Methoxyammonium chloride

Carcinogenicity

Description of key information

No reliable data are available for the test substance. The toxicological assessment is based on read-across to CAS 10039-54-0.

OECD 451, GLP, rat, oral: LOAEL = 0.2 mg/kg bw/d in males and NOAEL = 0.4 mg/kg bw/d in females for systemic effects (2001, reliability 1)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
------------
Species and strain:
Male and female Wistar rats Chbb:THOM (SPF) were supplied by Boehringer Ingelheim Pharma KG, Biberach/Riss, Germany at an age of 31 +/- 1 days. Only animals free from clinical signs of disease were used for the study.

Animal identification:
The rats were identified by ear tattoo .

Reasons for species selection:
Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

Housing and diet:
The rats were housed singly in type DK III stainless steel wire mesh cages supplied by Becker & Co ., Castrop-Rauxel, Germany (floor area about 800 cm2) . Underneath the cages, waste trays were fixed containing absorbent material (type 3/4 dustfree embedding, supplied by SSNIFF, Soest, Germany). The animals were housed in a fully air-conditioned room. Central air-conditioning guaranteed a range of 20 - 24°C for temperature and of 30 - 70 % for relative humidity. The day/night rhythm was 12 hours (12 hours light from 06.00 a.m. - 06.00 p.m., 12 hours dark from 06.00 p.m. - 06.00 a.m.).Technical failures led to transient, minor deviations from these ranges which had no influence on the results of the study. Documentation of the deviations is retained as raw data. Deviations from these ranges did not occur. The animal room was completely disinfected using a disinfector ("AUTEX", fully automatic, formalin-ammonia-based terminal disinfector). The floor and the walls were cleaned once a week. The cleansing liquid used was water containing about 0.1 % Incidin perFect® (supplied by Henkel, Duesseldorf, Germany).
The food used was Kliba basic maintenance diet rat/mouse/hamster, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland . Food and drinking water (Milli-Q-Pure water from water bottles and demineralized water) were available ad libitum.

Experimental procedure and time schedule
On the day of arrival the animals were subjected to an 11 day acclimatization period during which they received ground food and drinking water ad libitum. Prior to the start of the administration period, the animals were allocated to the test groups according to weight. The list of randomization instructions was compiled by a computer. At the start of the administration period (day 0), the rats were 42 +/- 1 days old . The body weight of the males was in the range of 171 g- 211 g (group mean: 189 g) and the body weight of the females was in the range of 131 g -166 g (group mean: 146 g) .

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

TEST SUBSTANCE PREPARATION
The test substance was administered as a solution in water (first 8 months filtered Milli-QPure water, after 8 months until the end of the study in demineralized water). The test substance was weighed in, depending on dose group, added to the drinking water, and mixed with a stirrer. The drinking water solutions were prepared twice a week.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses:
--------
The analytical investigations of the test substance preparations were carried out at the Analytical Laboratories of BASF Aktiengesellschaft, Ludwigshafen, Germany.
The stability of the test substance in demineralized water over a period of 4 days at room temperature was proven prior to the study.
As the test substance preparations were true solutions in water, no homogeneity analyses were performed.
The concentration of the test substance preparations was proven in samples taken at the start of administration period, after about 3, 6, 12 and 18 months, and one month before the end of the study.

Duration of treatment / exposure:

24 month

Frequency of treatment:

ad libitum

Post exposure period:

At the end of the 12 month (satellite groups) or 24 month (main groups) administration period all surviving animals were sacrificed after a fasting period (withdrawal of food for about 16-20 hours).

Dose / conc.:

0.3 mg/kg bw/day (nominal)

Remarks:

satellite group; males

Dose / conc.:

1.1 mg/kg bw/day (nominal)

Remarks:

satellite group; males

Dose / conc.:

4.5 mg/kg bw/day (nominal)

Remarks:

satellite group; males

Dose / conc.:

0.4 mg/kg bw/day (nominal)

Remarks:

main group and satellite group; females

Dose / conc.:

1.6 mg/kg bw/day (nominal)

Remarks:

main group and satellite group; females

Dose / conc.:

6.2 mg/kg bw/day (nominal)

Remarks:

main group and satellite group; females

Dose / conc.:

0.2 mg/kg bw/day (nominal)

Remarks:

main group; males

Dose / conc.:

1 mg/kg bw/day (nominal)

Remarks:

main group; males

Dose / conc.:

3.7 mg/kg bw/day (nominal)

Remarks:

main group; males

No. of animals per sex per dose:

50 animals per sex per dose in the main group and 10 animals per sex per dose in the satellite groups

Control animals:

yes, concurrent vehicle

Details on study design:

In a standard combined chronic toxicity/carcinogenicity toxicity study according to OECD TG 453, the test substance was administered to groups of 50 male and 50 female Wistar (Chbb:THOM, SPF) rats daily in the drinking water at dose levels of 0, 5, 20 and 80 ppm for 24 months (main groups), and additional to groups of 10 animals per sex and dose for 12 months (satellite groups) for evaluation of hematology parameters.
The doses administered corresponded to a mean daily intake in the main groups of about 0, 0.2, 1.0, and 3.7 mg/kg bw/d in males and 0, 0.4, 1.6, and 6.2 mg/kg bw/d in females; and in the satellite groups of about 0, 0.3, 1.1, and 4.5 mg/kg bw/d in males and 0, 0.4, 1.6, and 6.2 mg/kg bw/d in females.

Observations and examinations performed and frequency:

CLINICAL EXAMINATIONS
Clinical observations:
------------------
The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays. Once a week an additional comprehensive clinical examination (including palpation) was carried out.

Food consumption:
---------------
Food consumption was determined once a week over a period of 7 days during the first 13 weeks of the administration period, thereafter at 3-months intervals and prior to start of necropsy. The values were calculated as food consumption in grams per animal and day.

Water consumption:
----------------
Water consumption was determined once a week over a period of 7 days during the first 13 weeks of the administration period, thereafter at 4-weeks intervals and prior to start of necropsy. The values were calculated as water consumption in grams per animal and day.

Body weight data:
---------------
Body weight was determined before the start of the administration period in order to randomize the animals. During the conduct of the study, body weight was determined once a week during the first 13 weeks, thereafter at 4-week intervals; and prior to start of necropsy. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

Food efficiency:
-------------
Food efficiency (group means) was calculated based upon individual values for body weight and food consumption.

Intake of test substance:
---------------------
The mean daily intake of test substance (group means) was calculated based upon individual values for body weight and water consumption.

Sacrifice and pathology:

CLINICAL PATHOLOGY
Satellite groups :
Blood was taken from the retroorbital venous plexus in the morning from non-fasted animals without anesthesia. The blood sampling procedure and the subsequent analysis of the samples were carried out in a randomized sequence. The list of randomization instructions was compiled with a computer using a random number generator. The assays of blood parameters were performed under internal laboratory quality control conditions with commercial reference controls to assure reliable test results. The results of the clinical pathology examinations are expressed in units of the International System (SI).
The following examinations were carried out in all surviving animals per test group and sex:
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Technicon H 1 E model; Bayer, Munich, Germany):
• leukocytes
• erythrocytes
• hemoglobin
• hematocrit
• mean corpuscular volume
• mean corpuscular hemoglobin
• mean corpuscular hemoglobin concentration
• platelets
• differential blood count
Furthermore, reticulocytes and Heinz bodies were counted and methemoglobin was measured with an hemoximeter (OSM3, Radiometer, Copenhagen). Bone marrow smears were prepared from all animals killed in extremis or at scheduled necropsy. The smears were stained according to Wright, but not evaluated .
Main groups:
Blood was taken by tail puncture during the administration period from non-fasted animals without anesthesia. At the end of the study differential blood smears were prepared after decapitation from fasted, anesthetized animals.
Moreover, blood smears were prepared and evaluated from animals killed in extremis during the study. Bone marrow smears were prepared from all animals killed in extremis or at scheduled necropsy. The smears were stained according to Wright, but not evaluated. The differential blood count was evaluated visually. The data were transferred to the computer.

PATHOLOGY
Necropsy
The animals scheduled for necropsy were sacrificed by decapitation under C02-anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. The animals which died intercurrently or were sacrificed in a moribund state were necropsied as soon as possible after death and assessed by gross pathology.
Weight parameters
Weight assessment was carried out on all animals sacrificed at scheduled dates. The weights of the following organs were determined:
1 . Anesthetized animals
2 . Liver
3. Kidneys
4. Brain
5. Testes
6. Epididymides
7. Spleen
8. Adrenal glands
Organ / Tissue preservation list
The following organs / tissues were preserved in neutral buffered 4% formaldehyde:
1. All gross lesions
2. Brain
3. Pituitary gland
4. Thyroid glands/ parathyroid glands
5. Thymus
6. Trachea
7. Lungs
8. Heart
9. Aorta
10. Salivary glands (mandibular gland, sublingual gland)
11. Liver
12. Spleen
13. Kidneys
14. Adrenal glands
15. Pancreas
16. Testes, ovaries
17. Uterus, oviducts, vagina
18. Epididymides, prostate, seminal vesicle
19. Skin
20. Esophagus
21. Stomach (fore- and glandular stomach)
22. Duodenum, jejunum, ileum
23. Cecum, colon, rectum
24. Urinary bladder
25. Lymph nodes (mesenteric and iliac lymph node)
26. Female mammary gland
27. Skeletal muscle
2 . Sciatic nerve
29. Sternum with marrow
30. Bone marrow (femur)
31. Eyes
32. Femur with knee joint
33. Spinal cord (cervical, thoracic and lumbar cord)
34. Extraorbital lacrimal glands

Paraffin embedding, sectioning and staining
After the organs were fixed, the processing, the examination by light microscopy and the evaluation of findings were performed.
The hematoxylin-eosin stained slides were examined light-microscopically and assessed. A correlation between gross lesions and histopathological findings was performed. Of all animals that died intercurrently or were sacrificed in a moribund state the scope of histopathology was as indicated for the animals of the control group in the final sacrifice groups.
The spleen of the animals in the main group was cut in about five pieces and assessed, histopathologically.
Of few organs of single animals, the following special stains were performed: Elastica van Gieson (EvG) or Elastica Masson Goldner (EMG) for collagenic fibres, Periodic acid Schiff reaction (PAS) for mucopolysaccharides, Gomori for argyrophile structures, iron staining (Perls) for hemosiderin, Kresyl violet for basophilic foci, Toluidin blue for mast cells, and Kongo red (Korot/R) for amyloid.
Of few organs of single animals, the following immunhistochemical examinations were performed: calcitonin, ACTH, S100 for Schwann cells, CD 34 for lymphocyte antigen, actin for muscle, PCNA for cell proliferation, and neurone specific endolase (NSE).

Other examinations:

Determination of iron in spleen:
Spleen samples (ca . 100-200 µg) from the first ten animals without spleen findings and from all animals with spleen findings in each group were used to determine the iron concentration. Iron concentration was determined by inductively coupled plasma-atomic emission spetrometry (ICP-AES) after hydrolysis of spleens with sulfuric acid.

Statistics:

Clinical Examinations:
Parametric one-way analysis using the F-test (ANOVA) (two-sided). If the resulting p-value was equal or less 0.05, a comparison of each group with the control group using the DUNNETTs test (two-sided) was performed for the hypothesis of equal means.

Clinical Pathology:
Non parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using MANN-WHITNEY U-test (two sided) for the equal medians.

Pathology:
Non parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using the WILCOXON test for the hypothesis of equal medians.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No substance-related findings were observed. All abnormal clinical signs were equally distributed between control and treated animals and/or occurred in single animals, only.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

The number of animals which died or were sacrificed moribund until day 742 (i.e. 24 months of treatment) was the following:
0 ppm: males 26 %, females 26 %; 5 ppm: males 28 %, females 18 %; 20 ppm: males 30 %, females 38 %; 80 ppm: males 38 %, females 30 %.
Although the numbers are slightly higher in mid and high dose groups, this was assessed as being incidental an not related to treatment, due to the lack of a clear dose-response relationship.
- Satellite groups:
The number of animals which died or were sacrificed moribund until day 364 (i.e. 12 months of treatment) was the following:
0 ppm: males 10 %, females 0 %; 5 ppm: males 0 %, females 0 %; 20 ppm: males 0 %, females 0 %; 80 ppm: males 10 %, females 0 %.
No substance-related effects were observed, as each one male animal were sacrificed moribund in the control and high dose group.

Mortality until end of necropsy:
Most animals of the satellite groups survived until the scheduled end of the study. One high dose and control male were sacrificed moribund. The number of decedents in the main groups was the following:
From 50 animals examined in each dose group, the number of decedents in males was 13 in the 0 ppm group, 14 in the 5 ppm group, 15 in the 20 ppm group and 19 in the 80 ppm group. The number of decedents in females was 13 in the 0 ppm group, 9 in the 5 ppm group, 19 in the 20 ppm group and 15 in the 80 ppm group.
Although the number of decedents was slightly higher in the highest dose group, this was not regarded as treatment-related.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight was significantly decreased in mid dose males from day 35 to 56 ; in this group, also body weight change was significantly decreased from day 21 to day 63. Due to the lack of a dose-response relationship and the temporary occurrence, these deviations were assessed as being incidental and not related to treatment.
- Satellite groups:
No significant changes.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Intake of test substance
The mean daily test substance intake in mg/kg body weight over the entire study period was the following:
- Main groups: 5 ppm: males 0.2, females 0.4; 20 ppm: males 1.0, females 1.6; 80 ppm: males 3.7, females 6.2
In order to assure equally spaced intervals for calculation of the mean test substance intake, only the values of days 7, 35, 63, 91 and days 119 to 707 were taken into account.
- Satellite groups: 5 ppm: males 0.3, females 0.4; 20 ppm: males 1.1, females 1.6; 80 ppm: males 4.5, females 6.2
In order to assure equally spaced intervals for calculation of the mean test substance intake, only the values of days 7, 35, 63, 91 and days 119 to 343 were taken into account.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

- Main groups:
There were some few statistically significant deviations (increased or decreased values) in low and mid dose males. Due to the isolated occurrence and the lack of a doseresponse relationship, this was assessed as being incidental.
- Satellite groups:
There were some isolated statistically significant deviations (increased or decreased values) in low and mid dose males and high dose females. Due to the isolated occurrence and the lack of a dose-response relationship, this was assessed as being incidental.

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

effects observed, non-treatment-related

Description (incidence and severity):

There were some few significant deviations (increased and/or decreased values) in high dose males and all treated females. However, due to the isolated occurrence and the lack of a dose-response relationship, these deviations were assessed as being incidental and not related to treatment.
- Satellite groups:
No significant changes.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Satellite groups:
No test substance-related changes were observed in the results of the white blood cell count and the differential white blood cell count of the satellite animals of either sex in the course of the study. However, in the red blood cell parameters of the male and female rats of satellite group 3 various toxicologically relevant changes were found: In the high dose males, red blood cell counts were reduced at each time interval. On day 186, 276 and 362 hemoglobin concentrations and on day 186 and 362 hematocrit values were decreased in the males of test group 3 and mean corpuscular volume was enlarged in these animals on day 94. On day 186 hemoglobin concentration was also decreased in the males of test group 2. Due to the small magnitude of change the fall in hemoglobin in the mid dose males is considered to be fortuitous and not of toxicological concern. Additionally, in the peripheral blood of the males of test group 3 slightly increased number of Heinz bodies was found on day 94 and 186 and slightly increased incidence of Howell-Jolly bodies was seen on day 362. Females of the satellite group 3 had statistically significantly lower red blood cell counts and hemoglobin concentrations and enlarged mean corpuscular volume and higher platelet counts than concurrent controls at each time interval. On day 87, 179 and 355 hematocrit values were reduced and mean corpuscular hemoglobin was increased in the females of group 3. Furthermore, in the circulation of group 3 females slight increases in reticulocytes were found on day 87 and 355, slightly elevated number of Heinz bodies was observed on day 87 and 179 and slight increases in Howell-Jolly bodies were seen on day 355.
- Main groups :
There were no treatment-related changes in the differential white blood cell count of the male and female rats of the main groups at each time interval. Examination of erythrocyte morphology, however, revealed mild anisocytosis and microcytosis on day 368 and slightly increased incidence of Howell-Jolly bodies in the males of group 3 at each time interval. tn the peripheral blood of the females of group 3 increased polychromasia and elevated number of Howell-Jolly bodies were found on day 368 and 549 and at the end of the study.
- Prematurely killed animals :
Changes in white and red blood cells were observed in the peripheral blood of males and females killed in extremis. Alterations were noted both in non-treated animals as well as in treated rats. Thus, the observed changes are considered to be spontaneous, incidental or age-related. Accordingly, these findings are not associated with the test compound administered.

Clinical biochemistry findings:

not examined

Endocrine findings:

effects observed, non-treatment-related

Urinalysis findings:

not examined

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):

Absolute weights
When compared with control group 0, the mean absolute weights of the following organs were significantly increased or decreased:
- Satellite groups: Spleen (+30.4 % in males and +41.2 % in females) in the 80 ppm group
- Main groups: Spleen (+58.3 % in females) in the 80 ppm group and brain (-3% in males) in the 20 ppm group
The decreased absolute brain weight in males of group 2 is considered incidental. All other mean absolute weight parameters did not show significant differences when compared with the control group.

Relative Organ Weights
When compared with control group 0, the mean relative weights of following organs were significantly increased:
- Satellite groups: Spleen (+32.6 % in males and +40.7 % in females) in the 80 ppm group
- Main groups: Spleen (+58.7 % in females) in the 80 ppm group
All other mean relative weight parameters did not show significant differences when compared with the control group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Satellite groups:
All gross lesions occurred either singly or were biologically equally distributed over the control group and the treatment groups.
- Main groups:
Spleen: In top dose males, the number of animals with masses in the spleen was slightly increased: From 50 animals examined in each dose group, the incidence was 2 in the 0 ppm group, 3 in the 5 ppm group, 2 in the 20 ppm group and 5 in the 80 ppm group.
Urinary bladder: The number of males with concretion in the urinary bladder was decreased in treated animals. The incidence was 9 in the 0 ppm group, 6 in the 5 ppm group, 3 in the 20 ppm group and 2 in the 80 ppm group.
Heart: The number of females with calcification in the heart was decreased in treated animals. The incidence in males was 9 in the 0 ppm group, 5 in the 5 ppm group, 4 in the 20 ppm group and 2 in the 80 ppm group. In females, the incidence was 13 in the 0 ppm group, 4 in the 5 ppm group, 3 in the 20 ppm group and 3 in the 80 ppm group.
All other gross lesions occurred either singly or were biologically equally distributed over the control group and the treatment groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

- Satellite groups:
Spleen:
In the top dose group, congested vessels were observed in the spleen of eight males and in all females. This finding was characterized by dilated blood-filled vascular spaces. Hemosiderin storage occurred in all control and treated males and females, with higher degrees in males of groups 2 and 3 and in top dose females. Most of males showed extramedullary hematopoiesis in the spleen. The degree of this finding was higher in group 3 than in the control group. In females, the number of animals as well as the degree of severity was increased in the top dose group. The incidence for hematopoiesis in males was 9 in the 0 ppm group, 6 in the 5 ppm group, 7 in the 20 ppm group and 9 in the 80 ppm group. In females, the incidence for hematopoiesis was 2 in the 0 ppm group, 2 in the 5 ppm group, 2 in the 20 ppm group and 8 in the 80 ppm group.
One top dose female showed a minimal angiomatous hyperplasia in the spleen.
All other findings noted were either single observations or they were biologically equally distributed between control and treatment groups. All of them were considered to be incidental or spontaneous in origin and without any relation to treatment.
- Main groups:
Spleen :
Hyperplasia and ectasia of capillary-sized vessels, lined by a normal endothelium which lacked pleomorphism and mitotic activity was diagnosed as angiomatous hyperplasia. Small amounts of fibrous tissue separated the hyperplastic vessels. The number of males and females with angiomatous hyperplasia was increased in the top dose group. In females, the degree of severity of this finding was higher in 80 ppm dose group. The incidence is given in "Remarks on results including tables and figures".
An increased number of males and females with congested vessels in the spleen was observed in top dose animals. The finding was characterized by dilated, blood-filled vascular spaces. The incidence for congested vessels in males was 6 in the 0 ppm group, 6 in the 5 ppm group, 9 in the 20 ppm group and 33 in the 80 ppm group. In females, the incidence was 1 in the 0 ppm group, 0 in the 5 ppm group, 0 in the 20 ppm group and 33 in the 80 ppm group.
Hemosiderin storage occurred in nearly all control and treated animals, with higher degrees in top dose males and in females of dose groups 2 and 3. Many animals showed extramedullary hematopoiesis in the spleen. The degree of severity was higher in top males and females. In females of group 3, the incidence of this finding was slightly increased. The incidence for hematopoiesis in males was 35 in the 0 ppm group, 37 in the 5 ppm group, 29 in the 20 ppm group and 37 in the 80 ppm group. In females, the incidence was 27 in the 0 ppm group, 27 in the 5 ppm group, 22 in the 20 ppm group and 37 in the 80 ppm group.
Liver:
Many males and females showed (multi)focal or diffuse pigment storage in the liver. Most of the stored pigment turned out to be hemosiderin by representatively performed iron staining. (Multi)focal pigment storage occurred in comparable incidences in control and treated males. The number of females with (multi)focal pigment storage was decreased in dose group 3. Diffuse pigment storage in the liver was noted in higher incidences and higher degrees in top dose males and females. The incidence for pigment storage focal in males was 13 in the 0 ppm group, 18 in the 5 ppm group, 17 in the 20 ppm group and 17 in the 80 ppm group. In females, the incidence was 9 in the 0 ppm group, 7 in the 5 ppm group, 6 in the 20 ppm group and 2 in the 80 ppm group. The incidence for pigment storage diffuse in males was 3 in the 0 ppm group, 1 in the 5 ppm group, 0 in the 20 ppm group and 13 in the 80 ppm group. In females, the incidence was 25 in the 0 ppm group, 14 in the 5 ppm group, 19 in the 20 ppm group and 45 in the 80 ppm group.
Bone marrow:
Increased hematopoiesis was observed in some males and females with higher incidences in the top dose groups. The incidence for increased hematopoiesis in males was 4 in the 0 ppm group, 4 in the 5 ppm group, 4 in the 20 ppm group and 10 in the 80 ppm group. In females, the incidence was 5 in the 0 ppm group, 1 in the 5 ppm group, 1 in the 20 ppm group and 15 in the 80 ppm group.
Urinary bladder:
In most of males with macroscopically diagnosed concretion in the urinary bladder, the finding could be confirmed histopathologically. The number of males with concretion was decreased in the top dose group. The incidence for concretion in males was 10 in the 0 ppm group, 4 in the 5 ppm group, 7 in the 20 ppm group and 5 in the 80 ppm group.
Heart:
The number of females with (multi)focal calcification in the heart was decreased in the top dose group. The incidence for calcification in males was 5 in the 0 ppm group, 3 in the 5 ppm group, 4 in the 20 ppm group and 7 in the 80 ppm group. In females, the incidence was 16 in the 0 ppm group, 4 in the 5 ppm group, 3 in the 20 ppm group and 9 in the 80 ppm group.

All other findings noted were either single observations or they were biologically equally distributed between control and treatment groups. All of them were considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Satellite groups:
There were very few neoplastic findings which were biologically equally distributed over the control group and the treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
- Main groups:
Spleen:
Most of the macroscopically diagnosed masses in the spleen turned out to be hemangiosarcomas, histopathologically. The number of males with hemangiosarcomas in the spleen was slightly increased in treated males. The incidence is given in "Remarks on results including tables and figures". Of the affected animals, one male and one female of the control group, three males of group 2, and three top dose males died prematurely. In males, each one hemangiosarcoma of the control group and of group 1, as well as two hemangiosarcomas in the top dose group metastasized into other organs. Hemangiomas were observed in one female of group 1, one female of group 2 and in four top dose females.

Urinary bladder:
In the urinary bladder, transitional cell papillomas were observed in six control males, but none of the top dose males showed this finding. All other neoplastic findings occurred either singly or were biologically equally distributed over the control group and the treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Tumor bearing animals:
The numbers of animals with neoplasms, with benign, malignant, and systemic neoplasms, as well as the total numbers of primary, benign, malignant, and systemic neoplasms were comparable between control and top dose males and females. They were biologically equally distributed over the control and treatment groups. In top dose animals, the numbers of males and females with one primary neoplasm were slightly increased, whereas the numbers of males and females with two and more primary neoplasms were slightly decreased. This rather beneficial effect is considered incidental. The number of males with metastasized neoplasms, as well as the total number of metastasized neoplasms in males was slightly increased in the top dose group. In females, the number of animals with metastasized neoplasms, as well as the total number of inetastasized neoplasms was comparable between control and treatment groups.

Details on results:

Iron concentration in spleen
No difference in iron concentration in spleen was found in rats with and without findings in this organ indicating that the iron concentration did not contribute to the findings.

Dose descriptor:

LOAEL

Effect level:

0.2 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: haemangiosarcoma in spleen

Dose descriptor:

NOAEL

Effect level:

0.4 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: haemangiosarcoma in spleen

Neoplastic lesions in the spleen:
Hemangiosarcoma
Males: 5, 20, 80 ppm: 7/50, 9/50, 8/50 vs 4/50 controls
Females: 5, 20, 80 ppm: 1/50, 1/50, 3/50 vs 2/50 controls

Hemangioma
Males: 0/50 in all doses groups vs 0/50 controls
Females: 5, 20, 80 ppm: 1/50, 1/50, 4/50 vs 0/50 controls

Hemangiosarcoma plus Hemangioma
Males: 5, 20, 80 ppm: 7/50, 9/50, 8/50 vs 4/50 controls
Females: 5, 20, 80 ppm: 2/50, 2/50, 7/50 vs 2/50 controls

Precursor lesions:
Hyperplasia, angiomatous
Males: 5, 20, 80 ppm: 9/50, 4/50, 16/50 vs 4/50 controls
Females: 5, 20, 80 ppm: 13/50, 12/50, 34/50 vs 14/50 controls

Conclusions:

The oral administration of the test compound via the drinking water at 80 ppm caused hemolytic anemia, due to oxidative damage of erythrocytes and formation of methemoglobin. Target organ in pathology was the spleen. The number of hemangiosarcomas in the spleen was increased in all treated males. In four females of the high dose level, hemangioma in the spleen was seen.

The no observed adverse effect level (NOAEL) was 5 ppm (0.4 mg/kg bw/d) in females, but below 5 ppm (0.2 mg/kg bw/d) in males.

Executive summary:

In a standard combined chronic toxicity/carcinogenicity toxicity study according to OECD TG 453, the test substance was administered to groups of 50 male and 50 female Wistar rats daily in the drinking water at dose levels of 0, 5, 20 and 80 ppm for 24 months (main groups), and additional to groups of 10 animals per sex and dose for 12 months (satellite groups) for evaluation of hematology parameters. The doses administered corresponded to a mean daily intake in the main groups of about 0, 0.2, 1.0, and 3.7 mg/kg bw/d in males and 0, 0.4, 1.6, and 6.2 mg/kg bw/d in females; and in the satellite groups of about 0, 0.3, 1.1, and 4.5 mg/kg bw/d in males and 0, 0.4, 1.6, and 6.2 mg/kg bw/d in females.

There was no statistically significant body weight loss, and no significant effect on mortality in the treatment groups when compared to control groups, and furthermore, no treatment-related clinical signs were noted in either sex throughout the study. At termination, mean absolute and relative spleen weights were statistically significant increased in the 80 ppm females. Gross observations on the spleen showed a slightly increased number of males with macroscopically diagnosed masses at 80 ppm. Microscopy yielded further evidence of spleen injury in 20 ppm and 80 ppm males and females at sacrifice. The only neoplasms showing an increased incidence were tumours of the spleen in both male and female rats when compared to controls. The spleen tumours observed in male and female rats were diagnosed as hemangiosarcomas and hemangiomas. In male rats the incidence of hemangiosarcomas in the spleen was increased from 4/50 among controls and all treatment groups, to 7/50, 9/50 and 8/50 in the 5, 20, and 80 ppm groups, respectively. Two of the hemangiosarcomas of the 80 ppm dosed males and one of the control males metastasized into other organs. In females, there was a biologically equally distribution of hemangiosarcomas in the spleen over the control group animals and the treated animals (0/5/20/80 ppm: 2/1/1/3). Whereas the number of females with hemangiomas was slightly increased in the 80 ppm dose group (0, 5, 20, 80 ppm: 0/1/1/4). It was shown that there was a slightly increased incidence of hemangiosarcomas in the spleen of male rats at all hydroxylammonium sulfate treatment groups. Although, the incidence of hemangiosarcomas in the spleen seen in male rats was small, not dose-related and the difference to controls did not attain statistically significance, the increase of this tumour type was considered to present an effect of treatment. This was supported by the fact that the incidence of hemangiosarcomas in the spleen seen in all treatment groups was generally higher than concurrent intra laboratory historical controls which underlines the biologically significance of this finding. Tumours developed earlier in treated animals than in controls. Furthermore, the following findings occurred in the spleen: hyperplasia and ectasia of capillarys, lined by a normal endothelium which lacked pleomorphism and mitotic activity and separated by a small amounts of fibrous tissue. These findings diagnosed as angiomatous hyperplasia were seen in all dose groups and controls. However, high frequencies of these spleen lesions were observed in males and females at 80 ppm. Persistent hyperplasia may be indicative as site of tumour development. So, these findings of angiomatous hyperplasia in the spleen were considered as a precursor lesion of angiomatous tumours (hemangioma, hemangiosarcoma). In the satellite groups (treatment for 12 months), very few neoplastic findings were noted which were biologically equally distributed over the control group and the treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

 

Overall, this combined chronic toxicity/carcinogenicity study in the rat is acceptable and does satisfy the guideline requirement for carcinogenicity testing (OECD TG 453) in rats. Results of this standard study have shown that the test item is carcinogenic in rats after treatment with dosages of ≥5 ppm (equivalent to about ≥0.2 mg/kg bw/d) in males and of 80 ppm (equivalent to about 6.2 mg/kg bw/d) in females. In male rats an increased incidence of hemangiosarcomas was noted in all treated groups. Hemangiomas were observed in four females from the 80 ppm dose group. Both numbers of hemangiosarcomas in male rats and hemangiomas observed in female rats were outside the range of historical control background data. Angiomatous hyperplasia in the spleen considered as a precursor lesion of angiomatous tumours was observed at an increased number in males and females at 80 ppm. Therefore, 5 ppm (equivalent to about 0.2 mg/kg bw/d (= LOAEL) in males and 0.4 mg/kg bw/d (= NOAEL) in females) is considered to be the lowest dose level for tumour development in the spleen in male and female rats in this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

0.2 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The quality of the whole database is high

System:

immune system

Organ:

spleen

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Results of a combined chronic toxicity/carcinogenicity study in the rat have shown that the test item is carcinogenic in rats after treatment with dosages of ≥5 ppm (equivalent to about ≥0.2 mg/kg bw/d) in males and of 80 ppm (equivalent to about 6.2 mg/kg bw/d) in females. An increased incidence of hemangiosarcomas was noted in all treated groups. Both numbers of hemangiosarcomas in male rats and hemangiomas observed in female rats were outside the range of historical control background data. Angiomatous hyperplasia in the spleen considered as a precursor lesion of angiomatous tumours was observed at an increased number in males and females at 80 ppm. Therefore, the substance is classified for carcinogenicity Cat. 2 under Regulation (EC) No. 1272/2008, as amended for the seventeenth time in Regulation (EC) No. 2021/849.

Additional information

 

No reliable data are available for the test substance. The toxicological assessment is based on read-across to CAS 10039-54-0.

 

In a standard combined chronic toxicity/carcinogenicity toxicity study according to OECD TG 453, the test substance was administered to groups of 50 male and 50 female Wistar rats daily in the drinking water at dose levels of 0, 5, 20 and 80 ppm for 24 months (main groups), and additional to groups of 10 animals per sex and dose for 12 months (satellite groups) for evaluation of hematology parameters. The doses administered corresponded to a mean daily intake in the main groups of about 0, 0.2, 1.0, and 3.7 mg/kg bw/d in males and 0, 0.4, 1.6, and 6.2 mg/kg bw/d in females; and in the satellite groups of about 0, 0.3, 1.1, and 4.5 mg/kg bw/d in males and 0, 0.4, 1.6, and 6.2 mg/kg bw/d in females.

There was no statistically significant body weight loss, and no significant effect on mortality in the treatment groups when compared to control groups, and furthermore, no treatment-related clinical signs were noted in either sex throughout the study. At termination, mean absolute and relative spleen weights were statistically significant increased in the 80 ppm females. Gross observations on the spleen showed a slightly increased number of males with macroscopically diagnosed masses at 80 ppm. Microscopy yielded further evidence of spleen injury in 20 ppm and 80 ppm males and females at sacrifice. The only neoplasms showing an increased incidence were tumours of the spleen in both male and female rats when compared to controls. The spleen tumours observed in male and female rats were diagnosed as hemangiosarcomas and hemangiomas. In male rats the incidence of hemangiosarcomas in the spleen was increased from 4/50 among controls and all treatment groups, to 7/50, 9/50 and 8/50 in the 5, 20, and 80 ppm groups, respectively. Two of the hemangiosarcomas of the 80 ppm dosed males and one of the control males metastasized into other organs. In females, there was a biologically equally distribution of hemangiosarcomas in the spleen over the control group animals and the treated animals (0/5/20/80 ppm: 2/1/1/3). Whereas the number of females with hemangiomas was slightly increased in the 80 ppm dose group (0, 5, 20, 80 ppm: 0/1/1/4). It was shown that there was a slightly increased incidence of hemangiosarcomas in the spleen of male rats at all hydroxylammonium sulfate treatment groups. Although, the incidence of hemangiosarcomas in the spleen seen in male rats was small, not dose-related and the difference to controls did not attain statistically significance, the increase of this tumour type was considered to present an effect of treatment. This was supported by the fact that the incidence of hemangiosarcomas in the spleen seen in all treatment groups was generally higher than concurrent intra laboratory historical controls which underlines the biologically significance of this finding. Tumours developed earlier in treated animals than in controls. Furthermore, the following findings occurred in the spleen: hyperplasia and ectasia of capillarys, lined by a normal endothelium which lacked pleomorphism and mitotic activity and separated by a small amounts of fibrous tissue. These findings diagnosed as angiomatous hyperplasia were seen in all dose groups and controls. However, high frequencies of these spleen lesions were observed in males and females at 80 ppm. Persistent hyperplasia may be indicative as site of tumour development. So, these findings of angiomatous hyperplasia in the spleen were considered as a precursor lesion of angiomatous tumours (hemangioma, hemangiosarcoma). In the satellite groups (treatment for 12 months), very few neoplastic findings were noted which were biologically equally distributed over the control group and the treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

 

Overall, this combined chronic toxicity/carcinogenicity study in the rat is acceptable and does satisfy the guideline requirement for carcinogenicity testing (OECD TG 453) in rats. Results of this standard study have shown that the test item is carcinogenic in rats after treatment with dosages of ≥5 ppm (equivalent to about ≥0.2 mg/kg bw/d) in males and of 80 ppm (equivalent to about 6.2 mg/kg bw/d) in females. In male rats an increased incidence of hemangiosarcomas was noted in all treated groups. Hemangiomas were observed in four females from the 80 ppm dose group. Both numbers of hemangiosarcomas in male rats and hemangiomas observed in female rats were outside the range of historical control background data. Angiomatous hyperplasia in the spleen considered as a precursor lesion of angiomatous tumours was observed at an increased number in males and females at 80 ppm. Therefore, 5 ppm (equivalent to about 0.2 mg/kg bw/d (= LOAEL) in males and 0.4 mg/kg bw/d (= NOAEL) in females) is considered to be the lowest dose level for tumour development in the spleen in male and female rats in this study.