Dimethyl phosphonate

Endpoint summary

Description of key information

-Repeated dose toxicity: oral: NOAEL (male) =200 mg/kg/bw/day; NOAEL (female)= 100 mg/kg bw/day similar to OECD guideline 408; 6 concentrations (0, 25, 50, 100, 200, 400 mg/kg bw/day) orally administered to male and female rats.
-Repeated dose toxicity: oral: NOAEL (male/female) = 95 mg/kg/bw/day, OECD guideline 408; 6 concentrations (0, 95, 190, 375, 750, 1500 mg/kg bw /day) orally administered to mouse.
-Repeated dose toxicity: oral: NOAEL= 250 mg/kg bw/day; 15 days no guideline followed; 6 concentrations (0, 250, 500, 1000, 2000, or 3000 mg/kg bw/day) orally administered to rats.
-Repeated dose toxicity: oral: LOAEL= 250mg/kg bw/day; 15 days, no guideline followed; 6 concentrations (0, 250, 500, 1000, 2000, or 3000 mg/kg bw/day) orally administered to mice.
-Repeated dose toxicity: oral: LOAEL (male) =100 kg/bw/day; 2 years, similar to OECD guideline 451; 3 concentrations (males: 0, 100, 200 mg/kg bw/day; females: 0, 50, 100 mg/kg bw/day female rat) orally administered to rats.
-Repeated dose toxicity: oral: LOAEL (male/female) = 100 mg/kg bw/day; 2 years similar to OECD guideline 451; 3 concentrations (0, 100, 200 mg/kg bw /day) orally administered to mice.
-Repeated dose toxicity: inhalation and dermal: waiver

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Comparable to guideline study with deviations (No organ weight determination, no haematological/biochemical examinations, no urinalysis, no data on statistics). 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 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

No organ weight determination, no haematological/biochemical examinations, no urinalysis, no data on statistics

Principles of method if other than guideline:

13-week studies were conducted to evaluate the cumulative toxic effects of repeated administration of dimethyl hydrogen phosphite and to determine the doses in the 2-year studies.

GLP compliance:

not specified

Limit test:

no

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: 6-7 weeks old.
- Weight at study initiation: 184-194 g (male), 136-138g (female)
- Housing: 5 animals per cage (polycarbonate).
- Diet (e.g. ad libitum): Purina Lab Chow meal (St. Louis, MO); available ad libitum.
- Water (e.g. ad libitum): Acidified with HCl (pH 2.5) 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 oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Appropriate amounts of dimethyl hydrogen phosphite were mixed with corn oil. 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, 7.50, 15.01. 30.03, 60.06, 120.01 mg/mL
- Amount of vehicle (if gavage): 3.33 mL/kg.

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.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

5 days/week

Remarks:

Doses / Concentrations:
0, 25, 50, 100, 200, 400 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: fifteen-day repeated administration studies were conducted to determine doses for the 13-week studies.
Based on the mortality data and on the clinical signs, the high dose selected for the 13-week studies was 400 mg/kg.

Positive control:

No

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND FREQUENCY: 
- Clinical signs: yes (observed twice per day).
- Mortality: yes (observed twice per day).

- Body weight: yes (recorded weekly).

- Organ weight: no

- Food consumption: no

- Water consumption: no

- Haematology: no

- Biochemistry: no

- Urinalysis: no

Sacrifice and pathology:

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): Necropsy performed on all animals; the following tissues from vehicle control and 400 mg/kg group of rats: gross lesions, parathyroids, colon, esophagus, brain, sternebrae (including marrow), liver, lung and mainstem bronchi, stomach, thymus, pancreas, kidney, urinary bladder, eyes, mandibular lymph node, salivary glands, thyroid gland, small intestine, ovaries/ uterus or testis, trachea, spleen, adrenal glands, pituitary gland, mammary gland.
Eyes of vehicle control and 200 mg/kg groups of rats were examined.

Other examinations:

No further data

Statistics:

Not performed

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:

effects observed, treatment-related

Description (incidence and severity):

observed in male and female rats that received 400 mg/kg bw.

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:

no effects observed

Details on results:

TOXIC RESPONSE/EFFECTS BY DOSE LEVEL:
MORTALITY
-100 mg/kg bw, f: 2/10
-200 mg/kg bw, m: 1/10; f: 2/10
-400 mg/kg bw, m: increased mortality in male and female animals; 9/10; f: 8/10.
3/5 deaths that occurred in the 100 and 200 mg/kg groups may be due to the accidental introduction of gavage solutions into the lungs.


BODY WEIGHTS
Final mean body weights of males and females that received 400 mg/kg were depressed 46% and 39% relative to those of the vehicle control. The final mean body weight of females that received 200 mg/kg was depresses 14% relative to that of the vehicle controls.

OTHER EFFECTS
Degeneration of the lens was observed in the eyes of 4/9 females and 1/7 males that received 400 mg/kg. Acute diffuse inflammation of the cornea was observed in 1/9 females that received 400 mg/kg. The eyes of the next lower dose group (200 mg/kg) were examined histologically; eye lesions were not seen in either males (0/10) or females (0/9) (Eyes from all animals were not available for analysis to autolysis). Urinary bladder calculi were observed in 2/10 male rats that received 400 mg/kg.

Lesions were observed in the lungs of vehicle controls and all dosed groups.
Blood taken at the end of the studies was found to be positive by hemagglutination inhibition assay for pneumonia virus and by the complement fixation assay Sendai virus in 5/5 vehicle control females and 5/5 vehicle control males

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Remarks on result:

other: no adverse effects observed.

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Body weight decrease, increased urinary bladder calculi, eye changes, increased lung lesions, and increased mortality at 400 mg/kg bw

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Remarks on result:

other: no adverse effects observed.

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Body weight decrease at 200 mg/kg bw

Critical effects observed:

not specified

Table 1. Survival and mean body weights of rats in the thirteen-week gavage studies of dimethly hydrogen phosphite

		Mean Body Weights (a)
Dose (mg/kg)	Survival (b)	Initial	Final	Change (c)	Final Weight Relative to Vehicle Controls (percent)
MALE
0	10/10	186	308	+122	--
25	10/10	185	290	+105	94.2
50	10/10	188	266	+ 78	86.4
100	10/10	194	314	+120	101.9
200	(d) 9/10	184	298	+114	96.8
400	(e) 1/10	184	168	- 16	54.5
FEMALE
0	10/10	136	193	+57	--
25	10/10	137	195	+58	101.0
50	10/10	136	191	+55	99.0
100	(f) 8/10	138	185	+47	95.9
200	(g) 8/10	137	167	+30	86.5
400	(h) 2/10	135	117	-18	60.6

(a) Only group weights were taken by laboratory; no individual animal weight data are available.

(b) Number surviving /number in group

(c) Mean weight change of the group

(d) Week of death: 10

(e) Week of death : 3, 4, 4, 5, 5, 7, 8, 9

(f) Week of death: 7, 11

(g) Week of death: 9, 12

(h) Week of death: 2, 3, 3, 3, 3, 4, 5, 8, 10

Table 2. Numbers of rats with histopathologic lesions in the eye and lung in hte 13 -week gavage studies of dimethyl hydrogen phosphite

	Vehicle Control		100 mg/kg	200 mg/kg		400 mg/kg
Lesion	Male	Female	Female	Male	Female	Male	Female
Eye
No.animals examined microscopically	--	--	--	10	9	7	9
Degeneration, lens	--	--	--	--	--	1	4
Inflammation, chronic, diffuse cornea	--	--	--	--	--	--	1
Lung
No.animals examined microscopically	10	10	2	1	2	10	10
Inflammation, chronic, focal	4	1	--	--	--	--	--
Inflammation, chronic, diffuse	3	2	--	--	1	5	6
Congestion	--	--	2	1	1	--	1
Congestion, diffuse	--	--	--	--	--	3	1
Congestion, acute	--	--	--	--	--	1	--
Histiocytosis	--	--	--	--	--	5	--

Executive summary:

In one sub-chronic investigation male and female Fischer 344 rats were administered 0, 25, 50, 100, 200, 400 mg/kg bw/d dimethyl phosphonate for 5 days/week for 13 weeks via gavage, with a method similar to OECD guideline 408 with restrictions (No organ weight determination, no haematological/biochemical examinations, no urinalysis, no data on statistics). A decreased body weight gain was observed in female rats at 200 mg/kg bw/d and above and for male rats at 400 mg/kg bw/d. Mortality was increased at 400 mg/kg bw/d for both sexes. Eye changes (degeneration of the lens, acute diffuse inflammation of the cornea) and increased lung lesions (inflammation, congestion, histiocytosis) were found in male and female rats at 400 mg/kg bw/d. In male rats increased urinary bladder calculi were observed at 400 mg/kg bw/d. The NOAEL is 100 mg/kg bw/d for female and 200 mg/kg bw/d for male rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Comparable to guideline study with deviations (No organ weight determination, no haematological/biochemical examinations, no urinalysis, no data on statistics). Adopted according to OECD SIDS (public available peer reviewed source). The original source is available and has been reviewed.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral

Two sub-acute, two sub-chronic and two chronic oral investigations studying the repeated dose toxicity of dimethyl phosphonate were performed in rats and mice. In one good quality sub-chronic investigation male and female Fischer 344 rats were administered 25, 50, 100, 200, 400 mg/kg bw/day dimethyl phosphonate on 5 days/week for 13 weeks via gavage (NTP, 1985). A decreased body weight gain was observed in female rats at 200 mg/kg bw/day and above and for male rats at 400 mg/kg bw/day. Mortality was increased at 400 mg/kg bw/day for both sexes. Eye changes (degeneration of the lens, acute diffuse inflammation of the cornea) and increased lung lesions (inflammation, congestion, histiocytosis) were found in male and female rats at 400 mg/kg bw/day. In male rats increased urinary bladder calculi were observed at 400 mg/kg bw/day. The NOAEL is 100 mg/kg bw/day for female and 200 mg/kg bw/day for male rats. The present study was chosen as key study because it was well conducted and it was the most critical study in terms of general toxicology.

In well a conducted chronic study male Fischer 344 rats were administered 100, 200 mg/kg bw/day dimethyl phosphonate and female rats 50, 100 mg/kg bw/day, respectively, on 5 days/week for two years. At doses > or = 100 mg/kg bw/day male rats showed dose-related lung effects (interstitial pneumonia, alveolar/bronchiolar adenoma or carcinoma) and at 200 mg/kg bw/day increased cataract formation, and squamous cell carcinoma. Focal mineralization in the cerebellum was observed in males at 200 mg/kg bw/day (NTP, 1985).

Female rats showed forestomach hyperplasia and a statistically not significant, but dose-related increase in lung alveolar/bronchiolar carcinoma at doses > or = 100 mg/kg bw/d.

The LOAEL for male rats is 100 mg/kg bw/day and the NOAEL for female rats is 50 mg dimethyl phosphonate/kg bw/day.

In a sub-acute study B6C3F1 mice were treated with 250, 500, 1000, 2000, or 3000 mg/kg bw/day dimethyl phosphonate for 15 days. A NOAEL could not be derived from this study due to stomach lesions down to the lowest test concentration (epithelial ulcerations, glandular stomach ulcerations, acute/chronic gastritis, squamous atrophy, hyperplastic gastropathy, hyperkeratosis, submucosal and intra-epithelial abscesses, massive necrosis) (NTP, 1985).

In a sub-chronic investigation B6C3F1 mice were treated with 95, 190, 375, 750, 1500 mg bw/kg bw/day dimethyl phosphonate. At 190 mg/kg bw/day and above cardiac mineralization was seen in male mice and hepatocellular vacuolization in female mice (NTP, 1985). At 375 mg/kg bw/day the liver changes were also seen in male mice. Lung congestions were observed with higher incidence at 375 mg/kg bw/day in both sexes, and mortality was increased at this dose. Testicular atrophy was observed at 375 mg/kg bw/day. 750 mg/kg bw/day was lethal for all animals within 4 weeks.

The NOAEL is therefore 95 mg/kg bw/day for male and female mice.

In a chronic investigation male B6C3F1 mice (males and females were administered 100, 200 mg/kg bw/day for two years) showed calcification of testis at concentrations of > or = 100 mg/kg bw/day. At 200 mg/kg bw/day lower body weights and increased mortality was observed in males only (NTP, 1985). LOAEL for male and female mice was 100 mg/kg bw/day.

Inhalation

One subacute inhalation study (4-week exposure) was also conducted. However the test is not reliable because several relevant methodological deficiencies: at the commencement of the study the weights variation of animals used exceeded ± 20% of the mean weight (21% in males and 33% in females); only vehicle control used; the lowest concentration showed evidence of toxicity; analytical purity was not reported; test material administration was conducted in different ways in different test groups; exposure atmospheric sampling was not conducted properly; temperature at which the test was performed was between 24-27 °C; on test day 16 four male rats of group treated with 483.1 mg/m³ were not loaded into the chamber and did not receive exposure to the test material, due to a technician error. Therefore, this data source is not acceptable for assessment.

Male and female Sprague-Dawley rats (20/sex per group) inhaled 48.7, 142.1, 483.1, and 803.9 mg/m³ (12, 35, 119, and 198 ppm) dimethyl phosphonate vapour for 6 hours/day on 5 days/week (Mobil Oil Corporation, 1982). At all concentrations increased kidney weights were observed in male and female rats. Irritation of superficial ocular structures, mucosal irritation and keratitis were shown in all dose groups and in both sexes. The eye changes progressed to cataracts in dose groups of > or = 142.1 mg/m³. At doses > or = 142.1 mg/m³ cutaneous irritation was observed, the skin effects progressed to dermatitis at 483.1 mg/m³, and at 803.9 mg/m³ necrosis and acute purulent inflammation of the skin were main causes of deaths. At 142.1 mg/m³ inflammation of the anterior nares was visible in male and female rats. At 483.1 mg/m³ the external nares were affected, and at 803.9 mg/m³ red discoloration of the lungs and the nasal turbinates were observed in both sexes.

In male rats reduced body weight gains were observed at doses > or = 142.1 mg/m³. In the next higher dosage (483.1 mg/m³) body weight losses and increased mortality was shown in male and female rats.

Time to death varied between 7 and 26 days at 483.1 and 803.9 mg/m³. Hypospermatogenesis was observed in male rats at lethal doses of > or = 483.1 mg/m³. Hematopoiesis in the spleen occurred in 4/18 female rats at 803.9 mg/m³ only and was not observed in the controls or the lower doses. No historical control data were provided. The LOAEL derived for this study is 48.7 mg/m³ (12 ppm; corresponds to about 10 mg/kg bw/d). No NOAEL was achieved in this study. However, according to Annex VIII of the Regulation EC1907/2006, the short-term toxicity study (28 days) does not need to be conducted if a reliable sub-chronic (90 days) or chronic toxicity study is available, provided that an appropriate species and route of administration were used. An oral carcinogenicity study (103 weeks) and an oral sub-chronic study (13 weeks) were conducted in rats and mice.

As a route-to-route extrapolation is possible, a repeated dose toxicity study with dermal exposure does not need to be conducted. Therefore dermal and inhalation repeated dose toxicity do not need to be conducted

Justification for classification or non-classification

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified for repeated dose toxicity.