1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl 4-chloro-4-deoxy-α-D-galactose

Carcinogenicity

Description of key information

The carcinogenicity of the test item via oral application on rats was determined in a GLP study according to FDA guidelines similar to OECD guidelines. Sprague-Dawley CD rats were fed with diet containing test substance at a concentration of 3,000, 10,000 and 30,000 ppm continuously for up to two years. The test animals were derived from parents that had received the test substance at the same concentrations for four weeks prior to pairing and during gestation. Under the conditions of this study, treatment of rats with a diet containing up to 30,000 ppm test substance did not increase the incidence of tumours or influence the type of tumour observed. There were no changes that were considered to be toxic responses to the test item from dietary administration. 

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1985-1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

FDA: Toxicologic principles for the safety assessment of direct food additives and color additives used in food. Red Book 1982.

Principles of method if other than guideline:

Similar to standard OECD carcinogenicity assays, however, study animals orginate from parents who have also been dosed with the test compound for 4 weeks prior to mating and throughout gestation.

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3 batches were received by Sponsor for the study
Batch No. 167001 (Code No. 1-700)
Batch No. 167002 (Code No. JJ1-900; Code No. 1-700)
Batch No. 163003 (Code No. 1-601)

- Purity test date:
Batch No. 167001 (Code No. 1-700): 26 June 1985; 97.9%
Batch No. 167002 (Code No. JJ1-900; Code No. 1-700): 8 Jan 1985; 97.7%
Batch No. 163003 (Code No. 1-601): 8 Jan 1985; 99.4%

RADIOLABELLING INFORMATION--For Satellite Phase-Metabolism Study
- Radiochemical purity: 96-99%
- Specific activity: 0.541 mCi/mmol
- Locations of the label: C14
- Expiration date of radiochemical substance: Not provided

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Weeks 1-28, test material stored at room temperature in the dark.
At request of Sponsor, the test material was stored at 4 degrees Celsius in the dark from Weeks 29 until the end of the study.
- Stability under test conditions:
Samples were returned to Sponsor upon completion of study for analyses and the results indicated that there was no material variation in the composition or quality of the sample since receipt of the sample at the test laboratory at the beginning of the study. In addition, the Sponsor conducted a homogeneity and stability study of the test substance mixed with the rat chow diet. Analysis was done on days 1, 9 and 15 after mixing. It was concluded that the test substance was stable in the rat chow for at least 2 weeks when stored at 23 +/- 2 degrees Celsius.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
A 5% premix of test substance in ground rat diet was prepared as follows: A known quantity of test substance and aliquot of ground diet were admixed and passed through a cross-beater mill fitted with a 2 mm screen. The admixture was then mixed with a further quantity of ground diet for 15 minutes in a 50L mixer, to achieve the 5% concentration. This 5% premix was then diluted with further quantities of diet and mixed for 15 minutes in a 50L mixer to provide the required final test concentrations.

FORM AS APPLIED IN THE TEST (if different from that of starting material) :
Test substance solid/powder was admixed into rat diet.

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Acceptable species to assess toxicity and oncogenicity

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited; Treated rats were mated to produce an F1 generation and then these F1 generation animals were selected for the oncogenicity study.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 24-28 days
- Weight at study initiation: Males-70-76 grams as mean amongst test groups; Females-66-71 grams as mean amongst test groups
- Fasting period before study: None
- Housing: Type RC1 cages from North Kent Plastics Ltd. consisting of high density polypropylene body measuring 56 x 38 x 18 centimeters. Five rats of each sex were housed in each cage.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Time of birth until 24-28 days post partum

DETAILS OF FOOD AND WATER QUALITY:
Drinking water from the public supply was available via polyethylene bottles with chromium plated sipper tubes. The water met the World Health Organiziation and European Economic Community Standards for drinking water. Complete powdered rodent diet, Laboratory Animal Diet No. 2, was available to rats ad libitum. It contained no added antibiotic or other chemotherapeutic or prophylactic agent. Analyses from water and feed suppliers did not indicate any contamination.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 55
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16 November 1983 To: 27 November 1985

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Diet with test substance was prepared freshly each week.
- Mixing appropriate amounts with (Type of food): The 5% premix test rat diet was diluted with further quantities of rat diet and mixed for 15 minutes in a 50L mixer to provide the final required concentrations.
- Storage temperature of food: Test diet was stored in light-proof plastic storage bins in animal room at room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test material-diet admixture were analysed for stability by the Sponsor. Spot checks on the achieved concentration of test substance in the diet were performed by analysing all diets prepared for Weeks 1, 13, 26, 52, 78, and 104. See Table 1 in Results. Homogeneity was assessed by taking samples from six positions in the mix for each of the highest and lowest concentration mixes. The results of the assays demonstrated that the mixing process used provided a stable and homogeneous mix and that the concentrations achieved were satisfactory.

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

Daily, ad libitum through diet

Post exposure period:

None

Dose / conc.:

3 000 ppm (nominal)

Remarks:

0.3%

Dose / conc.:

10 000 ppm (nominal)

Remarks:

1%

Dose / conc.:

30 000 ppm (nominal)

Remarks:

3%

No. of animals per sex per dose:

50 animals/sex/dose

Control animals:

yes, plain diet

Details on study design:

- Toxicokinetic data : Metabolism studies (absorption and excretion) were performed during weeks 26, 52, 83 and 85-87 during treatment with a satellite group (10 males/10 females).
- Dose selection rationale: Not provided
- Rationale for animal assignment (if not random): Random assignment by animal number
- Rationale for selecting satellite groups: Random group of 10 females and 10 males selected to form a group separately from the oncogenicity study.
- Post-exposure recovery period in satellite groups: None, sacrificed during study
- Section schedule rationale (if not random): Random

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Initiation, Weekly for first 14 weeks and then two-weekly intervals thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/week: Yes; Food consumption was calculated weekly not daily
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes; Achieved dosages expressed as mg/kg bw/day were calculated at the same intervals as bodyweight for each group and sex.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes; Group mean food conversion efficiencies were calculated at weekly intervals for the first 14 weeks and at 26-weekly intervals thereafter until Week 78. Efficiencies were not conducted after Week 78 as bodyweight variations caused by approaching senescence and increasing mortality confound interpretation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Once each week for first 13 weeks and at approximately 13 weekly intervals thereafter

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Weeks 11, 25, 38, 51, 64, 77, 96, and 103
- Dose groups that were examined: Control and highest dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 11, 25, 38, 51, and 77 weeks. At 103 weeks, blood samples were withdrawn from 10 male and 10 femal rats with highest animal numbers from each group. Before termination (104 weeks), blood samples were withdrawn from all surviving rats not sampled after 103 weeks.
- Anaesthetic used for blood collection: Yes (light ether)
- Animals fasted: Yes
- How many animals: 10 males and 10 females from each group
- Parameters checked: Packed cell volume (PCV); Haemoglobin concentration (Hb); Erythrocyte count (RBC); Leucocyte (WBC), total; Leucocyte (WBC), differential; Platelet count; Reiculocyte count (Retics); Prothrombin time (PT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Same as that for haematology-11, 25, 38, 51, and 77 weeks. At 103 weeks, blood samples were withdrawn from 10 male and 10 femal rats with highest animal numbers from each group. Before termination (104 weeks), blood samples were withdrawn from all surviving rats not sampled after 103 weeks.
- Animals fasted: Yes
- How many animals:10 males and 10 females from each group (same animals as those drawn for haematology)
- Parameters checked: Urea; glucose; alkaline phosphate activity (AP); alanine amino-transferase activity (ALT); Aspartate amino-transferase activity (AST); total protein; electrophoretic protein fractions; total bilirubin concentration; sodium; potassium; chloride; calcium; magnesium; phosphorus. In addition, after 11 weeks of treatment, the plasma was examined for Triiodothyronine (T3) and Thyroxine (T4) concentration.

URINALYSIS: Yes
- Time schedule for collection of urine: Weeks 11, 25, 39, 51, and 77
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Volume; Calcium; Magnesium; Phosphorus

NEUROBEHAVIOURAL EXAMINATION: No data


OTHER: Organ Weights: Adrenal glands; brain; caecum (full and empty); heart; kidneys; liver; ovaries; spleen; testes; thymus; uterus

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; Examination of external surface; all orifices; cranial cavity; carcase; external surface of the brain; thoracic, abdominal and pelvic cavities and their viscera; cervical tissues and organs.

HISTOPATHOLOGY: Yes; Any abnormalities; adrenal glands; aortic arch; bone (femur with marrow); brain; caecum; colon; diaphragm; duodenum; eyes and optic nerve; fallopian tube; harderian glands; heart; ileum; jejunum; kidneys; lacrymal gland; liver; lungs and mainstem bronchi; lymph nodes; mammary gland; marrow smear; oesophagus; ovaries; pancreas; pituitary gland; prostate gland; rectum; salivary glands; sciatic nerves; seminal vesicles; skeletal muscle; skin; smooth muscle; spleen; spinal cord; stomach; testes; thymus; thyroid and parathyroid glands; trachea; tumours, tissue masses, and regional lymph nodes; turbinate bones; urinary bladder; uterus; vagina; and zymbals gland.

Other examinations:

BREEDING Phase: Test substance was administered continuously in the diet at concentrations of 3000, 10000 or 30000 ppm to groups of 70 male and 70 female rats. Animals were treated for 4 weeks before pairing and treatment was continued throughout pairing, gestation and lactation until termination of the F0 animals. Dams were allowed to rear their offspring to weaning and on day 23 post partum offspring (F1) were randomly selected from first 50 litters to provide animals for the carcinogenicity study and satellite (metabolism) study. Examinations of F0 males and females included: clinical signs, mortality, food consumption, bodyweight, mating performance (conception rate), fertility, and gestation parameters (gestation length, index, live birth index, viability index, litter sizes).

SATELLITE (Metabolism) Phase: To determine whether chronic dietary administration of test substance results in any adaptive changes in rat absorption, metabolism or excretion, 3 males and 3 females from the highest dose group were given a single oral aqueous dose of radiolabelled test material after 26, 52 and 85-87 weeks. Collection of urine and feces followed the dosing at all time intervals. At week 83, expired carbon dioxide was also collected for 24 hours. Selected urine samples were analysed by thin layer chromatography and autoradiogrpahy. All animals were continued to be fed control diet or diet plus test compound during the radio-chemical metabolism phase.

Statistics:

Significance of difference was first conducted betwee the two control groups. In most cases these groups did not differ (P>0.05) and subsequent comparisons were between each treatment group and the combined data for the two control groups. Wherever differences between the two control groups were established, each treated group was compared with each control group.

Time to event analysis of mortality was by Cox's test, both as an overall test for homogeneity of survival curves and for pair-wise comparisons against control. Tarone's extension of Cox's test was used to examine linear trend on dose and to assess deviation from linearity.

For body weight gain, haematology, blood chemistry and urine data, a series of Student's t-tests was performed using a pooled within-treatment error variance. A least significant difference was calculated at the 0.1%, 1% and 5% levels of significance. Differences in absolute, bodyweight-relative organ weights and analysis of covariance using the terminal bodyweight as covariate were assessed using Dunnett's test. A least significant difference was calculated at the 99.0 and 95.0% confidence levels. As the weights of the caecum (empty and full) for animals receiving the highest concentration were clearly higher than those for controls after 52, 78 and 104 weeks of treatment, analysis of covariance was not considered appropriate and they were therefore excluded from the analysis.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs recorded were not considered to be related to treatment. Ingestion of diet had no influence on the number of rats bearing palpable swellings.

Signs typical of a sialodacryoadenitis virus (SDA) infection, including swelling of the glands in teh throat, ocular discharge and occasional sneezing, were noted in Weeks 4 and 5 of treatment. The infection was mild and transitory in nature, and affected animals of either sex in control and treated groups. Following SDA infection, corneal opacity was observed in a small proportion of animals from all groups.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Survival among males receiving the lowest or highest concentrations was higher than that for controls and there was a signficant trend toward enhanced survival with increasing concentration but with a significant departure from linearity. In females, there were fewer deaths among rats receiving the intermediate or highest concentration than in control groups and further there was a significant trend towards higher survival with increasing dietary concentration without signficant departure from linearity. The survival of male rats after 104 weeks of treatment was 40%, 48%, 68%, 62% and 70% for the two control and the lowest, intermediate and highest concentration groups, respectively. Survival in the female groups was 58%, 56%, 50%, 80% and 80%, respectively.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain for both female and male rats receiving test substance at any concentration was significantly lower than for their respective combined control groups throughout the treatment period. Some evidence of a relationship to the dietary concentration of test substance was apparent for males. See Table 2.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Both male and female rats consumed less food at any concentration than those in their respsective combined control groups throughout the treatment period. However, no dose response was observed.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Efficiency of food conversion was essentially similar in control and treated animals during the first 14 weeks of treatment. After 15 weeks, efficiency of treated animals was lower than that of the controls.

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

effects observed, treatment-related

Description (incidence and severity):

The water intake for male and female rats at any concentration was higher than that of the combined control groups. In most cases, the degree of change was related to the dietary concentration of the test substance.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No evidence of treatment-related changes. The findings observed were those common in rats of this strain or those associated with the suspected sialodacryoadenitis virus infection during weeks 4 and 5 of the study.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A lower packed cell volume was noted among male rats receiving the highest concentration after 25, 38 and 51 weeks of treatment and among female rats receiving the highest concentration after 51 weeks, when compared with controls. This was generally associated with a lower haemoglobin concentration. Although these changes often attained statistical significance, the intergroup differences were small and the individual values were not abnormal. The variations were not considered to be of toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Lower ALT activity in male rats receiving the highest concentration when compared to controls was observed after 11, 38, 51, 77 and 103 weeks of treatment. Also noted among the rats receiving the low or intermediate concentration with intergroup differences showing some occasionial statistical significance. Significantly lower ALT activity was noted in treated females after Week 77 but not after week 103. Therefore, decreases in ALT were determined not to have toxicological significance.

Glucose levels were slightly lower than controls in female rats after 25, 38 and 51 weeks of treatment at any concentration. At 77 weeks but not 103 weeks, the highest dosed females also showed lower concentrations. However, these differences were small, not consistently observed and not noted in the males. After 38 weeks, glucose was slightly increased in males receiving low or high concentrations. These slight intergroup variations were not deemed to be of biological or toxicological significance.

Statistically significant lower T4 levels were noted in male groups after 11 weeks of treatment but no effects observed in females. Examination of the individual values for the males revealed 3 of 10 values in the controls were much greater than the range of all other values, when excluded, the controls were similar to that those at the highest concentration. No treatment-related histopathological changes were noted in the thyroid. The differences in males were considered to be unrelated to treatment.

Description (incidence and severity):

Rats of either sex receiving the highest concentration excreted more magnesium than controls after 11, 25, 39 and 51 weeks of treatment. A higher magnesium excretion was also noted for female rats receiving the intermediate concentration after 25 and 51 weeks of treatment; for male rats receiving the intermediate concentration after 51 weeks of treatment and for males receiving the low concentration after 25 and 51 weeks of treatment. This trend was reversed after 77 weeks of treatment with rats excreting less than their respective controls.

The volumes of urine excreted by female rats at any concentration tended to be lower than those for controls. This difference achieved statistical significance after 39 and 77 weeks of treatment. After 77 weeks of treatment, urine volumes of treated male rats were lower than controls.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistical analysis of group mean absolute organ weights for animals killed after 52, 78 or 104 weeks of treatment revealed higher full and empty caecum weights for males and females receiving the highest concentration when compared to their respective controls (except empty caecal weights of the males at 78 weeks). In addition, full caecum weights were higher than controls for rats receiving the intermediate concentration which were killed after 52 weeks of treatment. The increased caecal weight is consistent with changes observed in rats fed high levels of poorly absorbed material in the diet and caecal enlargement is considered a physicological response in the rat which is of no toxicological significance. There were no histopathological findings noted in the caecum considered to be related to treatment.

Several other statistically significant differences in absolute and bodyweight-relative organ weights were noted which was determined to be influenced by the lower bodyweight observed in the treated animals. Therefore, analysis of covariance, using the terminal bodyweight as the covariate, was undertaken as a means of adjusting organ weights for differents in bodyweights. Analysis of weights for animals killed after 52 weeks of treatment revealed higher kidney and brain weights in males receiving the highest concentration and higher liver weights in female rats receiving the test substance at any concentration compared with controls. The higher brain weight observed in males was considered to be of no significance as the absolute weight was unaffected by treatment. The higher kidney and liver weight was observed in one sex on one occasion. None of the organ weight differences were correlated with histopathological findings and therefore none of the intergroup differences were considered to be of toxicological significance.

Analysis of the weights for animals killed after 78 or 104 weeks of treatment did not reveal any differences between the group of rats receiving the highest concentration and their controls (besides the caecal weight which showed a clear treatment effect and were not included in the statistical analysis).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No macroscpoic abnormalities which were considered to be related to treatment were observed. A range of incidental findings were found which were considered to have occurred spontaneously and to be unrelated to the treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The incidences of a number of findings were higher among treated groups than among controls including: renal pelvic epithelial hyperplasia (generally graded as minimal) in all treated groups of females; renal pelvic mineralisation (graded as minimal or slight) in females fed at 10000 or 30000 ppm; adrenal cortical haemorrhagic degeneration in female rats fed at 30000 ppm; cataracts in eyes of treated male rats.

The incidence of hydronephrosis was higher among females that had received 3000 ppm than among controls. In the absence of any response at the highest concentration, this difference is not considered to be of biological significance.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment with test substance did not significantly increase the incidence of any neoplasm.

The following tumours were present at very low incidence (e.g. one or two) among animals that had received 30000 ppm, and were absent in the corresponding control groups: in males-ganglioneuroma in the brain, renal liposarcoma, pulmonary adenoma, thymoma, thyroid follicular cell carcionoma, and thoracic sarcoma of unknown origin; in females-renal squamous cell carcinoma, ovarian carcinosarcoma, pituitary adenoma, in the skin and subcutis an anaplastic carcinoma and a basal cell carinoma, histiocytic sarcoma and malignant lymphoma.

Details on results:

There was no increase in incidence of any gross abnormality attributable to the ingestion of the test substance at any dietary concentration.

Changes in the incidence of non-neoplastic findings (pelvic nephracalcinosis and epithelial hyperplasia) in the kidneys of females were associated with treatment but are consistent with renal changes that have been reported by other investigators in female rats with caecal enlargement. Microscopy revealed a statistically significant higher incidence of renal pelvic mineralisation in the females receiving 10000 and 30000 ppm and renal pelvic epithelial hyperplasia in all treated groups of females. In all cases (except one moderate grade), these findings were graded as minimal or slight. These two changes are related in that the epithelium of the renal pelvis reacts to the mineral crystals in the pelvis by physicological hyperplasia. It is probable that there is a single primary effect-the mineralisation. The authors indicate that renal disease, as exemplified by progressive nephropathy, was not found from treatment. Renal mineralisation a sex-related and not uncommon in control female rats as in this study.

Pelvic epithelial hyperplasia and mineralisation is a condition that is related in the rat to caecal hypertrophy. The mechanism by which they occur is not clearly understood. Caecal enlargement and pelvic nephrocalcinosis have been frequently reported as a consequence of feeding rats compounds that belong to a broad group of carbohydrates which includes natural sugars such as lactose, various polyols as well as chemically-modified starches and bulking agents. However, these findings are not considered to be of toxicological significance.

Caecal englargement is reflected in this study as increased full and empty caecal weights. The increased incidences of the renal changes and the caecal enlargement are both believed to be a response to the high dietary levels of the test substance which is poorly absorbed from the gut.

Relevance of carcinogenic effects / potential:

There was no increase in the incidence of any tumour in rats receiving the test substance at any concentration. All tumours that were only observed amoung treated groups were considered to have arisen by chance and to be unrelated to treatment. Test substance is not considered to be carcinogenic.

Key result

Dose descriptor:

NOAEL

Effect level:

> 30 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicological relevant effects noted at any dose level.

Remarks on result:

other: Pelvic epithelial hyperplasia and mineralisation is a condition that is related in the rat to caecal hypertrophy. However, these findings are not considered to be of toxicological significance.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

30 000 ppm

System:

gastrointestinal tract

Organ:

other: caecum

Treatment related:

no

Dose response relationship:

yes

Relevant for humans:

no

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

10 000 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Breeding Phase Result Summary:

-Test substance had no effect upon the general condition of the F0 animals with respect to clinical signs and mortality.

-Bodyweight gains of treated F0 males and F0 females prior to pairing and during gestation were significant reduced.

-During lactation overall weight gain of treated females was increased (from Day 1 post partum until commencment of the oncogenicity study, the concentration of tesst substance to females with litters in highest dosage group was reduced from 30000 to 20000 ppm).

-Food intake and food conversion efficience were initially reduced in all groups, but by 4th week of treatment there was no association of effect with dose level.

-Mating performance and fertility were unaffected by treatment.

-At 10000 and 30000 ppm the proportion of animals with gesstation length greater than 23 days was slightly increased. However, the gestation index was maximal in all groups and no incidences of dystocia were observed. Therefore, not considered to be biological significant.

-Number of offspring born and alive at Day 1 post partum and subsequent offspring survival before and after the Day 4 post partum cull were similar in all groups.

-Offspring bodyweight at Day 1 post-partum and Day 14 post partum was similar in all groups. However, bodyweight gain by offspring in all treated groups during the last week before weaning when offspring were eating diet directly was slightly but significantly reduced when compared with values for the combined control groups.

Satellite Metabolic Adaptation Study Summary:

-About 7% of the radiolabel was recovered in urine and with the exception of the anomalous Week 52 results, approximately 80% was recovered in faeces. The increased total recovery of radioactivity in urine and faeces at week 85/87 compared with week 52 suggests that the problem at week 52 may have been related to cage size. Therefore, there was no significant differences in urinary or faecal elimination attributed to sex, to prior treatment with the test substance in the diet or to age.

-Radiolabel was not detected in expired air from any animal at either 0 -6 hour or 6 -24 hour after dosing.

-No evidence of significant metabolism of test substance was found with the quanititative urine samplings and it was concluded that the principal route of elimination of orally administered test substance in rats was in the faeces, with a small amount excreted in the urine (about 7%).

-The test substance is eliminated essentially unchanged in the urine and faeces of male and female rats. No evidence of significant metabolism of the test substance outside the limits of the levels of impurities was found.

-No evidence of metabolic adaptation to the test substance in rats of either sex following chronic dietary exposure at 30000 ppm for more than 18 months was found.

 Table 1. Achieved Dosages (mg/kg bodyweight/day)

Weeks	Low-Male	Intermediate-Male	High-Male	Low-Female	Intermediate-Male	High-Female
1	565	1917	5707	553	1853	5489
13	150	511	1592	195	654	1975
1 -104	94	323	1005	135	458	1395

 Table 2. Bodyweight change-group mean values (g)-Weeks 0 -104

Males					Females
Control	Control	3000 ppm	10000 ppm	30000 ppm	Control	Control	3000 ppm	10000 ppm	30000 ppm
902	908	788^a	731^a	721^a	533	581	448^a	426^a	410^a

^aSignificantly different from combined controls, p<0.001

Conclusions:

The carcinogenicity of the test item via oral application on rats was determined in a GLP study according to FDA guidelines similar to OECD guidelines. Sprague-Dawley CD rats were fed with diet containing test substance at a concentration of 3,000, 10,000 and 30,000 ppm continuously for up to two years. The test animals were derived from parents that had received the test substance at the same concentrations for four weeks prior to pairing and during gestation. Under the conditions of this study, treatment of rats with a diet containing up to 30,000 ppm test substance did not increase the incidence of tumours or influence the type of tumour observed. There were no changes that were considered to be toxic responses to the test item from dietary administration.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

chronic

Species:

rat

System:

gastrointestinal tract

Organ:

kidney

other: caecum

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

According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments), the test substance should not be classified for carcinogenicity since there were no increased incidence of non-neoplastic or neoplastic findings following repeated oral exposure in rats.

Additional information

There was no increase in incidence of any gross abnormality attributable to the ingestion of the test substance at any dietary concentration.

Changes in the incidence of non-neoplastic findings (pelvic nephracalcinosis and epithelial hyperplasia) in the kidneys of females were associated with treatment but are consistent with renal changes that have been reported by other investigators in female rats with caecal enlargement.  Microscopy revealed a statistically significant higher incidence of renal pelvic mineralisation in the females receiving 10000 and 30000 ppm and renal pelvic epithelial hyperplasia in all treated groups of females.  In all cases (except one moderate grade), these findings were graded as minimal or slight.  These two changes are related in that the epithelium of the renal pelvis reacts to the mineral crystals in the pelvis by physicological hyperplasia.  It is probable that there is a single primary effect-the mineralisation.  The authors indicate that renal disease, as exemplified by progressive nephropathy, was not found from treatment.  Renal mineralisation a sex-related and not uncommon in control female rats as in this study.  

Pelvic epithelial hyperplasia and mineralisation is a condition that is related in the rat to caecal hypertrophy.  The mechanism by which they occur is not clearly understood.  Caecal enlargement and pelvic nephrocalcinosis have been frequently reported as a consequence of feeding rats compounds that belong to a broad group of carbohydrates which includes natural sugars such as lactose, various polyols as well as chemically-modified starches and bulking agents.  However, these findings are not considered to be of toxicological significance.

Caecal englargement is reflected in this study as increased full and empty caecal weights.  The increased incidences of the renal changes and the caecal enlargement are both believed to be a response to the high dietary levels of the test substance which is poorly absorbed from the gut.