Dimethyl phosphonate

Carcinogenicity

Description of key information

In a carcinogenicity study (equivalent to OECD guideline 451) dimethyl phosphonate showed clear evidence of carcinogenicity in male F344 rats and equivocal evidence in female F344 rats. Target organs were lungs and forestomach. No evidence of carcinogenicity was observed in male and female B6C3F1 mice.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with restrictions (no biochemical examinations; only two doses tested). Adopted according to OECD SIDS (public available peer reviewed source). The original source is available and has been reviewed.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

yes

Remarks:

(no biochemical examinations; only two doses tested)

GLP compliance:

not specified

Species:

rat

Strain:

other: F344/N

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, MI, USA)
- Age at study initiation: 7 weeks
- Weight at study initiation: 139 g (average weight; males); 111 g (average weight; females)
- Housing: 5 animals per cage were housed.
- Diet (e.g. ad libitum): NIH 07 Open Formula (Zeigler Bros, Gardners, PA); available ad libitum.
- Water (e.g. ad libitum): Acidified with HCl (pH 2.6) tap water; available ad libitum.
- Acclimation period: 2 weeks.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24 (max 28)
- Humidity (%): 30-70
- Air changes (per hr): 12-15 room air changes/h
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dimethyl hydrogen phosphite and corn oil were mixed to yield desired concentrations. Appropriate amount of dimethyl hydrogen phosphate and corn oil mixed in a graduated cylinder by inversion; mixtures were resuspended before dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil was chosen because of the potential for chemical hydrolysis in water.
- Concentration in vehicle: 0, 25, 50 mg/mL (males); 0, 12.5, 25 mg/L (females)
- Amount of vehicle (if gavage): 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses for dimethyl hydrogen phosphite in corn oil were performed on every eighth dose mixture to confirm that the correct concentrations were administered to the test animals. The method of analysis involved a methanolic extraction as a purification step and a gas chromatographic assay as a quantification step. In addition, samples were sent to the analytical chemistry laboratory for referee analysis twice each year during the 2-year study.

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

5 days/week

Post exposure period:

about 1 week

Remarks:

Doses / Concentrations:
0, 100, 200 mg/kg bw/day male rats; 0, 50, 100 mg/kg bw/day female rat
Basis:
actual ingested

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on survival and weight gain information obtained from the 13-week studies, the doses for male rats in the 2-year study were set at 100 and 200 mg/kg and for female rats at 50 and 100 mg/kg.
Doses for female rats were set lower than those for male rats because the females showed a more severe weight depression at 200 mg/kg in the 13- week studies.

Positive control:

No

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND FREQUENCY
- Body weight: yes (once per week for 13 weeks, 1 every 4 weeks thereafter).
- Food consumption: no
- Water consumption: no
- Clinical signs: yes
- Organ weight: no
- Mortality: yes (observed 2xd)
- Haematology: no
- Clinical chemistry: no
- Urinalysis: no

Sacrifice and pathology:

Necropsy performed on all animals. Tissue examined microscopically: tissue masses and gross lesions, regional lymph node, skin, blood smear, mandibular lymph node, mammary gland, salivary glands, thigh muscle, sciatic nerve, bone marrow, costochondral junction (rib), thymus, larynx, trachea, lungs and bronchi, heart, thyroid gland, parathyroids, esophagus, stomach, duodenum, jejunum, ileum, colon, cecum, rectum, mesenteric lymph node, liver, pancreas, spleen, kidneys, adrenal glands, urinary bladder, seminal vesicles/prostate, testes, or ovaries/uterus, nasal cavity, brain, pituitary gland, eyes, and spinal cord.

Other examinations:

No further examinations

Statistics:

SURVIVAL ANALYSES: The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Animals were censored from the survival analyses at the time they were found dead of other than natural causes or were found to be missing; animals dying from natural causes were not censored. Statistical analyses for a possible dose-related effect on survival used the method of Cox (1972) for testing two groups for equality and Tarone´s (1975) life table test for a dose-related trend. All reported P values for the survival analysis are two-sided.

CALCULATION OF INCIDENCE: The incidence of neoplastic or non-neoplastic lesions is given as the ratio of the number of animals bearing such lesions at a specific anatomic site to the number of animals in which that site was examined. In most instances, the denominators include only those animals for which the site was examined histologically. However, when macroscopic examination was required to detect lesions (e.g., skin or mammary tumours) prior to histologic sampling, or when lesions could have appeared at multiple sites (e.g., lymphomas), the denominators consist of the number of animals on which necropsies were performed.


ANALYSIS OF TUMOUR INCIDENCE: Three statistical methods are used to analyze tumour incidence data. The two that adjust for intercurrent mortality employ the classical method for combining contingency tables developed by Mantel and Haenszel (1959). Tests of significance included pairwise comparisons of high dose and low dose groups with vehicle controls and tests for overall dose-response trends.

For further detail see "Any other information on material and methods including tables".

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

Result (carcinogenicity): positive

BODY WEIGHTS AND CLINICAL SIGNS
Mean body weights of high dose male rats were 5% lower than those of the vehicle control males after week 24 and 10% lower after week 40. Low dose male rats and high dose female rats showed marginal depressions in weight gain compared with the corresponding vehicle controls; by the end of the studies, they weighed 4% to 5% less than the corresponding vehicle controls.

SURVIVAL
Survival of female rats was comparable among all groups. The survival of high dose male rats was significantly lower than that of the vehicle controls; the increased incidence of deaths in this group during the course of the experiment was attributed to the toxicity of the chemical.
Pneumonia was found in 0/10 vehicle control, 3/19 low dose, and 16/24 high dose male rats that died early in the study (non accidental deaths); thus, lung disease may have been a cause of the decreased survival in dosed male rats. Ten of 24 high dose male rats that died early in the study had lung tumours. The results of hemagglutination inhibition assays, complement fixation assays, and ELISA were negative for virus infection at 6, 12, 18, and 24 months.

PATHOLOGY
LUNG: The incidences of non-neoplastic and neoplastic lung lesions were increased in dosed male and female rats. The terms alveolar epithelium hyperplasia, adenomatous hyperplasia, and interstitial chronic pneumonia were used to diagnose a complex compound-related lesion characterized by hyperplasia of the alveolar epithelium and thickening of the septal walls around terminal bronchioles and adjacent alveoli.
The lesion was compound related and most severe in the high dose animals. The incidence of inflammatory cells did not appear to be increased. The interstitial pneumonia diagnosed in the vehicle controls was very mild, did not have a centriacinar distribution, and usually consisted of a focal collection of histiocytes and/or mild perivascular cuffing of lymphocytes.

Adenomatous hyperplasia was a focal expansile lesion characterized by extensive proliferation of well-differentiated pneumocytes. This lesion was considered hyperplastic rather than neoplastic because the underlying supporting tissues of the lung remained intact and cytomorphologic evidence of neoplasia was lacking. The expansile nature of the lesion plus proliferative infoldings into alveolar spaces distinguished this lesion from the commonly observed focal hyperplasia of the alveolar epithelium. The latter is usually seen as a minimal or mild lesion following type I pneumocyte injury.

Squamous cell carcinomas, alveolar/bronchiolar adenomas, alveolar/bronchiolar carcinomas, and alveolar/bronchiolar adenomas or carcinomas (combined) in males and alveolar/bronchiolar carcinomas in females occurred with significant positive trends. The incidences of squamous cell carcinomas, alveolar/bronchiolar adenomas, alveolar/bronchiolar carcinomas, and alveolar/bronchiolar adenomas or carcinomas (combined) in high dose male rats were significantly greater than those in the vehicle controls.

Alveolar/bronchiolar adenomas were characterized by focal areas of increased cellularity which caused compression of the adjacent parenchyma.
The cells formed solid, glandular, or papillary patterns and obliterated the underlying alveolar structure. There was little cellular atypia, and mitotic figures were uncommon.
Compared with adenomas, alveolar/bronchiolar carcinomas showed more cellular atypia, invasion of adjacent lung parenchyma, and scirrhous response. On gross examination, the alveolar/ bronchiolar carcinomas were yellow or white firm masses involving one or more lobes of the lung. Microscopically, these neoplasms were composed of polyhedric cells usually arranged in a papillary pattern, although tubular and solid trabecular patterns were also observed.
Cellular atypism and invasion of surrounding tissues were consistent features. The alveolar/bronchiolar carcinomas metastasized to the mediastinal tissues in three high dose males and one low dose male. No metastases were seen in the female rats with carcinoma of the lung.
Five high dose male rats had lung tumours composed entirely of squamous cells. For this reason, these tumours were diagnosed as squamous cell carcinomas.
Squamous cell carcinomas appeared grossly as white to green lung masses. Microscopically, these lung masses were characterized by squamous differentiation, cellular atypia, and invasion of surrounding tissues.
One of the animals with a squamous cell carcinoma also had an alveolar/bronchiolar carcinoma involving a separate lobe.
Interstitial pneumonia was found in 0/10 vehicle control, 4/19 low dose, anti 18/24 high dose male rats that died early in the studies (nonaccidental deaths); thus, pulmonary changes may have contributed in part to the decreased survival in the dosed male rats. The interstitial pneumonia was characterized by centriacinar alveolar epithelial hyperplasia and thickening of septal walls.
There did not appear to be an increased incidence of inflammatory cells; the lesion is considered to be compound related and noninfectious.

FORESTOMACH: In male rats, there were proliferative lesions of the forestomach. Diffuse to focal thickening of the squamous epithelium was diagnosed as hyperplasia. Lesions characterized by papillary projections lined by squamous epithelium with fibrovascular cores were diagnosed as squamous cell papillomas. When the squamous cells invaded the submucosa, the lesions were diagnosed as squamous cell carcinomas.
The squamous cell carcinomas were characterized by invasion of subjacent tissues and marked cellular atypia.
The incidences of hyperplasia in high dose rats of each sex and the incidence of hyperkeratosis in high dose males were greater than those in the vehicle controls. Squamous cell papillomas, squamous cell carcinomas, and squamous cell papillomas or carcinomas (combined) in male rats occurred with significant positive trends. The incidence of squamous cell papillomas or carcinomas (combined) in high dose males was significantly greater than that in the vehicle controls. Two forestomach neoplasms were seen in high dose female rats.
HEMATOPOIETIC SYSTEM: The incidence of mononuclear cell leukemia in low dose male rats was significantly greater than that in the vehicle controls by life table analysis (vehicle control, 9/50; low dose, 15/50; high dose, 13/50). No effects were observed in female rats (vehicle control, 6/50; low dose, 7/50; high dose, 7/50).


EYE: Cataracts were observed at an increased incidence in high dose male rats (vehicle control, 25/50, 50%; low dose, 19/50, 38%; high dose, 36/50, 72%). The following incidences were observed in females: vehicle control, 17/50 (34%); low dose, 13/50 (26%); high dose, 22/50 (44%). The incidences were not clearly related to cage placement.


CEREBELLUM: Focal mineralization in the granular layer of the cerebellum was present in 12/49 (24%) high dose male rats but not in any of the other groups of males or females. The mineralization was characterized by multiple spherical basophilic concretions up to 1 mm in diameter.
The concretions tended to occur in clusters in the granular layer. No association between the presence of concretions and cell damage was found, nor did the concretions appear to be associated with vessels.

LIVER: Neoplastic nodules in male rats occurred with a significant negative trend (vehicle control, 3/50; low dose, 0/50; high dose, 0/50; P= 0.022). The incidences of neoplastic nodules in female rats were comparable among groups (vehicle control, 0/50; low dose, 0/50; high dose, 1/50).

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Statistically significant mononuclear cell leukemia was observed at dose > 100 mg/kg bw/day; lung carcinoma in male rats.

Table 1. Incidences of lung lesions in rats in the two-year gavage studies of dimethyl hydrogen phosphite

MALE	Vehicle Control	100 mg/kg	200 mg/kg
Hyperplasia, alveolar epithelium	2/50	7/50	16/50
Hyperplasia, adenomatous	0/50	3/50	26/50
Pneumonia, interstitial chronic	7/50	19/50	43/50
Metaplasia, squamous	0/50	0/50	3/50
Alveolar/bronchiolar adenoma	0/50	0/50	5/50
Alveolar/bronchiolar carcinoma	0/50	1/50	20/50
Squamous cell carcinoma	0/50	0/50	5/50
FEMALE	Vehicle Control	50 mg/kg	100 mg/kg
Hyperplasia, alveolar epithelium	1/50	0/49	11/50
Hyperplasia, adenomatous	0/50	0/49	10/50
Pneumonia, interstitial chronic	4/50	5/49	33/50
Alveolar/bronchiolar carcinoma	0/50	1/49	3/50

Table 2. Analysis of lung tumors in rats in the two-year gavage studies of dimethyl hydrogen phosphite

MALE	Vehicle Control	100 mg/kg	200 mg/kg
Squamous Cell Papilloma (b)
Overall Rates	0/50 (0%)	0/50 (0%)	5/50 (10%)
Adjusted Rates	0.0%	0.0%	14.2%
Terminal Rates	0/39 (0%)	0/30 (0%)	1/26 (4%)
Life Table Tests	p=0.004	(c)	p= 0.020
Incidental Tumor Tests	p=0.034	(c)	p= 0.141
Alveolar/Bronchiolar Adenoma (d)
Overall Rates	0/50 (0%)	0/50 (0%)	5/50 (10%)
Adjusted Rates	0.0%	0.0%	15.2%
Terminal Rates	0/39 (0%)	0/30 (0%)	2/26 (8%)
Life Table Tests	p = 0.004	(c)	p=0.018
Incidental Tumor Tests	p = 0.017	(c)	p=0.074
Alveolar/Bronchiolar Carcinoma (e)
Overall Rates	0/50 (0%)	1/50 (2%)	20/50 (40%)
Adjusted Rates	0.0%	3.3%	63.5%
Terminal Rates	0/39 (0%)	1/30 (3%)	15/26 (58%)
Life Table Tests	p<0.001	p=0.448	p<0.001
Incidental Tumor Tests	p<0.001	p=0.448	p<0.001
Alveolar/Bronchiolar Adenoma or Carcinoma (f)
Overall Rates	0/50 (0%)	1/50 (2%)	24/50 (48%)
Adjusted Rates	0.0%	3.3%	71.8%
Terminal Rates	0/39 (0%)	1/30 (3%)	17/26 (65%)
Life Table Tests	p<0.001	p=0.448	p<0.001
Incidental Tumor Tests	p<0.001	p=0.448	p<0.001
FEMALE	Vehicle Control	50 mg/kg	100 mg/kg
Alveolar/Bronchiolar Carcinoma (g)
Overall Rates	0/50 (0%)	1/49 (2%)	3/50 (6%)
Adjusted Rates	0.0%	2.9%	8.8%
Terminal Rates	0/40 (0%)	1/35 (3%)	3/34 (9%)
Life Table Tests	p=0.047	p=0.473	p=0.094
Incidental Tumor Tests	p=0.047	p=0.473	p=0.094

(b) Historical incidence at this laboratory: 0%; historical incidence in NTP studies: 2/1, 143, 0.2% ± 0.58%

(c) No P value is presented because no tumours were observed in vehicle control and 100 mg/kg groups.

(d) Historical incidence at the test laboratory: 2/150, 1.3% ± 1.2%; historical incidence in NTP studies: 34/1, 143, 3.0% ± 1.9%

(e) Historical incidence at the test laboratory: 3/150, 2.0% ± 0.0%; historical incidence in NTP studies: 16/1, 143, 1.4% ± 1.5%

(f) Historical incidence at the test laboratory: 5/150, 3.3% ± 1.2%; historical incidence in NTP studies: 50/1, 143, 4.4% ± 2.4%

(g) Historical incidence at the test laboratory: 1/150, 0.7% ±1.2%; historical incidence in NTP studies: 10/1, 142, 0.9% ± 1.3%

Table 3. Incidences of forestomach lesions in rats in the two-year gavage studies of dimethyl hydrogen phosphite

MALE	Vehicle Control	100 mg/kg	200 mg/kg
Hyperkeratosis	0/50	1/50	8/50
Hyperplasia	8/50	16/50	32/50
Squamous cell papilloma	0/50	1/50	3/50
Squamous cell carcinoma	0/50	0/50	3/50
FEMALE	Vehicle Control	50 mg/kg	100 mg/kg
Hyperplasia	4/50	2/50	14/48
Squamous cell papilloma	0/50	0/50	1/48
Squamous cell carcinoma	0/50	0/50	1/48

Table 4. Analysis of forestomach tumors in rats in the two-year gavage studies of dimethyl hydrogen phosphite

MALE	Vehicle Control	100 mg/kg	200 mg/kg
Squamous Cell Papilloma
Overall Rates	0/50 (0%)	1/50 (2%)	3/50 (6%)
Adjusted Rates	0.0%	3.3%	10.8 %
Terminal Rates	0/39 (0%)	1/30 (3%)	2/26 (8%)
Life Table Tests	p=0.032	p=0.448	p= 0.067
Incidental Tumor Tests	p=0.052	p=0.448	p= 0.115
Squamous Cell Carcinoma
Overall Rates	0/50 (0%)	0/50 (0%)	3/50 (6%)
Adjusted Rates	0.0%	0.0%	10.1%
Terminal Rates	0/39 (0%)	0/30 (0%)	1/26 (4%)
Life Table Tests	p = 0.023	(a)	p=0.074
Incidental Tumor Tests	p = 0.066	(a)	p=0.196
Squamous Cell Papilloma or Carcinoma (b)
Overall Rates	0/50 (0%)	1/50 (2%)	6/50 (12%)
Adjusted Rates	0.0%	3.3%	20.0%
Terminal Rates	0/39 (0%)	1/30 (3%)	3/26 (12%)
Life Table Tests	p=0.002	p=0.448	p=0.006
Incidental Tumor Tests	p=0.006	p=0.448	p=0.025

(a) No P value is presented because no tumours were observed in 100 mg/kg and vehicle control groups.

(b) Historical incidence at the test laboratory: 0/147; historical incidence in NTP studies: 6/1, 114, 0.5%.

On the basis of the results we propose to classify dimethyl phosphonate as Category 2 (Carc.2, H351: Suspected of causing cancer), according to the EU-GHS.

Executive summary:

Dimethyl phosphonate was tested for carcinogenicity in doses of 100 and 200 mg/kg bw/d in male F344 rats and 50 and 100 mg/kg bw/d in female F344 rats respectively, with a method similar to OECD guideline 451 with deviations (no biochemical examinations; only two doses tested). The doses were administered orally via gavage on 5 days/week for 103 weeks.

A clear evidence of carcinogenicity was found for male rats and an equivocal evidence for female rats. In gross pathology and histopathology statistically significant squamous cell carcinoma in lung and alveolar/bronchial cell adenoma or carcinoma in male rats were found to be treatment related. In male rats in the highest dose group the incidence of alveolar/bronchiolar adenoma is 5/50 and the incidence of the alveolar/bronchiolar carcinoma is 20/50. In female rats a marginally increase in alveolar/ bronchial cell adenoma or carcinoma was assessed as to be dose-related (0/50, 1/49, 3/50), but was not statistically significant (NTP, 1985). Regarding the forestomach carcinogenicity, statistically significant hyperplasia, hyperkeratosis, and squamous cell carcinoma or adenoma (6/50) were observed in male rats in the highest dose group. In the forestomach of female rats hyperplasia was found in the 100 mg/kg bw dose group. The incidence of forestomach neoplasms was only slightly, and not statistically significantly increased. Statistically significant mononuclear cell leukemia was observed with higher incidences in male rats of the 100 mg/kg bw/d dose group. In the high dose group a slightly lower incidence was observed. The incidence of mononuclear cell leukemia in low dose male rats was significantly greater than that in the vehicle controls (vehicle control, 9/50; low dose, 15/50; high dose, 13/50). The incidence was at the upper limit of the historical control and confined to male animals (NTP, 1985).

In conclusion dimethyl phosphonate showed clear evidence of carcinogenicity in male F344 rats and equivocal evidence in female F344 rats. Target organs are lungs and forestomach.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with restrictions (no biochemical examinations; only two doses tested). Adopted according to OECD SIDS (public available peer reviewed source). The original source is available and has been reviewed.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

yes

Remarks:

(no biochemical examinations; only two doses tested)

GLP compliance:

not specified

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Breeding laboratories (Portage, MI, USA)
- Age at study initiation: 6-8 weeks
- Weight at study initiation: 23 g (males), 19 g (females)
- Housing: 5 animals per cage were housed
- Diet (e.g. ad libitum): NIH 07 Open Formula (Zeigler Bros, Gardners, PA); available ad libitum
- Water (e.g. ad libitum): Acidified with HCl (pH 2.6) tap water; available ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24 (max 28)
- Humidity (%): 30-70
- Air changes (per hr): 12-15 room air changes/h
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dimethyl hydrogen phosphite and corn oil were mixed to yield desired concentrations. Appropriate amount of dimethyl hydrogen phosphate and corn oil mixed in a graduated cylinder by inversion; mixtures were resuspended before dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil was chosen because of the potential for chemical hydrolysis in water.
- Concentration in vehicle: 0, 25, 50 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses for dimethyl hydrogen phosphite in corn oil were performed on every eighth dose mixture to confirm that the correct concentrations were administered to the test animals. The method of analysis involved a methanolic extraction as a purification step and a gas chromatographic assay as a quantification step. In addition, samples were sent to the analytical chemistry laboratory for referee analysis twice each year during the 2-year study.

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

5 days/week

Post exposure period:

about 1 week

Remarks:

Doses / Concentrations:
0, 100, 200 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

50

Control animals:

yes

Details on study design:

- Dose selection rationale: the results from these 13-week studies were used to select doses for the 2-year studies. Decreased survival and toxicity to the lung were seen at 375, 750, and 1.500 mg/kg in male and female mice; these effects were not seen at 190 mg/kg. The maximum dose for the 2-year studies was set at 200 mg/kg.

Positive control:

No

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND FREQUENCY
- Body weight: yes (once per week for 13 weeks, 1 every 4 weeks thereafter).
- Food consumption: no
- Water consumption: no
- Clinical signs: yes
- Organ weight: no
- Mortality: yes (observed 2xd)
- Haematology: no
- Clinical chemistry: no
- Urinalysis: no

Sacrifice and pathology:

Necropsy performed on all animals. Tissue examined microscopically: tissue masses and gross lesions, regional lymph node, skin, blood smear, mandibular lymph node, mammary gland, salivary glands, thigh muscle, sciatic nerve, bone marrow, costochondral junction (rib), thymus, larynx, trachea, lungs and bronchi, heart, thyroid gland, parathyroids, esophagus, stomach, duodenum, jejunum, ileum, colon, cecum, rectum, mesenteric lymph node, liver, gallbladder, pancreas, spleen, kidneys, adrenal glands, urinary bladder, seminal vesicles/prostate, testes, or ovaries/uterus, nasal cavity, brain, pituitary gland, eyes, and spinal cord.

Other examinations:

No further examination

Statistics:

SURVIVAL ANALYSES: The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Animals were censored from the survival analyses at the time they were found dead of other than natural causes or were found to be missing; animals dying from natural causes were not censored. Statistical analyses for a possible dose-related effect on survival used the method of Cox (1972) for testing two groups for equality and Tarone´s (1975) life table test for a dose-related trend. All reported P values for the survival analysis are two-sided.

CALCULATION OF INCIDENCE: The incidence of neoplastic or non-neoplastic lesions is given as the ratio of the number of animals bearing such lesions at a specific anatomic site to the number of animals in which that site was examined. In most instances, the denominators include only those animals for which the site was examined histologically. However, when macroscopic examination was required to detect lesions (e.g., skin or mammary tumors) prior to histologic sampling, or when lesions could have appeared at multiple sites (e.g., lymphomas), the denominators consist of the number of animals on which necropsies were performed.

ANALYSIS OF TUMOR INCIDENCE: Three statistical methods are used to analyze tumor incidence data. The two that adjust for intercurrent mortality employ the classical method for combining contingency tables developed by Mantel and Haenszel (1959). Tests of significance included pairwise comparisons of high dose and low dose groups with vehicle controls and tests for overall dose-response trends.

For further detail see "Any other information on material and methods including tables".

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

RESULT (carcinogenicity): B6C3F1 mice were treated with 100 and 200 mg/kg bw/d in the same way as described above.
Statistically significant increased numbers of hepatocellular adenomas were observed in the 100 mg/kg bw/d female group only. No evidence of carcinogenicity was concluded for B6C3F1 mice

BODY WEIGHTS AND CLINICAL SIGNS
After week 28, mean body weights of high dose male mice were 5% to 10% lower than those of vehicle controls. Mean body weights of dosed and vehicle control female mice were comparable.
Results of hemagglutination inhibition assay, complement fixation assays, and ELISA were negative for virus infection at 6, 12, and 24 months.

SURVIVAL
The survival of high dose male mice was significantly lower than that of the vehicle controls. No significant differences for survival were seen in dosed female mice.

PATHOLOGY
LIVER: Fatty metamorphosis was observed at increased incidences in dosed female mice (vehicle control, 0/50; low dose, 1/49, 2%; high dose, 4/50, 8%). The incidence of hepatocellular adenomas in low dose female mice was significantly greater than that in the vehicle controls. Hepatocellular carcinomas were observed in two female vehicle controls but not in any dosed females. The incidence of hepatocellular adenomas or carcinomas (combined) in the low dose female group was not significantly greater than that in the vehicle controls. The incidence of hepatocellular carcinomas in low dose male mice was significantly lower than that in the vehicle controls (vehicle control, 9/50; low dose, 2/47; high dose, 7/50; P=0.038), but the incidence of hepatocellular adenomas or carcinomas (combined) in the low dose group was not significantly different from that of vehicle controls (vehicle control, 19/50; low dose, 10/47; high dose, 13/50).

TESTIS
Focal calcification was observed at increased incidences in dosed male mice (vehicle control, 2/50, 4%; low dose, 9/47, 19%; high dose, 24/50, 48%). The lesions were more extensive in the dosed animals and appeared as circular-tooblong deposits that obliterated the underlying cellular features. The shape and location of the deposits suggest mineralization of seminiferous tubules.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: Effect type: carcinogenicity.

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Calcification of testis was still found at 100 mg/kg bw

Remarks on result:

other: Effect type: toxicity.

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Statistically significant increased numbers of hepatocellular adenomas were observed in the 100 mg/kg bw/d female group only.

Remarks on result:

other: Effect type: toxicity.

Executive summary:

Dimethyl phosphonate was tested for carcinogenicity in doses of 100 and 200 mg/kg bw/d in male and female B6C3F1 mice, with a method similar to OECD guideline 451 with deviations (no biochemical examinations; only two doses tested). The doses were administered orally via gavage on 5 days/week for 103 weeks.

There was no evidence of carcinogenicity in male or female B6C3F1 mice receiving dimethyl hydrogen phosphite at doses of 100 or 200 mg/kg for 103 weeks.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

100 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Comparable to guideline study with restrictions (no biochemical examinations; only two doses tested). Adopted according to OECD SIDS (public available peer reviewed source). The original source is available and has been reviewed.

System:

respiratory system: lower respiratory tract

Organ:

lungs

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

Taking in consideration that a clear evidence of carcinogenicity was found for male rats, an equivocal evidence of carcinogenicity was found for female rats, and no evidence of carcinogenicity was concluded in mice, the classification of dimethyl phosphonate as Category 2 (Carc.2, H351: Suspected of causing cancer), according to CLP classification criteria (Regulation (EC) No 1272/2008) is proposed.

Additional information

Dimethyl phosphonate was tested for carcinogenicity (method equivalent to OECD guideline 451) in doses of 100 and 200 mg/kg bw/d in male F344 rats and 50 and 100 mg/kg bw/d in female F344 rats respectively. The doses were administered orally via gavage on 5 days/week for 103 weeks. A clear evidence of carcinogenicity was found for male rats and an equivocal evidence for female rats. In gross pathology and histopathology statistically significant squamous cell carcinoma in lung and alveolar/bronchial cell adenoma or carcinoma in male rats were found to be treatment related. In male rats in the highest dose group the incidence of alveolar/bronchiolar adenoma is 5/50 and the incidence of the alveolar/bronchiolar carcinoma is 20/50. In female rats a marginally increase in alveolar/ bronchial cell adenoma or carcinoma was assessed as to be dose-related (0/50, 1/49, 3/50), but was not statistically significant (NTP, 1985). Regarding the forestomach carcinogenicity, statistically significant hyperplasia, hyperkeratosis, and squamous cell carcinoma or adenoma (6/50) were observed in male rats in the highest dose group. In the forestomach of female rats hyperplasia was found in the 100 mg/kg bw dose group. The incidence of forestomach neoplasms was only slightly, and not statistically significantly increased. Statistically significant mononuclear cell leukemia was observed with higher incidences in male rats of the 100 mg/kg bw/d dose group. In the high dose group a slightly lower incidence was observed. The incidence of mononuclear cell leukemia in low dose male rats was significantly greater than that in the vehicle controls (vehicle control, 9/50; low dose, 15/50; high dose, 13/50). The incidence was at the upper limit of the historical control and confined to male animals (NTP, 1985; Dunnick 1986).

B6C3F1 mice were treated with 100 and 200 mg/kg bw/d in the same way as described above. Statistically significant increased numbers of hepatocellular adenomas were observed in the 100 mg/kg bw/d female group only. No evidence of carcinogenicity was concluded for B6C3F1 mice (NTP, 1985; Dunnick 1986).

The International Agency for Research on Cancer concluded 1990 and 1999 that there is limited evidence for the carcinogenicity of dimethyl phosphonate in experimental animals. Therefore dimethylphosphonate is not classifiable as to its carcinogenicity to humans (Group 3) (OECD SIDS, 2004; IARC, 1990; IARC, 1999).

Overall, it can be concluded that dimethyl phosphonate showed clear evidence of carcinogenicity in male F344 rats and equivocal evidence in female F344 rats. Target organs are lungs and forestomach. No evidence of carcinogenicity is observed in male and female B6C3F1 mice.

Carcinogenicity: via oral route (target organ): cardiovascular / hematological: hematopoiesis; digestive: other; respiratory: lung