sodium 2-{N-[(3,5-difluorophenyl)carbamoyl]ethanehydrazonoyl}nicotinate

Endpoint summary

Description of key information

Repeated dose toxicity: oral
key study: A combined chronic toxicity/carcinogenicity feeding study in rats (104 weeks) was conducted with a structural analogue substance (free acid of the test item). The NOAEL of the study was considered to be 1500 ppm of the test item, equivalent to a mean daily test substance intake of approximately 69 or 93 mg/kg bw/day in male and female animals, respectively.
Supporting studies: Further supporting studies conducted with the structural analogue substance using a range of mammalian species (dogs, rats and mice) were conducted. These studies support the results of the key study.
Repeated dose toxicity: dermal
Key study: It was considered that 1000 mg/kg/day represents the no-observed adverse effect level (NOAEL) for the test item formulation in the rabbit when administered dermally for at least 21 days.
Supporting study: A further supporting study conducted on a structural analogue substance (free acid of the test item) supports the result of the key study.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1994-02-08 to 1997-03-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted with a structural analogous read-across substance (free acid of the registered substance) in compliance to GLP and similar to current guidelines. Please refer to IUCLID section 13 for read-across justification

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Breeding Laboratories, CH 4414-Füllingsdorf, Switzerland
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: body weight was within +/- 20 % of the mean value for each sex
- Housing: 4 animals of the same sex per cage
- Diet: KLIBA powdered diet no. 343 (Klingentalmuehle AG, Basel, CH-4303 Kaiseraugst, Switzerland) ad libitum, offered freshly weekly.
- Water: Municipal supply of Muttenz/Basel, ad libitum from polyethylene bottles, offered freshly each week.
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 23 +/-2°C
- Humidity: 40 to 80 %
- Air changes: 10 - 15 per hour
- Photoperiod: 12/12 (hours dark / hours light)

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
A premix was prepared weekly by mixing powdered test material with powdered diet. This premix was stored at room temperature until use. Final diets were prepared weekly by mixing the premix with additional powdered diet. The validity of the dose preparation procedure was certified by satisfactory results of dietary test substance analyses.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each batch of animal feed was analysed for chemical and microbiological contaminants before delivery to the facility. These analyses were routinely performed (including bacteriological counts). Test substance homogeneity in diet mixes was verified at beginning of the study on diet prepared for week 1 . Stability of test substance in the diet was demonstrated beforehand. During the study, diet mixes were analysed in weeks 0, 2, 3, 7, 11 ,14, 19, 26, 39, 42 and 52 for confirmation of test article content.

Duration of treatment / exposure:

104 weeks
52 weeks for satellite groups

Frequency of treatment:

daily with diet

Remarks:

Doses / Concentrations:
500, 1500, 5000 , 10000 ppm corresponding to 22, 69, 235 and 517 mg/kg bw/day for males and 29, 93, 323 and 696 mg/kg bw/day for females, respectively
Basis:

No. of animals per sex per dose:

104 weeks exposure: 52 animals per sex per dose
52 weeks exposure (satellite grous): 20 aninmals s per sex per dose
Batch health check animals: 12 animals per sex per dose
Sentinel health check animals: 10 animals per sex per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Based on previous experience with the test item, a broad dose range was applied (0 - 10000 ppm)
- Rationale for animal assignment: Animals assigned in a randomised manner
- Rationale for selecting satellite groups: satellite groups of 20 males and 20 females per dose group were allocated to interim sacrifice after 52 weeks of treatment.
- Post-exposure recovery period in satellite groups: none

Positive control:

Not applicable

Observations and examinations performed and frequency:

Health check
During the acclimatisation phase, all animals were examined by the study veterinarian, and blood was collected for differential white blood cell (WBC) counts, red blood cell (RBC) morphology and serology.
Macroscopic abnormalities. Lungs, liver, kidney, spleen, heart were fixed in buffered 10’% formalin and examined histologically in-house.
Sentinel animals were maintained as control animals and were sampled from blood (but were not fasten overnight before) on weeks 26, 36, 80 and 103, and serology investigation were performed on each occasion. WBC counts were also performed on week 103.

Clinical Observations and Mortality
All animals were checked for viability and signs of illness twice daily (except at weekends and bank holidays, when animals were checked only once). All animals were subjected to a detailed health check, which included a palpation, on a weekly basis. All findings were recorded with a computerised data capture system.

Ophthalmoscopy
Prior to treatment and to the scheduled sacrifices, the eyes of all rats at 0 and 10000 ppm were examined with an ophthalmoscope, following administration of mydriatic solution. In the absence of treatment-related changes, no ophthalmoscopic examinations were performed on animlas at 500, 1500, and 5000 ppm.

Bodyweight and Feed Consumption
Animals were weighed weekly, starting one week before the commencement of treatment Feed consumption was also determined weekly, by calculating the difference in feed given and feed remaining in the hopper at the end of the week. Weight determinations were made with a electronic balance in conjunction with a computerised data capture system. The efficiency of feed utilisation per week was assessed during the first 13 weeks of the study by calculating feed conversion ratios.

Sacrifice and pathology:

Clinical Pathology
During weeks 13, 25, 51, 77 and 103, blood and urine samples were collected from 10 males and 10 females of each main group for clinical pathology investigations. Rats were deprived of food and water during overnight urine collection. Urine was cooled by ice during collection. Blood samples were drawn following urine collection by venepuncture of sublingual vein under Metophane anaesthesia. Additional blood samples were collected at these same time points form 10 males and 10 females o each of the satellite groups for haematological investigations on (weeks 13, 25, 51). These satellite group rats were also deprived of food overnight (but not water) before blood sampling.

Blood smears for differential WBC counts were also prepared in weeks 50 and 102/103 form all survivors, excluding those allocated for the above mentioned week 51 and week 103 investigations. Blood smears were also prepared from animals killed in extremis during the study. Blood was drawn by superficial venesection of the tail vein without anaesthesia.

Heamatological investigations
The following investigations were performed using a Sysmex E-2500 automaed haematology analyser:
haematocrit, haemoglobin concentration, rrythrocytes count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpucuscular haemoglobin concentration, Platelets count, Leucocytes count.

Blood smears were prepared and following paramters were evaluated:
reticulocytes, banded neutrophils, segmended neutrophils, lymphocytes, monocytes, eosinophils, basophils.

Clinical Chemistry Investigations:
Total Bilirubin, total protein, albumin, total globulin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamyl transferase, creatine kinase, lactate dehydrogenase, triglycerde, toal cholesterol, creatinine, fastening glucose, urea, calcium, chloride, inorganic phosphate, sodium, potassium.

Rinalysis:
Volume, specific gravity, pH, protein, glucose, keones, occult blood, urobilinogen, leucocyts, nitrite, urine sediment analysis

Terminal investigations
Moribund animals which were killed for human reasons during the course of the study, and surviving animals killed at scheduled necropsies after 52 were all sacrificed by carbon dioxide asphyxiation.

Macroscopic Examination
All animals on study, whether sacrificed or found dead, were subjected to a complete post-mortem examination, which included examination of the following:
external surfaces
all orifices
the cranial cavity and brain
thoracic
abdominal and pelvic cavities with associated organs and tissues
the neck with its tissues.
All abnormalities, including all suspected tumours

Organ Weights
The following organs were weighed at scheduled necropsies by use of electronic balance:
spleen
adrenals
brain
liver
kidneys
testes
heart
ituitary
ovaries (including oviduct)

Organs were trimmed of fat and connective tissue before weighing. Organ to bodyweight and brain weight ratios were calculated

Histopathology
Samples of the following tissues were preserved in 10% buffered formalin (except for eyes, which were fixed in Davidson's solution) until histoprocessing:

Brain
Stomach
Uterus (+ Cervix)
Pituitary
Duodenum
Ovaries
Tongue
Ileum
Mammary glands
Trachea
Jejunum
Skin
Thyroids
Caecum
Lymph nodes
Parathyroids
Colon
Salivary glands
Oesophagus
Rectum
Sciatic nerve
Thymus
Kidneys
Spinal cord (3 levels)
Heart
Adrenals
Femur (+ stiffle joint)
Lungs
Testes
Skeletal muscle
Liver
Epididymis
Sternum (+ bone marrow)
Spleen
Seminal vesicles
Eyes (+ optic nerve)
Pancreas
Prostate
Skull
Aorta
Urinary bladder
Lacrymal
Glands
(Harderian and exorbital)
Vagina
Gall bladder
All abnormalities

Statistics:

All animal data (as bodyweights, feed consumption and clinical observations) were recorded on-line and processed by a VAX-computer based software package (PSA, Scientific Computer Consultants Inc., Ringwood, NJ 07456, USA). Organ weights data were recorded manually and processed by PSA. Means, standard deviations and statistical analyses of the various data were calculated, where appropriate.
The following statistical tests were performed:
- Parametric data: ANOVA followed by Dunnets test.
If ANOVA failed, the following non-parametric methodology was used:
- Count data: Chi-squar, followed by Fishers exact test.
- Non-Parametric data: Kruskall Wallis, followed by Mann-Whitney-U.

Details on results:

52-week interim evaluation

1000 ppm
Reduced bodyweight gain (-20% vs. control) and reduced food utilisation efficiency in both sexes. Lower glucose and triglyceride levels in males in week 13 and 25 (approximately - 35 and -50 %, respectively) and 51 (triglyceride only), whereas phosphate was slightly increased (+17 %, p< 0.01) at the same time points.

5000 ppm
Reduced bodyweight gain in male rats only (male rats; -9% vs. control, p<0.01) (female rats; -4% vs. control. p>0.05), lower triglyceride levels in males in week 13, 25 and 51 and higher phosphate levels in week 13 in male rats only.
1500 ppm
Slight bodyweight gain deficit was observed in males only (-6% vs. controls, p<0.05).

500 ppm
No effects were observed

Results after termination of the study

Analysis of diets
 The results of the diet analyses demonstrated adequate exposure of the animals to the test substance at dietary concentrations well within the limits that are considered tolerable for feeding studies (i.e. ±20% of nominal for single analytical results, ±10 % of nominal for averaged concentrations over the whole dosing period), with the exception of Dose 500 ppm week 0.

Health check
The investigations performed during the acclimatisation phase did not reveal any signs of disease and the animals were regarded to be generally healthy and suitable for the study. The serological investigations on the presence of pathogens in sentinel animals were largely negative. Antibodies againstBacillus piliformis,which can cause Tyzzer's disease in adolescent rats, were detected in 11/19 rats in week 26, but were no longer detected in week 36. An immune response toB piliformisis observed frequently in the Han Wistar strain of rat, but outbreaks of nTyzzer's disease have not been observed in recent years. Accordingly, this finding was not
considered to be of clinical relevance. Furthermore, all macroscopic and microscopic findings observed in sentinel animals were considered to be incidental and commonly diagnosed in rats of this strain and age. No lesions were noted that were considered due to an infectious disease.

Mortality
The overall group distribution of deaths during the 104 week treatment period did not distinguish
treated animals from controls. Less males in the treated groups died compared with the control
mortality. Among females, there was a slightly higher mortality among treated groups compared
with the controls although the incidences did not show any dose relationship and the survival
was within the expected range.

Clinical observations
The incidence and severity of clinical signs, which were mainly related to skin and fur, or of palpable masses, did not distinguish treated animals from controls.

Ophthalmoscopy
All changes detected were considered as incidental otr as spontaneous background lesions, and therefore not related to treatment.

Body weights
Treatment-related and statistically significant effects on body weight development were observed in males at 10000 ppm, 5000 ppm and 1500 ppm in males and in females at 10000 ppm.
The body weight gain deficit resulted in mean body weight values at termination (week 104) of 23%, 11% and 8% lower than the control value for males at 10000 ppm, 5000 ppm or 1500 ppm, respectively, and 21% lower than the control value for females a 10000 pp. However, the differences only developed in the last three month of the study and did not attain a level of statistical significance. Therefore, the biological relevance of these differences is in doubt.

Fod consumption
The overall mean food consumption values did not distinguish treated animals form controls. Significant reductions in mean food consumptions which were observed in males and females at 10000 ppm (weeks 18-19, 73-74,77 80, 87, 90 and 91-92) are considered possibly treatment-related. Other significant deviations form weekly control feed consumption means were occasional and within the range of normal biological variation, and were therefore considered to be incidental in nature.

Test material intake
Mean daily test item intakes at 500, 1500 and 5000 ppm were linearly related to dietary concentration. The reduced weight gains at 10000 ppm caused disproportionally higher test item exposure levels. As a result of body weight development with largely unchanged food intake over time. the exposure levels within treatment groups gradually decreased in the course of the treatment period.

Haematology
There were no changes in haematology parameters that distinguished treated animals from controls. A l l statistically significant deviations (e.g. males: platelets, lymphocytes, monocytes segmented neutrophils, eosinophils, banded neutrophils, WBC, MCV, MCHC' females: haematocrit, haemoglobin, WBC, monocytes, eosinophils, banded neutrophils platelets lymphocytes, segmented neutrophils) were not clearly dose-dependent. These values were within the range of observed control values. Therefore, the effects were attributed to normal biological variation and considered to be incidental in nature. Reticulocyte counts showed a slight increase at week 25 only in females treated at 10000 ppm. On all other occasions, the differences between reticulocyte values for controls and the males and females receiving 10000 ppm were not statistically different. During red blood cells morphology evaluation, a slight increase of polychromasia and anisocytosis was noticed at weeks 25 and 51 in females treated at 10000 ppm or 5000 ppm and a slight increase of anisocytosis at week 13. Minor differences between control and high dose groups for male animals were evident only at week 13 of treatment. However, it is stressed that such increases were only marginal, and not strictly dose-related. Therefore, these differences are considered to have little or no biological relevance.

Clinical chemistry
Blood chemistry investigations gave no clear indication o f a treatment-related effect in anv organ
System. However, the following statistically significant deviations from control mean values were not excluded as possible treatment-related effects:
-Lower triglyceride levels in males at 5000 and 10000 ppm in weeks 13, 25, 51 and 77 with a dose-related trend. However, there were no indications of triglyceride accumulation in liver parenchyma at 5000 and 10000 ppm and the biological relevance of the lower plasma triglyceride levels is, therefore, uncertain.
-Lower glucose levels in males at 10000 ppm in weeks 13, 25, 51 and 77.
-Higher phosphate levels in males at 5000 ppm in week 13, and in males at 10000 ppm in weeks 13 and 25.
All other statistically significant deviations from control mean values were, considered to be mincidental in nature.

Urinalysis
Throughout the study, a slight tendency was present for increased numbers of amorphous urate and/or uric acid crystals m the urinary sediment of both males and females at 10000 ppm or 5000 ppm and at week 25 only in males at 1500 ppm. These crystals are the most common form of crystals seen in normal urine of an acidic pH. Since there were no relationships to dose or time these observations are considered to be of no biological relevance. Furthermore, no indication of a renal dysfunction was obvious from the blood chemistry results or the histopathology examinations.

Organ weight
At the 52-week interim sacrifice, all statistically significant deviations from the control mean absolute and relative organ weights were attributed to reduced body weights at 5000 and 10000.
After 104 weeks of treatment, final bodyweights for males and females at 10000 ppm and for males at 5000 ppm were statistically significantly lower than control values. Absolute and relative adrenal gland and kidney weights for females at 10000 ppm were higher than control values, with the relative weights attaining a level of Statistical significance No histological correlates could be found at microscopic examination.
Other values which attained a level of Statistical significance were considered to be the consequence oft he reduced bodyweights at 10000 ppm and 5000 ppm.

Histopathology
Macroscopic Findings
A number of macroscopic findings were diagnosed in rats that died or were sacrificed in extremis during the course of the study, and in those rats that were sacrificed on schedule after 52 or 104 weeks of treatment. The type, incidence and severity of these gross lesions were not considered to distinguish treated animals from controls.

Microscopic Findings
The non-neoplastic changes that were observed microscopically mainly affected the endocrine, reproductive and large parenchymatous organs. They were generally categorized as inflammatory, degenerative or hyperplastic lesions, and their incidence, type and severity did not distinguish treated from control rats. The type, incidence and organ distribution of neoplasm were similar in both treated and control rats and the diagnosed neoplastic lesions were not considered to be related to the administration of the test substance.

Dose descriptor:

NOAEL

Effect level:

69 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Based on changes in body weight and blood chemistry parameter.

Dose descriptor:

NOAEL

Effect level:

93 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Based on changes in body weight and blood chemistry parameter.

Dose descriptor:

NOAEL

Remarks:

After 52-weeks interim toxicity evaluation

Effect level:

80 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Based on body weight and haematology parameters

Dose descriptor:

NOAEL

Remarks:

After 52-weeks interim toxicity evaluation

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Based on body weight and haematology parameters

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

69 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Recommended study to simulate a likely route of exposure. The study was performed with a structural analogous read-across substance (free acid of the registered substance).

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995-09-26 until 1995-10-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance to GLP and according to current guidelines.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Ltd, Bicester, Oxon, England
- Age at study initiation: approximately 14 weeks
- Weight at study initiation: 2.2 to 2.7 kg
- Housing: individually, in Macrolon size 3 plastic cages
- Diet: standard laboratory diet SDS Rabbit Diet SQC, ad libitum
- Water: ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 21°C
- Humidity: 41 to 72%
- Photoperiod: 12/12 (hours dark / hours light)
- Air changes (per hr): approximately 19

Type of coverage:

occlusive

Vehicle:

water

Details on exposure:

TEST SITE
- Area of exposure: dorsal region
- % coverage: 10%
- Type of wrap if used: Impervious bandage "Elastoplast"

REMOVAL OF TEST SUBSTANCE
The test substance remained on the back of each rabbit for approximately 6 hours each day after which time the dressings were removed and the treated skin washed with warm (30 to 40 °C) water and gently blotted dry.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2 mL/kg bw
- Constant volume or concentration used for each dose group: yes
- For solids, paste formed: yes

VEHICLE
distilled water to for moistening the powder and to ensure good contact with the skin.
USE OF RESTRAINERS FOR PREVENTING INGESTION: no

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

not performed

Duration of treatment / exposure:

6 h per day for 21 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
nominal per unit body weight

No. of animals per sex per dose:

5 animlas per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The appropriate weight of test substance was spread evenly over the prepared skin of Group 2 to 4 rabbits inclusive. As the test substance is a powder, it was moistened using distilled water to ensure good contact with the skin. The control group was treated in a similar manner receiving water alone.
The treatment site was occluded by covering with an impervious bandage consisting of "Elastoplast" adhesive dressing backed with impervious "Sleek" plaster.
The test substance remained on the back of each rabbit for approximately 6 hours each day after which time the dressings were removed and the treated skin washed with warm (30 to 40 °C) water and gently blotted dry. Each animal received a constant dosage based on its most recently recorded body weight. Animals were treated once daily for at least twenty-one consecutive days. Treatment of all animals not scheduled for sacrifice on Day 22 was continued up to 24 hours prior to sacrifice.

Positive control:

not applicable

Observations and examinations performed and frequency:

Clinical signs
All animals were observed three times daily for signs of ill health, behavioural changes or toxicosis. Any observed changes were recorded.
All animals were checked early in each working day and again in the late afternoon to look for dead or moribund animals. This allowed a post mortem examination to be undertaken during the working part of that day.

Local dermal irritation
Local irritation was recorded immediately prior to the first daily application of the test substance and subsequently daily. Reactions (erythema and oedema) resulting from treatment were assessed on a numerical basis according to a Draize scoring system as follows:

Erythema and eschar formation:
No erythema 0
Slight erythema 1
Well-defined erythema 2
Moderate erythema 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) 4

Oedema formation:
No oedema 0
Slight oedema 1
Well-defined oedema (area well defined by definite raising) 2
Moderate oedema (raised approximately 1 millimetre) 3 Severe oedema (raised more than 1 millimetre and extending beyond
the area of exposure) 4
Any other lesion not covered by this scoring system, was described.

Bodyweight
All rabbits were weighed prior to dosing on Day 1 (Week 0) and subsequently at weekly intervals throughout the study.
Food consumption
The quantity of food consumed by each rabbit was measured at weekly intervals throughout the study.

Removal of blood samples
Food was withdrawn overnight prior to collection of samples. Blood was withdrawn from the median artery of the ear of all rabbits prior to termination (Week 3). Further removal of blood samples for re-analysis was carried out for individual animals, and the results of these analyses are presented in the appendices.

The collected blood samples were divided as follows:
EDTA anticoagulant tubes for haematological investigations
Citrate anticoagulant tubes for coagulation test
Heparin anticoagulant microtainer tubes for biochemical tests

Haematology
Packed cell volume (PCV)
Haemoglobin (Hb)
Red cell count (RBC)
Mean corpuscular haemoglobin concentration
Mean corpuscular volume
Mean corpuscular haemoglobin

Total white blood cell (WBC) count
Differential WBC count
Neutrophils
Lymphocytes
Eosinophils
Basophils
Monocytes
Large unstained cells
Cell morphology: the most common morphological changes (anisocytosis, micro/macrocytosis, variation in colour, hypo/hyperchromasia, left shift, atypical/blast cells) were recorded.
Platelet count
Thrombotest (TT)

Biochemistry
The following parameters were analysed with a Hitachi 737 Clinical Chemistry Analyser
Glucose
Total protein
Albumin (Alb)
Globulin (Glob)
Urea nitrogen
Creatinine
Alkaline phosphatase
Alanine aminotrnasferase
Aspartate aminotransferase
Bilirubin
Sodium
Potassium
Calcium
Chloride
Inorganic phosphorus
Cholesterol

Sacrifice and pathology:

Termination
All animals surviving treatment were randomly killed on Day 22, Day 23 or Day 24 by means of an intravenous overdose of pentobarbitone sodiu and a complete autopsy undertaken. Specified organs were weighed and relevant tissue samples were fixed for microscopic examination.

Organ weight
The following organs from each animal were dissected free of fat and weighed:
adrenals
ovaries
brain
spleen
kidneys testes (with epididymides)

Macroscopic pathology
The macroscopic appearance of the tissues of all rabbits was recorded and samples of the following tissues were preserved in 10% buffered formalin:
adrenals
aorta
brain (medullary, cerebellar
and cortical sections)
caecum colon duodenum
eyes (Davidson's fluid as fixative)
gall bladder
heart
ileum
jejunum
kidneys
larynx
liver
lungs
lymph nodes (cervical and mesenteric)
mammary glands
oesophagus
ovaries
pancreas
pharynx
pituitary
prostate
salivary
gland
sciatic nerve
skeletal muscle
skin (treated and untreated)
spleen
sternum (for bone and marrow sections)
stomach
testes (including epididymis)
thymus (where present)
thyroid (with parathyroid)
tongue
trachea
urinary bladder
uterus (with cervix)
vagina
any macroscopically abnormal tissue

Microscopic pathology
Fixed tissue samples required for microscopic examination were prepared by embedding in paraffin wax - sections were cut at 4 µm and stained with haematoxylin and eosin.
Microscopic examination of prepared slides (from tissues indicated under "Macroscopic pathology" were carried out for all rabbits of Group 1 (control group) and Group 4 (high dosage group) killed on Day 22/23/24.

Statistics:

All statistical analyses were carried out separately for males and females.
The following sequence of statistical tests were used for bodyweight gains, food consumption, organ weight and clinical pathology data:
If the data consisted predominantly of one particular value (relative frequency of the mode exceeded 75%), the proportion of values different from the mode were analysed by Fisher's exact test followed by Mantel's test for a trend in proportions.
Otherwise:
Bartlett's test was applied to test for heterogeneity of variance between treatments. If significant heterogeneity was found at the 1% level, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried out followed by Williams' test for a dose related response.
If significant heterogeneity of variance was present and could not be removed by a logarithmic transformation, the Kruskal-Wallis analysis of ranks was used. This analysis was followed by the non-parametric equivalent of Williams' test (Shirley's test).
Covariate analysis of organ weight data (with final bodyweight as covariate) was also performed using adjusted weights for organs where a correlation between organ weight and bodyweight was established at the 10% level of significance.
Significant differences between control animals and those treated with the test substance were expressed at the 5% (* p <0.05) or 1% (** p <0.01) level.

Details on results:

CLINICAL SIGNS
There were no mortalities. No treatment-related clinical signs were noted for any of the animals throughout the study.

DERMAL REACTIONS
No dermal reactions were seen during the first five days of the study. Dermal irritation, accompanied by yellow staining and cracking, developed during the latter part of Week 1 and during Weeks 2 and 3 for all treatment groups, and the incidence and severity was dosage related. For some animals treated at the low dosage of 100 mg/kg/day dermal irritation (slight to well defined erythema accompanied by slight to well defined oedema) was recorded. Irritation was accompanied by yellow staining and cracking. Most rabbits of the intermediate dosage of 300 mg/kg/day, dermal irritation (slight to moderate erythema accompanied by slight to well defined oedema) was observed. Dermal irritation was accompanied by yellow staining and cracking and occasionally by sloughing. At the high dosage of 1000 mg/kg/day dermal irritation (slight to moderate erythema accompanied by slight to moderate oedema) was recorded. Dermal irritation was accompanied by yellow staining and cracking and occasionally by sloughing.There were no dermal reactions seen for any animals from the control group.

BODYWEIGHT
There were no statistically significant differences from control noted for bodyweight gains in any of the treatment groups. Bodyweight gains showed considerable variation for all groups of rabbits. This is not uncommon for laboratory rabbits and in the absence of any dosage relationship, differences from control were considered to reflect variation.

FOOD CONSUMPTION
There were no statistically significant differences from control noted for the food consumption in any of the treatment groups. Food consumption for low dosage group female rabbits was slightly higher than control and lower than control for intermediate dosage group male rabbits. These small differences from control were considered to reflect variation and not an effect of treatment.

HAEMATOLOGY
There were no differences from control in the haematological parameters measured that were considered to be related to treatment. Statistically significantly higher than control monocyte levels were recorded for male rabbits of the high dosage group. The magnitude of this change was small and a treatment related effect was considered unlikely. nLymphocyte levels were statistically significantly lower than control for female rabbits treated at 1000 mg/kg/day. However, there was wide variation for individual values and the total white blood cell count was not affected. A treatment-related effect was considered unlikely.


BIOCHEMISTRY
There were no differences from control in the biochemical parameters measured.

ORGAN WEIGHTS
There were no differences from control in the organ weights measured.

MACROSCOPIC PATHOLOGY
The macroscopic examination performed at termination revealed yellow staining of the skin in some rabbits at all dosage levels.

MICROSCOPIC PATHOLOGY
Minimal or trace diffuse acanthosis was seen in the majority of male and female rabbits from all treated groups.

A related minimal or trace diffuse inflammation of the superficial dermis was observed in a proportion of male and female rabbits of the 1000 and 300 mg/kg/day treatment groups and male rabbits of the 100 mg/kg/day treatment group. These changes were considered to be dose-related.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Dose descriptor:

NOEL

Effect level:

< 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed.

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

Recommended study to simulate a likely route of exposure.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: oral

1) Key study - Read-across - Combined chronic toxicity/carcinogenicity feeding study in rats

This study was chosen as a key study as it represents the study with the longest exposure time (104 weeks). In addition, when applying assessment factors for interspecies and exposure time, this study leads to the worst case reference value (DNEL). All supporting studies confirm the findings of the key study and lead to the marginal higher DNELs.

Oral administration of a structural analogue substance (free acid of registered substance) to male and female rats at dietary concentrations of 0, 500, 1500, 5000 and 10000 ppm, resulted in mean daily intakes of 22, 69, 235 and 517 mg/kg bodyweight for male, and 29, 93, 323 and 696 mg/kg bodyweight for females, respectively. Treatment had no influence on the survival of the rats nor on their appearance or behaviour. No ophthalmoscopic ocular changes were detected that could be related to treatment. Significant reductions in body weight gain occurred in males at 10000 ppm and 5000 ppm and 1500 ppm, and in females at 10000 ppm without any reductions in food intakes. Hence, a reduction in food utilisation efficiently is considered to be the cause of the reduced mean body weights in these groups. There were no changes to haematology or urinalysis parameters that distinguished treated animals from controls. Blood chemistry investigations indicated lower glucose levels in males at 10000 ppm in weeks 13, 25, 51 and 77, lower triglyceride levels in males at 10000 ppm and 5000 ppm in weeks 13 25, 51 and 77, and higher phosphate levels in males at 5000 ppm in week 13 and in males at 10000 ppm in weeks 13 and 25. The cause of these changes was not determined in this study.

Terminal investigations in animals sacrificed after 52 weeks or those sacrificed after 104 weeks of treatment showed that: - No histopathological finding could be correlated with the changes in mean body organ weights observed at 10000 or 5000 ppm. - The neoplastic or non-neoplastic lesions that were diagnosed were commonly observed findings in rats of this strain and age, and were not related to the administration of the test article.

Conclusion: Based on the bodyweight gain deficits in males, the "no-observable-adverse-effect-level (NOAEL) of the study was considered to be 1500 ppm of the test item, equivalent to a mean daily test substance intake of approximately 69 or 93 mg/kg bw/day in male and female animals, respectively. Furthermore, the study provided no evidence for carcinogenic consequences of lifetime exposure to the test substance.

Moreover, based on the observations made after the 52 weeks intermim period, the"no-observable-adverse-effect-level" (NOAEL) in the first year of the study was considered to be 1510 ppm of SAN 835 H TC, equivalent to a mean daily test substance intake of approximatively 80 or 100 mg/kg/day in male and female animals, respectively

2) Read-across - 13-Week Dietary Toxicity Study in Rats with a 4-Week Recovery Period

A 13 week dietary toxicity study was conducted in order to characterise the subchronic toxicity of a structural analogue test item (free acid of the registered substance) to rats for and to assess recovery over a 4-week recovery period. The study was conducted in compliance with GLP and according to OECD Guideline 408. Groups of 10 male and female rats were used for following dose groups:

0, 1000, 5000, 10000, 20000 ppm (corresponding to 0, 68, 352, 725, 1513 mg/kg bw/day for males and 0, 81, 431, 890, 1750 mg/kg bw/day for females, respectively). Recovery groups (control and high dose) using 10 animals per sex per dose were used.

Bodyweight gain and food consumption were recorded weekly and food conversion ratios and chemical intake calculated.

Ophthalmologic mexamination was performed before and at the end of the study. Clinical pathology investigations were performed after 4 and 12 week, and included: HCT, Hb, RBC, RBC indices, platelets, reticulocytes,total and differential WBC counts; blood glucose, bilirubin, total protein and albumin, cholesterol, hepatic marker enzymes, urea, electrolytes and creatinine; urinary volume, SG, pH, protein, glucose, ketones, urobilinogen, bilirubin, occult blood and examination of sediment. At necropsy, organ weights were recorded and a full spectrum of tissues from high level and control rats was examined histologically. From low and mid-dose animals and from withdrawal group animals, a limited spectrum of tissues including target tissues was also examined.

Results

At 20000 ppm (equivalent to 1513 mg/kg/day in males, 1750 mg/kg/day in females):

A marked retardation of weight gain partly associated with reduced food consumption was seen in both sexes. This proved reversible on withdrawal of treatment. The efficiency of food utilisation was impaired. Slight anaemia (lower RBC) in males, elevated platelet counts in females, and elevated WBC counts in both sexes were seen. Of these, only male RBC counts showed no recovery. Lower globulin values, and elevated cholesterol levels, were seen in both sexes. Values for serum phosphate and chloride tended to be lower than those of controls. At withdrawal, depressed globulin values in males only were found. Urinary pH was lower in males only, and increased incidences of oxalate, uric acid, and triple phosphate crystals were seen in urinary sediment in females only, reversible on withdrawal. Necropsy gave no clear indication of target tissues. Histopathologically, an increased incidence of foamy macrophages in the lung and an increased incidence of testicular seminiferous tubule atrophy was seen.

At 10000 ppm (equivalent to 725 mg/kg/day in males, 890 mg/kg/day in females):

Reduced bodyweight gain was seen in both sexes, without an obvious reduction in food consumption. The efficiency of food utilisation was impaired. A slight anaemia (lower RBC) in males, elevated platelet counts in females, and increased WBC counts in both sexes were found. Elevated cholesterol values were seen in males only. Urinary pH was lower, again only in males; it could be inferred that incidences of oxalate, uric acid and triple phosphate crystals in urinary sediment were marginally increased in females only. Histopathologically, an increased incidence of foamy macrophages in the lung was found.

At 5000 ppm (equivalent to 352 mg/kg/day in males, 431 mg/kg/day in females):

No reaction to treatment was detected.

At 1000 ppm (equivalent to 68 mg/kg/day in males, 81 mg/kg/day in females):

No reaction to treatment was detected.

Conclusion

Treatment with the test item for 13 weeks resulted in a marked retardation in growth partly associated with reduced food consumption, testicular atrophy and accumulations of foamy macrophages in the lung. These findings appeared mostly reversible within 4 weeks. A level of 10000 ppm is considered an appropriate high dose level (MTD) for longer term study. 10000 ppm was the lowest level at which treatment-related effects were detected and is therefore the low effect level in this study. The no-effect level was 5000 ppm.

3) Read-across - A 13-Week Pilot Feeding Study in Mice

This 13 week pilot feeding study study was conducted on a structural analogue substanice (free acid of the registered substance) in order to determine suitable dosage levels for a postulated carcinogenicity study in mice. Groups each of 10 male and 10 female CD-I mice received diet containing 350, 1750, 3500, or 7000 ppm test item for a total duration of 13 weeks. Bodyweights and food consumption were monitored weekly, symptomatology at least weekly. At termination, all mice were sacrificed by carbon dioxide asphyxiation and necropsied. The weights of major organs were recorded, but no histopathology was performed.

Results were as follows: At 7000 ppm (equivalent to 1225 mg/kg/day in males, 1605 mg/kg/day in females):

Food consumption among females during the first week of treatment only was less than that of the controls to a degree which achieved statistical significance. This finding appeared to be without toxicological consequences, and this dosage level is therefore considered to be a no-effect level. At 3500 ppm (613 mg/kg/day in males, 787 mg/kg/day in females):

Food consumption among females during the first week of treatment only was less than that of the controls to a degree which achieved statistical significance. This finding appeared to be without toxicological consequences, and this dosage level is therefore considered to be a no-effect level. At 1750 ppm (287 mg/kg/day in males, 369 mg/kg/day in females):

Food consumption among females during the first week of treatment only was less than that of the controls to a degree which achieved statistical significance. This finding appeared to be without toxicological consequences, and this dosage level is therefore considered to be a no-effect level. At 350 ppm (58 mg/kg/day in males, 84 mg/kg/day in females):

There was no reaction to treatment.

Conclusion: Treatment at 7000 ppm showed no clear toxic effect. This dosage level achieved the upper limit for carcinogenicity testing (1000 mg/kg/day), and is therefore recommended as an appropriate high dose level for the postulated carcinogenicity study.

4) Read-across - 13-Week Feeding study in dogs

A 13 week dietary toxicity study was conducted in order to characterise the subchronic toxicity of a structural analogue test item (free acid of the registered substance) to beagel dogs for 13 weeks and to assess recovery over a further 4-week recovery period (without treatment). The study was conducted in compliance with GLP and according to OECD Guideline 409. Groups of 4 male and female dogs were used for following dose groups:

0, 1500, 10000, 30000 ppm corresponding to 0, 58, 403, 1121 mg/kg bw/day for males and 0, 59, 424, 1172 mg/kg bw/day for females, respectively.

At 30000 ppm (1121 mg/kg/day in males and 1172 mg/kg/day in females) the test item caused anaemia and a marked erythropoietic response in bone marrow, liver and spleen. Additional compensatory extramedullar^ haemopoiesis was observed in the lungs, lymph nodes and kidneys of one female with a severe anaemia. This dog lost weight during treatment and also showed degenerative changes in the liver, presumably as a result of hypoxia. Treatment-related stress at this dosage was indicated by myeloid hyperplasia in the bone marrow of two females, sinusoidal granulocytosis in the liver of two males and three females, and thymic involution in all four females and one male, in addition, non-specific skin changes, urothelial hyperplasia and cystitis in the urinary bladder were observed in all animals in this group.

At 10000 ppm (403 mg/kg/day in males and 424 mg/kg/day in females), the test item caused an erythropoietic response of bone marrow and liver which apparently compensated probable haematological effects, since no changes in peripheral haematological parameters could be demonstrated. The (adaptative) response of the bone marrow is considered indicative of a toxic effect, however, and this dosage is viewed as a "low-observable adverse effect level" (LOAEL).

The test item dose of 1500 ppm (58 mg/kg/day in males and 59 mg/kg/day in females), was determined to be a "no-observable effect level (NOEL) in the present study.

5) Read-across - 52-Week Feeding study in dogs

A 52 week dietary toxicity study was conducted in order to characterise the subchronic toxicity of a structural analogue test item (free acid of the registered substance) to beagel dogs. The study was conducted in compliance with GLP and according to OECD Guideline 452. Groups of 4 male and female dogs were used for following dose groups:

0, 750, 7500, 15000 ppm corresponding to 0, 26, 299, 529 mg/kg bw/day for males and 0, 28, 301, 538 mg/kg bw/day for females, respectively.

Results

15000 ppm: Lower weight gains and less efficient food utilisation (females, only). Hematological parameters indicated reticulocytosis. Clinical chemistry and urinalyses values were indistinguishable from control values, Necropsy revealed reddish discoloration of the femoral diaphysis. Histopaihological examination revealed compensatory erythroid hyperplasia in the bone marrow arid increased hemosiderin deposits in kidneys, spleen and liver.

7500 ppm: Less efficient food utilisation (females, only) in week 0-13. Hematological parameters indicated reticulocytosis. Clinical chemistry and urinalyses values were indistinguishable from control values. Necropsy revealed a reddish discoloration of the femoral diaphysis. Histopathological examination revealed compensatory erythroid hyperplasia in the bone marrow and increased hemosiderin deposits in kidneys, spleen and liver.

750 ppm:No treatment related changes were observed.

Conclusion

The lowest effect level (LEL) was identified at 7500 ppm (299 mg/kg/day in male dogs ; 301 mg/kg/day in female dogs). At this dose level, SAN 835 H TC caused reticulocytosis, erythroid hyperplasia in the bone marrow, and increased hemosiderin deposits in the liver, kidneys and spleen. The adaptive responses of the bone marrow are considered indicative of a toxic effect to erythrocytes.

The no-observable effect level (NOEL) ie the present study is 750 ppm (equivalent to 26 mg/kg/day in the male dogs ; 28 mg/kg/day in female dogs).

Repeated dose toxicity: dermal

1) Key study: 21 day dermal toxicity study in the rabbit

This study was performed to assess the systemic toxicity of the test item to the rabbit. The methods followed GLP and OECD guideline 410. The test item was administered, as supplied, by dermal application, once daily to groups of ten rabbits (five males and five females) for at least twenty one consecutive days at dosages of 100, 300 or 1000 mg/kg/day. The powder was moistened with distilled water at 2 ml/kg bodyweight on the treatment site. At the end of the six hour exposure period the bandages were removed and any residue was washed off with warm (30 - 40°C) water. A further group of ten (five male and five female rabbits) was held as a control treated in a similar manner receiving distilled water alone at 2 ml/kg bodyweight. Clinical signs, dermal reactions, bodyweights and food consumption were recorded during the study. Blood samples for clinical investigations were taken on Day 20 or 21 and the animals were killed and examined macroscopically on Day 22, 23 or 24. Histopathological examination of specified tissues was then initiated.

Dermal reactions:

A dosage-related degree and incidence of dermal reactions were seen: At the high dosage group (1000 mg/kg/day) dermal irritation (slight to moderate erythema, acompanied by slight to moderate oedema) accompanied by yellow staining and cracking developed during the latter part of Week 1 and during Weeks 2 and 3. For most animals of the intermediate dosage group (300 mg/kg/day) and for some animals of the low dosage group (100 mg/kg/day) dermal irritation (slight to moderate erythema accompanied by slight to observed accompanied by yellow staining and cracking, and occasionally sloughing was. There were no dermal reactions seen for animals from the control group.

Microscopic pathology:

Minor treatment-related changes were found in the treated skin samples of rabbits from all treatment groups. These lesions were diffuse acanthosis and diffuse inflammation of the superficial dermis.

Other findings

There were no differences from control in any other parameters measured, namely clinical signs, body weight, food consumption, haematology, biochemistry, organ weights and macroscopic pathology, that were considered to be related to treatment with SAN 836 H 86 SP 401 DP Formulation.

Conclusion The treatment related findings in the skin of dermal irritation and related microscopic changes were considered to represent an adaptive response of the skin to an irritant compound and therefore it was considered that 1000 mg/kg/day represents the no-observed adverse effect level (NOAEL) for the test item formulation in the rabbit when administered dermally for at least 21 days.

2) Supporting study: Read-across - 21 day dermal toxicity study in the rabbit

This study was performed to assess the systemic toxicity of a structural analogue (free acid of the registered substance) to the rabbit. The methods followed GLP and OECD guideline 410. The test item was administered, as supplied, by dermal application, once daily to groups of ten rabbits (five males and five females) for at least twenty one consecutive days at dosages of 100, 300 or 1000 mg/kg/day. The powder was moistened with distilled water at 2 ml/kg bodyweight on the treatment site. At the end of the six hour exposure period the bandages were removed and any residue was washed off with warm (30 - 40°C) water. A further group of ten (five male and five female rabbits) was held as a control treated in a similar manner receiving distilled water alone at 2 ml/kg bodyweight. Clinical signs, dermal reactions, bodyweights and food consumption were recorded during the study. Blood samples for clinical investigations were taken on Day 20 or 21 and the animals were killed and examined macroscopically on Day 22, 23 or 24. Histopathological examination of specified tissues was then initiated. The following comments are made in summary: Dermal reactions No dermal reactions were seen during the first week of the study. Dermal irritation, accompanied by brown staining, developed during Weeks 2 and 3 for all treatment groups. However, the incidence and severity was only slight and was not dosage-related and, in the absence of any histopathological change, was not considered to be an adverse effect. Other findings There were no mortalities or differences from control in any other parameters measured, namely clinical signs, bodyweight, food consumption, haematology, biochemistry, organ weights, macroscopic and microscopic pathology, that were considered to be related to treatment.

Conclusion: It was considered that 1000 mg/kg/day represents the no-observed-adverse-effect level (NOAEL) for the test item in the rabbit when administered dermally for at least 21 days.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Most reliable study (GLP and guideline study).

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
In accordance with column 2 of REACH Regulation EC (No) 1907/2006 Annex IX, the test repeated dose toxicity after inhalation (section 8.6) does not need to be conducted as a repeated dose toxicity studies for oral and dermal applications are available. Furthermore, the test item did not cause any toxic effects in an acute inhalation toxicity study conducted on rats. In addition, due to the solid nature and the very low vapour pressure (estimated to be 2.54E-14 Pa at 25 °C) exposure to the test item via the inhalation route in unlikely. Thus, no test on repeated dose inhalation toxicity has to be conducted and risk assessment is based on oral and dermal studies.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Most reliable study (GLP and guideline study).

Justification for classification or non-classification

Based on the results of the results of the repeated dose toxicity studies, the test item was not classified and labelled according to Directive 67/548/EEC (DSD) and to Regulation (EC) No 1272/2008 (CLP).