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

Carcinogenicity

Description of key information

The read-across test item (free acid of registered product) did not reveal any carcinogenic properties in the condcuted testsin two species (rat and mouse).

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

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

Adequacy of study:

key study

Study period:

1994-10-19 to 1997-02-7

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 451 (Carcinogenicity Studies)

Deviations:

no

GLP compliance:

yes

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River WIGA Deutschland GmbH
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: body weight was within +/- 20 % of the mean value for each sex
- Housing: 5 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: 12 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 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, 6, 12, 25, 38 ,51, 64 and 77 for confirmation of test article content.

Duration of treatment / exposure:

78 weeks
52 weeks for satellite groups

Frequency of treatment:

daily with diet

Post exposure period:

none

Remarks:

Doses / Concentrations:
0, 700, 3500, 7000 ppm corresponding to 0, 100, 517, 1037 mg/kg bw/day for males and 0, 98, 500, 1004 mg/kg bw/day for females, respectively
Basis:
nominal in diet

No. of animals per sex per dose:

78 weeks exposure: 50 animals per sex per dose
52 weeks exposure (satellite grous): 10 aninmals s per sex per dose
Batch health check animals: 10 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 - 7000 ppm)
- Rationale for animal assignment: Animals assigned in a randomised manner
- Rationale for selecting satellite groups: satellite groups of 10 males and 10 females per dose 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
One day after delivery the batch health check animals were sacrificed and necropsied. Blood was collected for differential white blood cell (WBC) counts and serology. Macroscopic abnormalities, lungs, liver, kidneys, spleen and heart, were fixed in buffered 10 % formalin and examined histologically. During the acclimatisation phase, all animals were examined by the study veterinarian. Since these investigations did not reveal any signs of disease the animals were regarded to be generally healthy and suitable for the study. In order to verify the health status of the animals during the study serology and histopathology was also performed on the sentinel animals. For that purpose each 3 male and 3 female mice of the sentinel group were sacrificed in week 30 (instead of week 26) and 53, and the survivors at the end of the study (week 78). Histopathology was performed on selected tissues (heart, lungs, spleen, kidneys, liver and macroscopic abnormalities) from the animals sacrificed on schedule whereas all organs and tissues as listed below in section histopathology were investigated in the moribund sentinel animals killed in extremis in weeks 69 and 70. Detailed results are not reported but maintained as raw data.

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.

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.

Test substance intake
Achieved intake of test substance was calculated per cage using bodyweight and feed consumption data, and the nominal dietary test material concentration.

Haematology
Blood smears were prepared from all non-fasted surviving animals (with exception of the sentinel group) in week 51 (blood drawn by superficial vene section of the tail vein), and in week 78 during terminal sacrifice (blood obtained by punction of the vena cava after anaesthesia). The smears were evaluated for white blood cell (WBC) differential counts and red cell morphology.
Blood smears were also prepared from animals killed in extremis (vena cava blood) and evaluated in house using light microscopy. The results of these specific evaluations are maintained within the raw data but were not reported.

Sacrifice and pathology:

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 and 78 weeks, 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
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:
- Non-Parametric data: Kruskall Wallis, followed by Mann-Whitney-U.

Details on results:

After 52-week interim evaluation

Mortality and clinical signs.
Thirty-two male and 12 female mice died or were sacrificed in extremis during this 52-week treatment period. The group distribution of these early deaths and the incidence of clinical signs and palpable masses did not distinguish treated animals from controls.

Body weight
Deviations of mean weight gain data to controls in the males (+ 8 to - 8 %) and in the females (-10 to -13 %) were considered unrelated to the dose and within the range of normal biological variation.

Food consumption
Food consumption was not affected by treatment at any dose level, nor were group feed conversion ratios (calculated for weeks 0 to 13).

Haematology
Only minor differences were noted between test and control animals (band forms neutrophils and eosinophils in the 7000 ppm males and monocytes in the 3500 ppm females). In all three cases these cell types appeared in very low frequencies, which exaggerated the statistical significance. These differences were, however, considered to be of no biological significance.

Macroscopic findings
A number of macroscopic observations were made in mice that died or were sacrificed in extremis and in those mice that were sacrificed on schedule. The type, incidence and severity of these gross lesions were not considered to distinguish treated animals from controls. With the exception of increased relative mean spleen weight In the high dosed females (mainly due to infiltration by a malignant lymphoma in one animal of this group) no significant deviations of absolute/relative organ weights from control means were observed in any other group.

Histopothoiogy
Revealed a number of non-neoplastic changes in mice of both treated and control groups, which were inflammatory, degenerative and hyperplastic in nature and affected the endocrine, reproductive, and large parenchymatous organs. A few neoplastic lesions were diagnosed, the type, incidence and organ distribution of which did not distinguish treated from control animals.

After temination of the study

Mortality and clinical signs: 108 Male and 107 female mice died or were sacrificed in extremis in the course of the study. The group distribution of these deaths and the incidence of clinical signs and palpable masses revealed no changes that could be attributed to treatment.

Bodyweight development, feed consumption and feed conversion ratios were not affected by treatment during the first 52 weeks of the study. A slight but significant reduction in bodyweight change was observed in females from the 7000 ppm group over the entire study period.

Haematology: Differential white blood cell counts and red/white cell morphology analyses in week 51 and 78 revealed no treatment-related changes.

Macroscopic findings: The type, incidence and severity of gross lesions observed at post-mortem examinations were not considered to distinguish treated animals from controls.

Histopathology revealed a wide range of non-neoplastic and neoplastic changes in all groups of mice. The type, incidence and organ distribution of these changes did not distinguish treated animals from controls.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No treatment related adverse effects observed

Remarks on result:

other: Effect type: toxicity (migrated information)

Dose descriptor:

NOEL

Remarks:

after 52-week interim evaluation

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related adverse effects observed

Remarks on result:

other: Effect type: toxicity (migrated information)

Dose descriptor:

NOEC

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No evidence of carcinogenicity during this study.

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

chronic

Species:

mouse

Quality of whole database:

The study is in compliance with GLP and respective testing guidelines. The study was performed with a structural analogous read-across substance (free acid of the registered substance).

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available experimental data the test substance is not classified and labelled for carcinogenicity according to Regulation (EC) No 1272/2008 (CLP) and Directive 67/548/EEC.

Additional information

Read-across- Carcinogenicity feeding study in mice

The dietary concentration of 7000 ppm of the read-across test item (free acid of the registered substance) equivalent to a mean daily test substance intake of approximately 1000 mg/kg bw/day was considered to be a "no-observable-adverse effect-level" (NOAEL). 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-effect-level" (NOEL) in the first year of the study was considered to be equivalent to a mean daily test substance intake of approximatively 1080 mg/kg/day.

Read-across- Carcinogenicity feeding study in rats

Oral administration of the read-across test item (free acid of the 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

Justification for selection of carcinogenicity via oral route endpoint:
Carcinogenicity study recommended to simulate likely route of exposure.