many animals: 10 / sex / group
- Parameters checked: extra determinations; Heinz body, Met-Hb, red cell morphology.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at 4 weeks of treatment.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- How many animals: 10 / sex / group

OTHER: Histopathology (brain, heart, liver, kidneys, spleen, bone marrow, testes, epididymides, ovaries, uterus, adrenal glands) was carried out from all mice.

Statistics:

univariate one-way analysis of variance, Student’s t-Test, Williams’ t- Test, median test

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No treatment-related mortality or clinical observations were noted. One control male and one male at 300 ppm died spontaneously following blood sampling at the study termination.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no significant differences between groups in body weight.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption was increased in both sexes at 1600 ppm during weeks 3 and 4.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

There was a dose related increase in Heinz body and methemoglobin formation in all treated mice, with statistically significant increases at 400 and 1600 ppm. A slight decrease in erythrocyte count, hemoglobin concentration and hematocrit values in males at 1600 ppm, along with slight reticulocytosis in all animals at 1600 ppm.
Morphological changes were also observed in the erythrocytes as increased anisocytosis in the males at 100 ppm and in both sexes at 400 and 1600 ppm and increased polychromatophilia in all animals at 1600 ppm.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The clinical biochemistry data showed significantly reduced alkaline phosphatase levels in the females at 400 and 1600 ppm.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Spleen weights increased in both sexes at 1600 ppm (males: 65% abs, 74 % rel to body weight ratio, 65 % rel to brain ratio. Increases in spleen weight in females: 33 % abs, 31% rel to body weight ratio, 30% rel to brain weight ratio)) supporting the hematological observations. Increased absolute heart (13%) and kidney (9%) and relative heart (11%) and kidney weights (8%) and heart to brain weight ratio (11%) in the group 4 females and relative heart weight of the group 4 males (8%) were also observed.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Dose related extramedullary hematopoiesis, mainly erythropoiesis, in the spleen was noted. Increased erythropoiesis (increased red cell precursors) in bone marrow was seen in 4/10 males at 1600 ppm. These observations were referred to by the investigators as reactive and compensatory processes in response to the hemolysis caused by the test compound.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

Remarks on result:

other: Equivalent to 22 and 26 mg/kg bw/d in males and females respectively

Key result

Dose descriptor:

LOAEL

Effect level:

400 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

400 ppm

System:

other:

Organ:

other: Erythrocyte (Met-Hb formation)

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

A NOAEL of 100 ppm, corresponding to 22 and 26 mg/kg bw/d in males and females respectively, can be determined for this study.

Executive summary:

In this 28-day feeding study was administered in the diet to groups of NMRI mice for 28 days. The study was comprised of 4 groups, each containing 10 male and 10 female mice. The nominal DESMEDIPHAM TECHNICAL concentrations were 0, 100, 400, 1600 ppm. There was no treatment-related mortality.  No signs of toxicity  related to treatment were observed.  Mean food consumption values of groups 1, 2 and 3 were comparable.  Mean body weight gain of all groups was comparable.  Mean food conversion of group 4 animals during treatment weeks 2 to 4 was greater than that of groups 1, 2 and 3.  Haematology findings indicated haemolytic anaemia, characterised by a dose-dependent increase in Heinz body and methaemoglobin formation in groups 3 and 4.  A trend to increased Heinz body formation was also noted in group 2.  Other changes were indicated by a slight decrease in erythrocyte count, haemoglobin concentration and haematocrit value of group 4 males, and by slight reticulocytosis in group 4.  Similarly, morphological changes in the erythrocytes were indicated by increased anisocytosis in the group 3 males and group 4,, and by increased polychromatophilia in group 4.  Mean spleen weight was increased in group 4. Increased hemopoiesis in the spleen was noted in two group 2, five group 3 and thirteen group 4 mice. Increased erythropoiesis in the bone marrow was noted in four group 4 males. These findings reflect reactive and compensatory processes in response to hemolysis caused by DESMEDIPHAM TECHNICAL. A NOAEL of 100 ppm, corresponding to 22 and 26 mg/kg bw/d in males and females respectively, can be determined for this study based on hematological effects (increase in Heinz body and methemoglobin formation in all treated mice, with statistically significant increases at 400 and 1600 ppm. A slight decrease in erythrocyte count, hemoglobin concentration and hematocrit values in males at 1600 ppm, along with slight reticulocytosis in all animals at 1600 ppm) and increased hematopoiesis in the spleen mainly at mid and high doses.

Reference 10

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reason / purpose for cross-reference:

reference to same study

Reference 11

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1983-01-17 to 1984-11-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

After the study was performed, a new version of the OECD Test Guideline 408 has been adopted 21st september, 1998. Examination of reactivity to stimuli examinations was only addressed in a hearing test. Spleen, epididymides, uterus and thymus should have been weighed in all animals according to current guideline. With these exceptions the main study fulfils the data requirements. accessory sex organs should have been examined histopatologically.

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Version / remarks:

August 22, 1978, and those revised and distributed in 1982.

Deviations:

yes

Remarks:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han (outbred), SPF quality

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: 65 - 78 g for males and 49 - 64 g for females.
- Housing: in groups of five in Makrolon type-4 cages with wire mesh lids and standard sterilised granulated softwood bedding.
- Diet: rat/mouse maintenance diet ad libitum
- Water: Tap water was available in mater bottles ad libitum.
- Acclimation period: 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature: The room temperature was 22 +/- 2 degrees centigrade.
- Humidity: 55 +/- 10 %
- Air changes: The animal room was air-conditioned, with 15 air changes per hour.
- Photoperiod: 12 hours of artificial fluorescent light each day.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Remarks:

Via diet

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): bi-meekly.
- Mixing appropriate amounts with (Type of food): Microgranulated feed was mixed with the test article using a Buehler mixer. Water was added to the feed preparations at a 1:10 volume/weight ratio to ensure pelleting.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of the test article in the feed pellets was determined before starting the test. At monthly intervals during the study, samples from all groups mere checked for dose level and homogeneity. The mean concentrations of desmedipham in feed samples were between 97.8 - 101.3% of the target dose levels and homogeneity was within ±15% for all dose levels.

Duration of treatment / exposure:

90 days

Frequency of treatment:

continuously

Dose / conc.:

0 ppm

Remarks:

Control

Dose / conc.:

300 ppm

Remarks:

Achieved test material intakes were 24 mg/kg bw/day for males and 27 mg/kg bw/day for females, respectively.

Dose / conc.:

1 200 ppm

Remarks:

Achieved test material intakes were 97 mg/kg bw/day for males and 109 mg/kg bw/day for females, respectively.

Dose / conc.:

4 800 ppm

Remarks:

Achieved test material intakes were 415 mg/kg bw/day for males and 378 mg/kg bw/day for females, respectively.

No. of animals per sex per dose:

10/sex/group

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The selection of the dose levels was based on the results of the preliminary study to the multiple generation reproduction study with Desmedipham in rats.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

Body weight, organ weights and clinical laboratory data were evaluated by statistical analysis (one-way ANOVA, Student’s t-Test, Williams’s test).


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Samples for hematology, clinical biochemistry and urinalysis were taken from all animals after 13 weeks treatment.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Samples for hematology, clinical biochemistry and urinalysis were taken from all animals after 13 weeks treatment.
General remark:
Plasma levels of creatine kinase (CK) and lactate dehydrogenase (LDH) activity mere not recorded at 13 weeks for technical reasons.

URINALYSIS: Yes
- Time schedule for collection of urine: Samples for hematology, clinical biochemistry and urinalysis were taken from all animals after 13 weeks treatment.

Sacrifice and pathology:

All animals were subject to gross necropsy.
HISTOPATHOLOGY: Yes

Histopathological examination was performed on all control and high dose animals. From the low and mid dose rats only gross lesions, liver, kidneys, lungs, heart, and thyroid glands were examined microscopically.

Statistics:

The following statistical methods were used to analyze body weights, organ weights and clinical laboratory data:
Univariate one-may analysis of variance was used to assess the significance of intergroup differences if the variables could be assumed to follow a normal distribution.
Student's t-test, based on a pooled variance estimate, was used for intergroup comparisons (i.e. single treatment groups against the control group).
Williams's test was used to determine the lowest dose group which was significantly different from the control group, if a monotone (increasing or decreasing) dose-response relationship existed.
The difference of the overall mean of the treated groups to the control group was tested for significance if some evidence existed for differences between treatment groups and the control group, and if a monotone dose-response relationship and significant t-tests were absent.
The median test was used to assess the significance of intergroup differences if the variables were defined on a discrete scale.
The adjustment for multiple testing was performed by comparing the expected and-observed number of significant results obtained.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No treatment related effects were noted on clinical signs, or hearing test. However, amber discoloration of the urine was noted in animals at 4800 ppm, and this was probably caused by desmedipham and/or its metabolites.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One animal (male) of group 4 died during blood sampling on the scheduled day of necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weight gain of the group 4 animals was reduced when compared with that of the control group. The same finding was noted in the group 3 females from week 10 to termination.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean food consumption of the group 4 females was reduced by approximately 20 - 30 % when compared with that of the control group.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

The mean food conversion of the group 4 males was slightly higher than that of the control group.

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

effects observed, treatment-related

Description (incidence and severity):

The values of the group 4 females were reduced. During week 11, a reduction of about 20% was observed when compared with that of the control group.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The ophthalmological examination results of groups 2, 3 and 4 were similar to that of the control group.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The hematological effects indicated hemolytic anemia and a compensatory erythrogenic response. Methemoglobin formation was dose-related and statistically significantly increased in all treated groups. Statistically significant decreases in red blood cell counts (5-24%), hemoglobin (4-19%), MCHC (2-8%) and hematocrit (4-9%) and increase in MCV and reticulocytosis (38%-328 %) were observed in all treated groups. At 1200 and 4800 ppm, dose-related increases in Heinz body formation, MCV, MCH and platelets were observed. Coagulation time (thromboplastin time) was increased in males at 300 and 1200 ppm and partial thromboplastin time in females at 4800 ppm.

Morphological changes were observed in the erythrocytes as polychromatophilia and anisocytosis in both sexes at 1200 and 4800 ppm and polychromatophilia in males at 300 ppm. Poikilocytosis, spherocytosis, increased Howell-Jolly bodies and increased nucleated erythrocytes (normoblasts) were apparent in both sexes at 4800 ppm.

Total leukocyte counts (WBC) were increased in males at 4800 ppm and in females at 1200 and 4800 ppm. In the differential counts number of lymphocytes were increased and number of monocytes were decreased in treated females, statistically significantly at 300 and 4800 ppm.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Significant increases in enzyme activities of aspartate- and alanine aminotransferases, alkaline phosphatase and gamma-glutamyl transferase were observed in females at 4800 ppm. The levels of urea, total bilirubin, total cholesterol, calcium, phosphorus, sodium, potassium and chlorine were significantly increased in females at 4800 ppm. Dose-related alterations in the plasma protein electrophoretic pattern (decrease in albumin, albumin/globulin ratio, a-1-globulin, a-2-globulin/males, and gamma-globulin/females) were noted in the test animals at 1200 and 4800 ppm. Additionally, plasma cholinesterase and brain at 300, 1200 and 4800 ppm and erythrocyte acetylcholinesterase levels were reduced significantly in females at 1200 and 4800 ppm.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The absolute organ weights of brain (5%), heart (19%), liver (11%), kidneys (21%), ovaries (33%) and adrenal glands (31%) were reduced significantly in females at 4800 ppm, as were kidney weights in males at 300 (10%) and 4800 ppm (12%). Organ weight to body weight ratios of brain (females:33%, males: 21%), heart (females: 14%, males: 21%), adrenal (females: 12%, males: 13%), liver (females: 25%, males: 32%) and kidney (females: 12%, males: 10%) were increased in both sexes, and relative testes weights were increased in males at 4800 ppm (25%). These changes were most likely related to the decreased body weights at 4800 ppm. At 1200 ppm organ weight to body weight ratios of brain (9%), heart (14%), liver (18%) and kidney (12%) increased in females.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In most group 4 males, the spleen was enlarged and dark red to black in color.
A small number of other gross lesions were observed in various organs in rats of all groups.

Neuropathological findings:

no effects observed

Description (incidence and severity):

No treatment related effects were noted on a hearing test.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological findings were mainly related to the observed hematological changes and were consistent with compensatory erythrogenic responses. In the liver, extramedullary hematopoiesis was noted in 2/20 animals at 1200 ppm and in 6/20 animals at 4800 ppm, and a dose-dependent increase in brown iron-positive pigmentation of Kupffer cells was observed in mid and high dose groups.

A slightly increased incidence in round cell infiltration (lymphoid cells) in liver was observed in both males (0/10, 3/10, 5/10 and 1/10 at 0, 300, 1200 and 4800 ppm, respectively) and females (1/10, 2/10, 3/10 and 2/10 at 0, 300, 1200 and 4800 ppm, respectively). In the spleen, increases in hematopoiesis, mainly erythropoiesis, was noted at all three doses of desmedipham. It was noted that in most group 4 males, the spleen was enlarged and dark red to black in color. In the kidneys, brown, partly ironpositive pigmentation of tubular epithelial cells was increased at 1200 and 4800 ppm. The observed increase in hematopoiesis in spleen and the increased pigmentation in liver and kidneys were related to the hematological effects caused by desmedipham.

An increased incidence in follicular hyperplasia of the thyroid glands was observed in both males and females at 1200 and 4800 ppm. This finding was characterized by reduction of follicular size, increased height of follicular epithelium, and by pale scant colloid. Levels of T4 or T3 were not determined.

Slightly increased incidences in animals with perivascular cuffing (lymphoid cell infiltration) and alveolar foam cells (swollen macrophages) in lungs were mainly observed in males.

Dose descriptor:

LOAEL

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

Remarks on result:

other: Equivalent to 24 mg/kg bw/day for males and 27 mg/kg bw/day for females

Dose descriptor:

NOAEL

Effect level:

< 300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

Remarks on result:

other: Equivalent to: 24 mg/kg bw/day for males and 27 mg/kg bw/day for females

Conclusions:

Desmedipham caused methemoglobinemia and compensatory erythrogenic responses in all treated rats at dose levels of 300, 1200 and 4800 ppm. Increased follicular hyperplasia in thyroid glands was observed at 1200 and 4800 ppm in both males and females. The LOAEL was 300 ppm in this study, based on effects on hematology.

The NOAEL, based on hematological effects, was less than 300 ppm, equivalent to 24 mg/kg bw/day for males and 27 mg/kg bw/day for females. The study was acceptable.

Executive summary:

In this oral toxicity study, Desmedipharm was administered to SPF-bred Wistar rats in the diet for 13 weeks. The study was comprised of 4 groups, each containing 10 male and 10 female rats. The following nominal concentrations of the test article were administered: 0 (group 1), 300 (group 2), 1200 (group 3), 4800 (group 4) ppm. The mean concentrations of desmedipham in feed samples were between 97.8 - 101.3% of the target dose levels and homogeneity was within ±15% for all dose levels. Samples for hematology (extra determinations; Heinz bodies, methemoglobin, cell morphology, cholinesterase levels), clinical biochemistry and urinalysis were taken from all animals after 13 weeks treatment. Histopathological examination was performed on all control and high dose animals. From the low and mid dose rats only gross lesions, liver, kidneys, lungs, heart, and thyroid glands were examined microscopically.

Desmedipham caused methemoglobinemia and compensatory erythrogenic responses in all treated rats at dose levels of 300, 1200 and 4800 ppm. Increased follicular hyperplasia in thyroid glands was observed at 1200 and 4800 ppm in both males and females. The LOAEL was 300 ppm in this study, based on effects on hematology.
The NOAEL, based on hematological effects, was less than 300 ppm, equivalent to 24 mg/kg bw/day for males and 27 mg/kg bw/day for females. The study was acceptable.

Reference 12

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reason / purpose for cross-reference:

reference to same study

Reference 13

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reason / purpose for cross-reference:

reference to same study

Reference 14

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1985-02-04 to 1985-11-31

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Pre-dates test guideline but broadly comparable to guideline.

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Version / remarks:

1982

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1981

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rat, KFfl-han., outbred, SPF quality. Standard species/strain with historical control data available.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age (at delivery): 6 weeks.
Body weight and range (at pre-test) males-. 68-100 g females-. 58- 86 g
Identification Cage number and individual ear tags.
Randomization Computer-generated random algorithm.
Acclimation 7 respectively 5 days under test conditions, after veterinary examination.
Conditions Optimum Hygienic Conditions behind a barrier system. Air-conditioned with 10 - 15 air changes per hour, and hourly monitored environment with temperature 22 +/-2 degrees centigrade, relative humidity 55 +/-1QX, 12 hours artificial fluorescent light/12 hours dark, music/light period.
Accommodation Groups of 5 in Makrolon type-4 cages with sterilized standard softwood bedding (Lignocell, Schill A6, Switzerland). Bedding was autoclaved at 120 degrees centigrade for 60 minutes before use.
Diet Pelleted standard Kliba 343 rat maintenance diet ('Kliba', Klingentalmuehle. AG, Switzerland) ad libitum. Results of analysis for contaminants are included in the report (Attachment, see pp. 243-249).
Water Tap water, ad libitum. Results of analyses for contaminants are included in the report (Attachment, see pp. 239-242).

Route of administration:

oral: feed

Details on route of administration:

DESMEDIPHAM TECHNICAL mas mixed with micro-granulated feed. Water <1:10 volume/weight ratio) was added to aid pelleting. The pellets were dried for 48 hours before storage.

Vehicle:

water

Details on oral exposure:

Method Oral, by feed admixture.
Frequency Ad libitum
Duration of acclimation period: 5 days under test conditions, with veterinary examination.
Duration of treatment 13 weeks.
Duration of recovery 4 weeks.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and content of the test article in the feed were determined before test initiation and at monthly intervals. Analyses were performed in the RCC Analytical Chemistry Laboratory, according to a method supplied by the sponsor. Stability analyses were performed in RCC Project 015153. An additional stability test was initiated and performed at the beginning of the treatment phase.

Duration of treatment / exposure:

13 weeks.

Frequency of treatment:

Continuous in diet.

Dose / conc.:

0 ppm

Remarks:

Control: basal diet

Dose / conc.:

6 ppm

Dose / conc.:

30 ppm

Dose / conc.:

60 ppm

Dose / conc.:

300 ppm

No. of animals per sex per dose:

25

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Based upon a previous 13-week feeding study in rats no clear NOAEL established.
- Fasting period before blood sampling for clinical biochemistry: Samples for hematology, clinical biochemistry and urinalysis were taken from all scheduled animals of each sex and group which were fasted overnight (18-hour period) at 4/5, 9 and 12/13 weeks of treatment, and from all animals of the recovery group prior to sacrifice, at 16/17 weeks.

Positive control:

Not required for this study type

Observations and examinations performed and frequency:

Observations for clinical signs and symptoms of toxicity were performed twice daily, a.m. and p.m. In addition to the daily observations, each rat had a weekly detailed clinical examination which included a palpation for tissue masses. A description of any lesion or mass observed at any examination was recorded and the subsequent progress was monitored.
FOOD CONSUMPTION The food consumption was recorded for 7-day periods and the mean daily food consumption calculated. These data were recorded weekly.
WATER CONSUMPTION Water consumption was monitored by daily visual appraisal of the water bottles. During week 11, the water consumption of all animals was measured quantitatively.
BODY WEIGHTS The body weight of each animal was recorded weekly.
OPHTHALMOSCOPIC EXAMINATIONS Qphthalmoscopic examinations were performed on ten animals per sex and group. A description of any abnormality was recorded. Examinations were performed at pretest, at 13 weeks of treatment and at the end of the recovery period.
ORGAN WEIGHTS
The following organ weights were recorded of all animals necropsied at the end of treatment and after the recovery period-. adrenal glands, brain, heart, kidneys, liver, ovaries, pituitary gland, spleen, testes, thyroid gland.

NECROPSY AND HISTOPATHOLOGY
Necropsy was performed on all rats. Tissue specimens were fixed in 4% neutral phosphate buffered formalin, processed and embedded in paraffin. Sections were cut at an approximate thickness of 4 micrometers and stained with hematoxylin and eosin.
Sections of the following organs/tissues were examined microscopically (number of sections per organ) •.
Adrenal glands <2>, aorta (thoracic, 1>, bone (sternum, 1), bone marrow (sternum, 1> , brain (3) , cecum (1>, colon (1) , duodenum (1), epididymides (2), esophagus (1), eyes (2), Harderian glands (2), heart (1), ileum (1), jejunum (1), kidneys (2), liver (2), lungs (2), lymph nodes (mandibular, mesenteric, 1), mammary gland (1), ovaries (2>, pancreas (1) , parathyroid glands (uiherever possible, 2), pituitary gland (1), prostate (1), rectum (1), salivary glands (1), sciatic nerve (i), seminal vesicles (2), skeletal muscle (1), skin (1), spinal cord (1>, spleen (1), stomach (1), testes (2), thymus (wherever passible, 1), thyroid gland (2), tongue (1), trachea (1), urinary bladder 01 > , uterus (3), and all gross lesions.
From all rats, lungs, liver, kidneys, and gross lesions were examined. The other organs were examined only from the rats of groups 1 and 5 that were killed at the end of the administration period (K0).

Sacrifice and pathology:

Fifteen rats per sex and group were killed after a treatment period of 13 weeks and 10 rats per sex and group were killed after an additional treatment-free period of 4 weeks.

Statistics:

The following statistical methods were used to analyze the body weights, food consumption, water consumption, organ weights and clinical laboratory data: Univariate one-way analysis of variance was used to assess the significance of intergroup differences. If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups. The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution. For the overall spontaneous mortality data, the Fisher's exact test for 2x2 tables was applied. Group means were calculated for continous data and medians were calculated for discrete data (scores) in the summary tables. Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical symptom or sign of toxicity was evident.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No treatment-related death was observed during this study one male of the control group (no. 9) died spontaneously during this study. The cause of death remains unclear.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight gain of all treated animals was unaffected by the treatment with DESMEDIPHAM TECHNICAL.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food consumption of the treated and the control groups were comparable.
The mean daily nominal test article consumption during the reported 13 weeks of treatment was 0.5, 2.6, 5.2 and 26 mg/kg body weight for males and 0.5, 2.7, 5.6 and 27 mg/kg body weight for females of treatment groups 2, 3, 4 and 5, respectively.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

The mean water consumption of the treated and control groups was comparable.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment-related findings were noted.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The assessment of hematological data indicated slight toxic methemoglobinemia and a compensatory erythrogenic response during the 13 weeks of treatment. The effects observed were-.
- slightly increased methemoglobin formation for male and female rats of group 5 at each time point during the treatment,
- minimally increased reticulocyte count for both sexes of group 5 at 9 weeks and for male rats of group 5 at 12/13 weeks of treatment.
Recovery phase-.
- slightly increased methemoglobin level for male rats of group 5 at 16/17 weeks,
- minimally increased reticulocyte count for both sexes of group 5 at 16/17 weeks.
These results indicate that DESMEDIPHAM TECHNICAL induces a slight oxidative attack on circulating erythrocytes with an accompanying increase in circulating reticulocytes . The effects were not cumulative to the extent that the body's normal adaptive mechanisms (such as increased erythropoiesis) would be overloaded, resulting in irreversible damage, nor were Heinz bodies observed. At the termination of the treatment-free period, reversible changes were noted in the methemoglobin levels for both sexes of group 5, however, for the male rats the findings were significantly greater than those of the controls .
All other statistical differences in the results of the hematological parameters were considered to be incidental and of normal biological variation

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The assessment of biochemical data indicated slightly lower T4 values for both sexes of group 5 at 12/13 weeks of treatment, and for the male rats of group 5 at termination of the treatment-free period, at 16/17 weeks. All other statistical differences in the results of the biochemical parameters were considered to be incidental and of normal biological variation

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Description (incidence and severity):

The assessment of urinalysis data indicated no treatment related changes at 4/5 weeks and 12/13 weeks of treatment, nor at termination of the treatment-free period at 16/17 weeks. All differences in the results of the urinalysis parameters were considered to be incidental and of normal biological variation.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the application period - after 13 weeks - a minimal decrease in the mean absolute and organ to brain weight ratio was noted for the pituitary of female rats in groups 4 and 5. These weight changes are interpreted as being incidental because no clear dose relationship could be seen, and neither gross nor histological alterations were present. This is also true for the altered liver weight in group 3 and 4 female rats as for the altered heart weight of the females in group 4. Because of a technical error, the organ weights at the end of the recovery period were not considered to be an accurate reflection of the actual organ weights and therefore not included in this report. However, the organ weights of the animals sacrificed after the treatment period showed no treatment-related differences between the control and treated groups. No treatment-related macro- and microscopic findings were noted in either the 13-week or 17- week sacrifice interval.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related macroscopic findings were observed. A few spontaneous gross lesions were encountered in both control and treated rats.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

A few spontaneous microscopic lesions were observed at a random incidence in various organs of both control and treated rats.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

30 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

Remarks on result:

other: Equivalent to achieved mean intakes of 2.6 and 2.7 mg/kg bw/d un males and females respectively

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

60 ppm

System:

other: Target system / organ toxicity Other: Erythrocyte (Met-Hb formation)

Organ:

other: Erythrocyte (Met-Hb formation)

See results tables attachment for details.

Conclusions:

A NOAEL of 30 ppm (2.6-2.7 mg/kg bw/d) was determined for this study based on haematological changes at 60 ppm.

Executive summary:

In this 13-week oral toxicity (feeding) study, DESMEDIPHAM TECHNICAL was administered in the feed to Wistar rats. The study was comprised of five groups, each containing 25 male and 25 female rats.  Ten animals per sex and dose group were observed for an additional recovery period of four weeks.  No treatment-related death was observed. One male of the control group died spontaneously during this study. No treatment-related clinical signs of toxicity were noted.  Body weights, food and water consumption were unaffected by treatment. The mean daily test article consumption was 0.5, 2.6, 5.2 and 26 mg/kg bw/d for males; 0.5, 2.7, 5.6 and 27 mg/kg bw/d for females.  Ophthalmoscopy did not reveal any treatment-related findings.  Haematological data indicated slight toxic methemoglobinemia and a compensatory erythrogenic response during the 13 weeks of treatment.  Clinical chemistry data indicated slightly lower T4 values for both sexes of group 5 at 12/13 weeks of treatment, and for the male rats of group 5 at termination of the recovery phase.  Urinalysis data indicated no treatment-related changes.
No treatment-related organ weight changes were observed either after 13 weeks or after 17 weeks of the experiment.  No treatment-related macroscopic and microscopic findings were observed after 13 and 17 weeks, respectively.  A NOAEL of 30 ppm (2.6-2.7 mg/kg bw/d) was determined for this study based on haematological changes at 60 ppm.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

2.6 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Good quality oral repeated dose toxicity studies are available in the rat, mouse and dog.

System:

cardiovascular

Organ:

other: Red blood cell (metHb formation)

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The classification of desmedipham for STOT-RE has been considered by RAC on the basis of the same dataset.  The RAC Conclusion (2019) is summarised below:

Effects indicative of regenerative haemolytic anaemia (such as reduced erythrocyte count, reduced haemoglobin (Hb), increased methaemoglobin (MetHb), presence of Heinz bodies, enlarged spleen, haemosiderin deposition in the spleen, liver and kidney, increased reticulocytes, increased extramedullary haematopoiesis) were seen across studies and species.  Haematological effects have been observed in studies of various durations, from 10-day PNDT studies to 2-year studies. The CLP regulation provides guidance values for 90-day studies. For studies of a different duration, guidance values can be extrapolated using Haber’s rule.  Haematological measurements show that the effective doses for Hb reduction and MetHb increase are the same regardless of whether exposure duration is 3 months or 2 years.  Comparison of studies shows that the degree of methaemoglobinaemia after 3 months is similar to that after 10 days of exposure. This information indicates that the effective dose for haematotoxic effects of desmedipham does not decrease with time (i.e. does not follow Haber’s rule). Therefore, RAC did not consider extrapolation of guidance values using Haber’s rule appropriate in this particular case and the default guidance value of 100 mg/kg bw/d was used in the assessment. In addition, studies of longer duration were given more weight in the assessment than short-term studies (in line with CLP guidance, 3.9.2.3.2).  Specific guidance on classification of substances causing haemolytic anaemia is available, according to which, if a haemolytic substance induces one or more serious health effects within the critical range of doses, classification is warranted. It is sufficient for classification that only one of these criteria is fulfilled.  It was concluded for desmedipham that none of the individual criteria was fulfilled.  While acknowledging the clear haematotoxic potential of desmedipham, RAC did not find the effects below the guidance values sufficiently adverse to meet the criteria for classification as outlined in the CLP guidance. 

RAC also concluded that the effects of desmedipham on the thyroid and nervous system do not justify classification.  RAC therefore concluded that no classification was warranted for STOT RE.

No change to the harmonised classification is proposed.