Dimethyl methylphosphonate

Repeated dose toxicity: oral

Administrative data

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: scientifically acceptable study (QC)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1978

Report date:

1978

Materials and methods

Principles of method if other than guideline:

Evaluation of subacute toxicity in rats after one month treatment with test substance in diet for 3 months

GLP compliance:

no

Remarks:

well documented study (QC)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl methylphosphonate

EC Number:

212-052-3

EC Name:

Dimethyl methylphosphonate

Cas Number:

756-79-6

Molecular formula:

C3H9O3P

IUPAC Name:

dimethyl methylphosphonate

Details on test material:

- Name of test material (as cited in study report): 80 021/B
No additional data

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: healthy Sprague Dawley derived rats bred on the premises
- Age at study initiation: 35 and 49 days at the start of the study
- Weight at study initiation: means of 165 g in males and 144 g in females
- Housing: rats were caged in groups of 5
- Diet (e.g. ad libitum): A commercial diet BP nutrition (UK) Limited, Rat and Mouse No. 1 expanded
- Water (e.g. ad libitum): drinking water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2 °C
No additional data

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Powdered diet was mixed with dry extract of malt (Diamalt OCD) in the ratio of 85% food to 15% malt extract by weight. A premix of the powdered diet and compound was prepared for each dose level. The premix was then thoroughly mixed with the bulk diet to produce the required concentrations of test substance, 10% water was added and the mixture passed through a mincer. The pellets so produced were dried for approximately 12 hours at a temperature not exceeding 47°C.

- Medicated diet was freshly prepared at the start of the study and fed on an ad lib basis.

- Control animals received diet prepared in the same manner but without the addition of the test compound.

Duration of treatment / exposure:

at least 93 days

Frequency of treatment:

continuously in diet

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 65, 195 and 638 mg/kg bw in males and 0, 71, 208 and 681 mg/kg bw in females
Basis:
other: calculated equivalent average daily intake

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 1000, 3000 and 10000 ppm in diet
Basis:
nominal in diet

No. of animals per sex per dose:

25 in the control and 10000 ppm groups; 20 in 1000 and 3000 ppm groups

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on the results of the 15-day study
- Rationale for selecting satellite groups: recovery evaluation in the control and in highest dose groups (5 males and 5 females per group were retained without treatment as a recovery experiment, after the end of the main experiment)
- Post-exposure recovery period in satellite groups: 28 days

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: on all rats pre-test and; on 10 females + 10 males from groups 1 and 4 during weeks 5, 9 and 12; on 5 males + females from groups 1 and 4 during week 17.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during week 5, 9 and 13 (from retro-orbital plexus); during week 17 for recovery animals
- Anaesthetic used for blood collection: Yes, under light anaesthesia (not specified)
- Animals fasted: No data
- How many animals: alls
- Parameters examined: heamoglobin (HB), erythrocytes (RBC), packed cell volume (PCV), platelets (PL), total white cell count (WBC), neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B), monocytes (M), prothrombin time (PRO TIME), erythrocyte sedimentation rate (ESR)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see above
- Animals fasted: No data
- How many animals: all
- Parameters examined: blood sugar, urea, serum glutamic pyruvic transaminase (s.G.P.T.), serum amyloid P (S.A.P.)

URINALYSIS: Yes
- Time schedule for collection of urine: weeks 5, 9 and 13
- Metabolism cages used for collection of urine: No data
- Animals fasted: data
- Parameters examined: pH, specific gravity, protein, glucose, bilirubin, ketones, blood, sediment

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: on weeks 5, 6-7, 8, 10, 12, 15, 16 and 17
- Dose groups that were examined: 4 animals/sex in test groups 1 and 4 on week 5; 4 animals/sex in test groups 1, 3 and 4 on weeks 6-7, 8, 10 and 12; and 4 animals/sex in test groups 1, and 4 on recovery weeks 15, 10 and 17
- Battery of functions tested: motor activity; animals were placed on a rat treadmill operating at 12 r.p.m. up to 120 seconds. Animals which completed 2 consecutive attempts of 120 seconds were considered to be normal in their function. Each rat was allowed up to 4 attempts; the mean of the 2 best attempts was used for assessing their performance.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; an autopsy examination was carried out where possible on animals which died or were killed during the course of the study and on all animals which survived until the end of the test. The following organs were weighed: adrenals, brain, heart, kidneys, liver, and gonads. Percentage organ weights were calculated with reference to brain weight and body weight.

HISTOPATHOLOGY: Yes; the following organs and tissues were preserved for histopathological examination: adrenals, oesophagus, aorta, ovaries, bone marrow, pancreas, brain, pituitary gland, caecum, prostate gland, colon, salivary gland, duodenum, skeletal muscle, eyes (+ optic nerve), skin, gross lesions, spinal cord (cervical, thoracic, lumbar), heart, ileum, spleen, jejunum, stomach (non-glandular, glandular), kidneys, testes + epididymus, liver, thymus gland, lungs, thyroid glands, lymph nodes (axillary & mesenteric), tongue, mammary gland, trachea, nerve (sciatic, both), urinary bladder, uterus.
Tissues were fixed in the following solutions: (1) CNS and peripheral nerue (10% buffered formalin; (2) bone marrow (Zenker formol); (3) other organs and tissues (Bouins fluid); (4) eyes (Davidson's fluid). Sections were cut a 5 µm and stained with haematoxylin and eosin. Frozen sections from liver and kidney were stained with Sudan IV in propylene glycol for fat. Kidney sections were also stained by the Masson method.

Other examinations:

none

Statistics:

Haematology, clinical chemistry, organ weights and terminal body weights were analyzed with the Mann Whitney U test. (Level of significance P<0.05). The neurotoxicity tests were analysed using Sugiura's two sample test (similar to the non-parametric Mann-Whitney U-test), and a modified Kruskal-Wallis one-way analysis of variance (also a non-parametric test). Both tests include the modifications necessitated by the fact that the samples are "censored" or truncated at one end, i.e. 120 seconds. At the assessments where the Kruskal-Wallis test has indicated significant differences between the three treatment groups being compared, a multiple comparison technique has been used to determine which of the three groups are causing the difference.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
- Apart from some differences in neuromuscular functions (see below), there were no clinical symptoms noted that were caused by administration of the compound

- One control male rat died after a terminal blood sampling accident (day 92) and another female (in the high dose group) was killed (day 23) after progressive emaciation caused by malformation in the growth of the incisor teeth which prevented an adequate intake of food.
Neither of these 2 mortalities was caused by compound administration. No death occurred during the recovery period

BODY WEIGHT AND WEIGHT GAIN
Body weight gains in all treated groups were comparable to controls throughout the study, and terminal weights were within expected limits for this colony. No abnormalities were observed after the recovery period.

FOOD CONSUMPTION
Food intake was comparable for all groups (Table 1; only high dose females tended to show a slightly less food intake when compared to controls but this was within the normal limits for this colony)

OPHTHALMOSCOPIC EXAMINATION
No adverse effects were noted (during main study and recovery period)

HAEMATOLOGY
No adverse effects were noted (during main study and recovery period)

CLINICAL CHEMISTRY
No adverse effects were noted (during main study and recovery period)

URINALYSIS
No adverse effects were noted (during main study and recovery period)

NEUROBEHAVIOUR
- An effect on neuromuscular function was definitely seen in male rats (group 4) receiving 10000 ppm of test substance in the diet. There was possibly a marginal effect towards the end of the study in female rats at the same dose level. At 3 000 ppm only male rats were examined. While there were no significant decreases (P<0.05) during the study, there was a tendency for treadmill times to be shorter during the 12th week, which suggests that a marginal effect may be present particularly as the rats increased in weight.
- Recovery: in the examination for neurotoxic effect there were no significant differences between control (group 1) and 10000 ppm (group 4) females. In the males there were significant differences but there was a tendency for improve treadmill times. However, because of the small groups it is not possible to be categorical on these effects.

ORGAN WEIGHTS
There was a dose related increase in kidney weights in males both as absolute and relative weights (Table 2). A slight increase in liver weights both absolute and relative was also seen in male rats at 10000 ppm. No other differences noted were considered to be caused by the administration of the compound (during main study and recovery period).

GROSS PATHOLOGY
No gross lesions were seen at autopsy that could be attributed to the administration of the compound (during main study and recovery period).

HISTOPATHOLOGY: NON-NEOPLASTIC
- On histopathological examination renal tubular regeneration was seen in all treated groups. This was minimal to moderate (21/40 rats) at 1 000 ppm and minimal to marked at 3 000 ppm (24/40 rats) and 10000 ppm (21/40 rats).
- Intracellular inclusions in the hepatocytes were seen in 14/40 rats at 1000 ppm, 12/40 rats at 3000 ppm and 15/40 rats at 10000 ppm. Minimal centrilobular hepatocellular hypertrophy was also seen in 1/40 rats at 1000 ppm, 3/40 rats at 3 000 ppm and 16/40 rats at 10 000 ppm.
- No other changes were noted that could be attributed to the administration of the compound.
- After recovery, there was evidence of renal tubular regeneration (7/10 rats) and occasional cellular inclusion in hepatocytes (3/10 rats), though to a slightly lesser degree than in the main study.

Effect levels

Dose descriptor:

LOEL

Effect level:

65 - 71 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: the test substance caused dose-dependent kidney and liver effects

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Mean Total Food Intake (g/rat) over the test period

Test group	Dose level (ppm)	Mean Total Food Intake (g/rat)
		Male	Male
1	0	2152	1602
2		2142	1521
3		2097	1537
4		2057	1464

 

Table 2: Body and organ weights at necropsy

Treatment group	Dose level (ppm)	Mean weight (g)
		Body weight			Left adrenal weight			Right adrenal weight			Left gonad weight			Right gonad weight
		Male	Female	Both sex	Male	Female	Both sex	Male	Female	Both sex	Male	Female	Both sex	Male	Female	Both sex
1	0	523	313	421	0.0297	0.0382	0.0339	0.0282	0.0379	0.0330	2.9991	0.0533	1.5639	3.0445	0.0550	1.5881
2	1000	517	297*	407	0.0310	0.0387	0.0349	0.0302	0.0367	0.0335	3.0793	0.0540	1.5667	3.0788	0.0519	1.5654
3	3000	497	300	399	0.0326*	0.0388	0.0357	0.0297	0.0377	0.0337	3.1164	0.0523	1.5844	3.1613	0.0515	1.6064
4	10000	506	286***	393	0.0316	0.0379	0.0349	0.0316*	0.0359	0.0338	3.1013	0.0493	1.5361	3.1553	0.0491	1.5624

 

Table 2 continue: Organ weights at necropsy

Treatment group	Dose level (ppm)	Mean weight (g)
		Left kidney weight			Right kidney weight			Heart weight			Liver weight			Brain weight
		Male	Female	Both sex	Male	Female	Both sex	Male	Female	Both sex	Male	Female	Both sex	Male	Female	Both sex
1	0	1.3384	0.9067	1.1281	1.3525	0.9226	1.1431	1.5628	1.1284	1.3512	15.5031	10.0983	12.8699	2.2514	2.0849	2.1703
2	1000	1.4996*	0.8933	1.1919	1.5882***	0.9105	1.2494	1.5796	1.1111	1.3454	15.6496	9.6074	12.6285	2.2255	2.0786	2.1520
3	3000	1.5672***	0.8924	1.2298	1.6386***	0.9135	1.2761	1.5709	1.1177	1.3443	15.8636	9.5372	12.7003	2.2193	2.0540	2.1343
4	10000	1.8513***	0.9126	1.3699	1.8838***	0.9234	1.3913	1.5742	1.0242	1.2922	15.0127***	9.9048	13.8548	2.1593***	2.0002**	2.0777**
*: P<0.05; *: P<0.01; *: P<0.001