Alkyl phosphites

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP standards and in accordance with OECD guideline 407 and EU method B.7. Some details on the test material are missing

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

and OECD guideline 407.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test animals

Species:

rat

Strain:

other: Crl:CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Inc. Kingston Facility, Stone Ridge, NY
- Age at study initiation: 7 weeks
- Weight at study initiation: Males 192.6-212.9 grams. Females 161.0-185.1 grams.
- Fasting period before study: Not stated
- Housing: Single house during the study period, suspended stainless steel and wire mesh with aborbent paper below cages.
- Diet: PMI Certified Rodent Diet Meal 5002 (ad libitum).
- Water: Automatic watering system (ad libitum)
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24.4
- Humidity (%): 40-70% relative humidity
- Air changes (per hr): Not stated
- Photoperiod (hrs dark / hrs light): 12 hours light (0700 to 1900 hours) and 12 hours dark (1900 to 0700 hours) by automatic timer.

IN-LIFE DATES: From: June 11, 1996 To: July 23, 1996

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
Test substance was dosed via oral gavage.

VEHICLE
- Corn Oil.
- Concentration in vehicle: 0, 10, 50 and 150 mg/kg of animal body weight.
- Amount of vehicle (if gavage): 5 ml/kg ( 7 days a week).
- Lot/batch no. (if required): SEP2096B and OCT2696B
- Purity: Not stated

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

SUMMARY: Samples of the test substance in corn oil were received by the Analytical Chemistry Laboratory for analysis. The samples were diluted with acetone and were analysed for uniformity, stability, and concentration verification using High Performance Liquid Chromatography (HPLC).

PREPERATION: Samples of the test substance in corn oil were received at nominal concentrations of 0.2, 1.0, and 3.0, and 15 % w/v from the Compound Preparation Department. Sample aliquots were diluted 2:10 or 2:50 with acetone. A corn oil control was diluted (simulating sample preparation) and analysed with each sample set.

STANDARDIZATION: A series of standards of the test substance diluted in acetone was prepared. The final concentrations of the standards bracketed the nominal diluted sample concentrations and a multi-point calibration was used to quantify the samples.

Duration of treatment / exposure:

28 days

Frequency of treatment:

7 days per week for a period of 4 weeks

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
10 mg/kg
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
50 mg/kg
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
150 mg/kg
Basis:
actual ingested

Doses / concentrations 4

Remarks:

Doses / Concentrations:
150 mg/kg (satellite group, dosed for 28 days and then allowed to recover for 14 days)
Basis:
actual ingested

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected on the basis of two seperate rangefinding studies, as detailed in results section.
- Rationale for animal assignment: Random
- Rationale for selecting satellite groups: Dosed with high dose for 28 days and then allowed to recover for 14 days.
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

No positive control was included.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily monday to friday, once daily saturday and sunday.
- Cage side observations checked in table were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0, 7, 14, 21 and 27 and on the day of death for all animals. Additionally body weights were recorded for the satellite animals on Days 35 and 41.

FOOD CONSUMPTION

-Food consumption was measured on Day 7, 14, 21 and 27 during the test period. Additionally food consumption was measured for the satellite animals on Days 35 and 41. Food consumption values for weeks 4 and 6 were based on a 6 day interval due to fasting of the animals on Day 27 and 41 for blood collection on Days 28 and 42, respectively.

OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 28 (Day 42 for satellite group)
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: Yes
- How many animals: All animals


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 28 (Day 42 for satellite group)
- Animals fasted: Yes
- How many animals: All animals


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment related clinical signs of toxicity observed in any of the animals at any dose level tested. Observations were limited to a low incidence of dental abnormalities and/or red ocular discharge. One 10 mg/kg dose female was observed with red material around her snout on Day 21, but this finding was probably the result of maloccluded incisors. All findings were considered incidental and unrelated to treatment with the test material.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Unscheduled mortality was limited to one satellite female which died following an accidental injury. This death was not considered treatment-related. All
remaining animals survived to scheduled terminal sacrifice on Day 28 or Day 42 (satellite group).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no statistically significant differences in mean body weights between the treated and control animals of either sex. All animals displayed increases in body weight over their initial values. Mean body weight of the satellite animals (not included in statistical analysis) was comparable to controls and other treated groups.

Mean body weight gain was statistically significantly lower in the mid dose males compared with controls during the Day 2 1/27 interval. This difference was not considered toxicologically significant since body weight and weight gain were not affected at the high dose level.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No statistically significant differences between mean food consumption between control and treated groups.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in quantitative or semiquantitative hematological parameters between treated and control animals of either sex. In general, the mean hematological values of the satellite animals (not included in statistical analysis) were comparable to controls and/or other treated groups on Day 28.

Following the recovery period, there were several statistically significant but relatively small differences in quantitative hematological parameters in satellite group animals from Day 28 to Day 42. These changes included a statistically significant increase in mean red blood cells in males (6%), an increase in hematocrit in females (5%), and a decrease in mean corpuscular hemoglobin concentration (2%) in females. These small differences were not considered clinically significant. In addition, there were statistically significant differencesin semi-quantitative hematology parameters, including an increase in mean percentage of monocytes in males and females, and mean percentage and absolute eosinophils in females. In the absence of correlating findings at main study termination, these changes were considered incidental and unrelated to treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no differences in serum chemistry parameters between treated and control animals of either sex at main study termination which were considered the result of treatment with the test material. In general, the mean serum chemistry values of the satellite animals (not included in statistical analysis) were comparable to controls and/or other treated groups on Day 28.

There was a statistically significant decrease in mean calcium of the 50 mg/kg males at study termination compared with controls on Day 28. Additionally, there was a statistically significant decrease in mean blood urea nitrogen in the 150 mg/kg dose females. In the absence of other correlating fmdings, these difference were not considered clinically significant.

Following the recovery period, there were several statistically significant differences in serum chemistry parameters of the satellite group animals from Day 28 to Day 42. These included statistically significant increases in mean sodium (1%), albumin (3%), and total protein (4%) of the male animals, and mean calcium (5%), total protein (7%), and cholesterol (40%) of the female animals. In addition, there was a statistically significant decrease in mean phosphorous (12%) in the male animals from Day 28 to 42. These generally small differences were not considered clinically significant since they were limited to the recovery phase.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in mean absolute organ weight, organ-to-body weight ratio, or organ-to-brain weight ratio between treated and control animals of either sex.

At the recovery group termination (Day 42), there were no statistically significant differences in mean organ-to-body or organ-to-brain weight ratios between the Day 42 satellite group weights and Day 28 control or high dose weights.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The majority of animals were free of observable abnormalities at the postmortem examination. One female each in the 10, 50 and 150 mg/kg dose group was observed with whitened areas in the stomach.

There was a low incidence of ocular discharge and/or maloccluded incisors, and one satellite male was observed with small, flaccid testis. These findings were considered incidental and unrelated to treatment with the test material.

There were no significant postmortem findings in the one satellite animal which succumbed following an accidental injury. This death was not related to treatment with the test material.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related microscopic changes were observed in the stomach of high dose (150 mg/kg) males and females. Microscopically, a minimal to moderate amount of submucosal edema and/or inflammation was observed in the forestomach of three male and four female rats of the high dose group. This change was characterized by a thickening of the submucosal area due to accumulation of edema fluid occasionally accompanied by a mostly polymorphonuclear inflammatory cell infiltration. There was no evidence of this change in any of the satellite animals following the 14 day recovery period.

No treatment-related microscopic changes were observed in any of the tissues examined from male and female rats of the low (10 mg/kg) or mid (50 mg/kg) dose groups. One other change observed at a very low incidence in the stomach was focal areas of submucosal edema/inflammation in the glandular area of the stomach. This was histologically different from the aforementioned treatmentrelated effect and was observed in only two control rats.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

1

Key result

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

>= 150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no systemic findngs up to highest dose tested

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

RATIONALE FOR DOSE SELECTION

 

Dose levels for the 28 Day study (0, 10, 50, and 150 mg/kg) were selected based on the results of two separate dose rangefinding studies. In the first rangefinding study, the test material was administered to 2 rats/sex/group at dose levels of 100, 250, 500, and 1000 mg/kg. Both females in the 1000 mg/kg dose group were observed with adverse clinical signs (dyspnea, staining, soft stool, decreased food consumption, emaciation, rales, and/or poor condition) and subsequently died prior to scheduled termination (Day 7). Additionally, evidence of body weight gain suppression and decreased food consumption were observed in the 1000 mg/kg dose group. There were no significant findings at necropsy.

 

In the second rangefinding study, the test material was administered to 4 ratslsex/group at dose levels of 0, 50, 150, 500, and 1000 and mg/kg. All males and three of four females in the 1000 mg/kg group, and one male and one female in the 500 mg/kg group were observed with adverse clinical signs (dyspnea, staining, stool abnormalities, decreased food consumption, emaciation, rales, and/or poor condition etc.) and subsequently died prior to scheduled termination (Day 14). Additionally, evidence of body weight gain suppression and decreased food consumption was observed in the 1000 mg/kg dose group and possibly in the 500 mg/kg group. At necropsy, there was evidence of stomach irritation in one 50 mg/kg male, one 150 mg/kg female, four 500 mg/kg males, three 500 mg/kg females, and all 1000 mg/kg animals. The gross stomach changes ranged from roughened mucosa in the 50 and 150 mg/kg dose groups to more severe changes in the 1000 mg/kg dose group such as sloughing and discoloration of the mucosa. Thus, the severity of stomach effects appeared to be dose-dependent.

 

Together, the above rangefinding studies showed that dose levels 500 mg/kg were not acceptable for the 28 Day study due to lethality. Although no lethality occurred at levels<500 mg/kg over the course of one or two weeks of dosing, gross stomach lesions were observed at rangefinding levels as low as 50 mg/kg. Therefore, in consideration of the longer (4 week) dosing period of the main study, a high dose level of 150 mg/kg was considered appropriate to avoid lethality and minimize excessive stomach irritation.

 

ANALYTICAL CHEMISTRY

 

UNIFORMITY OF TEST SUBSTANCE IN CORN OIL

Sample ID	Rep 1	Rep 2	Rep 3	Mean	SD	RSD
0.2%	0.144	0.132	0.117	0.131	0.0135	10.3%
1.5%	1.33	1.19	1.27	1.26	0.0702	5.6%
15%	15.9	16.1	15.6	15.9	0.252	1.58%

STABILITY OF TEST SUBSTANCE IN CORN OIL

Sample ID	Day 0	Day 2	% Change Day 0-2
0.2%	0.131	0.132	+0.763%
15%	15.9	12.4	-22.0%

Sample ID	Day 0	Day 3	% Change Day 0-3	Day 6	% Change Day 0-6
0.2%	0.206	0.174	-15.5%	0.162	-21.4%
3.0%	3.08	2.31	-25.0%	2.06	-33.1%

Results are reported in % w/v.

CONCENTRATION VERIFICATION OF TEST SUBSTANCE IN CORN OIL

Sample ID	Run 1	Run 2
0.2%	0.206	0.208
1.0%	1.26	10.4
3.0%	3.08	2.74

Results are reported in % w/v.

Satisfactory uniformity was observed. The relative standard deviation ranged from 1.58% to 16.7%. The test material was shown to be stable at room temperature for at least 2 to 3 days. Concentration verification analyses indicated that the test material concentration was within 9% of the nominal value with the exception of the 1.0% week one sample which was 26% higher than the nominal value.

Applicant's summary and conclusion

Conclusions:

In conclusion, oral administration of the test material under conditions of this study elicited no signs of systemic toxicity. Treatment-related adverse effects were limited to local stomach irritation observed in the high dose (150 mg/kg) males and females which was reversible after a 14 day recovery period. There were no adverse effects with respect to clinical signs, body weights, body weight changes, food consumption, clinical parameters, or organ weights. Based on the microscopic changes observed in the stomachs at the high dose level, a No Observable Adverse Effect Level (NOAEL) for local effects was established at 50 mg/kg of the test material in both males and females. Excluding microscopic effects in the forestomach attributed to the irritant nature of the test material, a systemic NOAEL in males and female rats would be 150 mg/kg.

No circumstances occurred that would have affected the quality or integrity of the data.

Executive summary:

This study was conducted to evaluate the potential toxicity of the test material when administered orally by gavage to rats for a period of 28 days. The study consisted of a carrier control (corn oil) group, three experimental groups, and a satellite recovery group. All groups contained 5 male and 5 female rats. Prior to start of the study, two separate dose rangefinding studies were conducted for the purpose of dose level selection. Based on results of the rangefinding studies, dose levels of 10,50,and 150 mg/kg were selected for the 28 Day study (a high dose level of 150 mg/kg was selected to avoid lethality and minimize excessive stomach irritation). Individual doses were adjusted weekly based on the most recent body weights. Satellite group animals were treated with the test material for 28 days at 150 mg/kg and then observed for an additional period of 14 days for reversibility, persistence or delayed occurrence of toxic effects.

 

Clinical observations were performed daily throughout the study. Body weights were recorded at pretest, at dose initiation (Day 0), on Days 7, 14, 21 and 27, on the day of death, and on Days 35 and 41 for satellite animals. Food consumption was measured weekly. Hematology and serum chemistry studies were performed on all animals on Day 28 and on all surviving satellite animals on Day 42. A full macroscopic postmortem examination was performed on all animals and required organs were preserved. Selected organs were weighed at study termination and a range of tissues was examined microscopically. The microscopic examination was extended to the stomachs of all rats based on the known irritating properties of the test material and on the presence of gross stomach lesions in a few rats at necropsy.

 

There were no treatment-related deaths during the study. Unscheduled mortality was limited to one satellite female which died following an accidental injury on Day 23. All remaining animals survived to scheduled sacrifice on Day 28 or 42 (satellite group).

 

No treatment-related clinical signs of toxicity were observed and there were no statistically significant and/or biologically meaningful differences among the groups with respect to mean body weights, body weight gain, food consumption, hematological parameters, serum chemistry parameters, absolute organ weights, organ-to-body weight ratios, or organ-to-brain weight ratios. Similarly, there were no meaningful changes in these parameters following the recovery period.

 

There were no gross or microscopic observations indicative of a systemic toxic effect of the test material. Gross stomach lesions were observed in one female each in the 10, 50 and 150 mg/kg dose groups. However, treatment-related microscopic findings were limited to 150 mg/kg males and females and characterized by submucosal edema and/or inflammation of the forestomach. These changes were not observed in the satellite animals following the 14 day recovery period indicating that this change was reversible.

 

In conclusion, oral administration of the test material under conditions of this study elicited no signs of systemic toxicity. Treatment-related adverse effects were limited to local stomach irritation observed in the high dose (150 mg/kg) males and females which was reversible after a 14 day recovery period. There were no adverse effects with respect to clinical signs, body weights, body weight changes, food consumption, clinical parameters, or organ weights. Based on the microscopic changes observed in the stomachs at the high dose level, a No Observable Adverse Effect Level (NOAEL) was established for local effects at 50 mg/kg of the test material in both males and females. Excluding microscopic effects in the forestomach attributed to the irritant nature of the test material, a systemic NOAEL in males and female rats would be 150 mg/kg.