Dodecylphosphonic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

other: screening OECD 421 study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

from 2012-03-02 to 2012-10-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study (OECD 421). The results of mortality/clinical signs, food consumption, body weight, gross pathology and histopathology were used to support the results observed in the key study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 421

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

2011-07-19

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Octylphosphonic acid

EC Number:

225-218-5

EC Name:

Octylphosphonic acid

Cas Number:

4724-48-5

Molecular formula:

C8H19O3P

IUPAC Name:

octylphosphonic acid

Test material form:

other: cream waxy granular solid

Details on test material:

- Name of test material (as cited in study report): octylphosphonic acid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: River Laboratories France, L’Arbresle, France
- Age at study initiation: 11 weeks old
- Weight at study initiation: Males: 419 g (range 389 g to 447 g) ; Females: 218 g (range: 195 g to 242 g)
- Housing: The animals were individually housed, except during pairing and lactation, in polycarbonate cages (Tecniplast 2154, 940 cm²) with stainless steel lids, and containing autoclaved sawdust (SICSA, Alfortville, France).Toward the end of gestation and during lactation with their litter, autoclaved wood shavings (SICSA Alfortville, France) was provided as nesting material, a few days before delivery and during the lactation period. Each cage contained an object for the environmental enrichment of the animals. The cages were placed in numerical order on the racks.
- Diet: free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 55776558 (SSNIFF Spezialdiäten GmbH, Soest, Germany) which was distributed weekly
- Water (e.g. ad libitum): free access to bottles containing tap water (filtered with a 0.22 µm filter)
- Acclimation period: 5 days before the beginning of the treatment period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%,
- Air changes (per hr): about 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: 2012-03-22 To: 2012-05-05

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. The test item was ground to a fine powder, using a mortar and pestle, and then mixed with the required quantity of vehicle. No correction factor was applied. The dose-levels of the test item were expressed in terms of the test item as supplied.
The dose formulations were prepared for up to 11 days (frequency based on the results of the stability study CIT/Study No. 38540 AHS).
The dose formulations were stored at room temperature, protected from light, and delivered to the study room in brown flasks.

VEHICLE
- Justification for use and choice of vehicle (if other than water): No data
- Concentration in vehicle:12, 30 and 80 mg/kg
- Amount of vehicle (if gavage): 5 mL /kg
- Lot/batch no. (if required): MKBG9425V and MKBF8603V
- Purity: No data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test item in samples of each control and test item dose formulation prepared for use in weeks 1, 3 and 6 was determined. These analyses were performed prior to administration of the dose formulations to the animals.
Acceptance criterion: measured concentration = nominal concentration ± 15%

The test item concentrations in the administered dose formulations analyzed in weeks 1, 3 and 6 were within an acceptable range of -13.6% to -3.5% when compared with the nominal values (± 15%).

Duration of treatment / exposure:

The dose formulations were administered daily according to the following schedule:
In the males:
- 2 weeks before pairing,
- during the pairing period,
- until sacrifice (at least 5 weeks in total),

In the females:
2 weeks before pairing,
during the pairing period,
during gestation,
during lactation until day 4 p.p. inclusive,
until sacrifice for female with no delivery.

Frequency of treatment:

Once a day

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
12 mg/kg
Basis:
other: Nominal concentration

Doses / concentrations 2

Remarks:

Doses / Concentrations:
30 mg/kg
Basis:
other: Nominal concentration

Doses / concentrations 3

Remarks:

Doses / Concentrations:
80 mg/kg
Basis:
other: Nominal concentration

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, following the results of a previous 4-week toxicity study (1989). In this study, the test item was given in oil formulations at 12, 60 or 300 mg/kg/day to Wistar rats. Severe effects, principally on the kidneys, stomach and thymus, and two deaths were reported at 300 mg/kg/day. At 60 mg/kg/day, there were similar findings in kidneys and stomach but to a lesser degree. The dose-level of 12 mg/kg/day was considered as the NOEL in the 4 week toxicity study (HOFF, 1989).

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
The body weight of each male will be recorded on the first day of treatment (day 1), then once a week until sacrifice.
The body weight of each female will be recorded on the first day of treatment (day 1), then once a week until mated (or until sacrifice for females with no evidence of mating), on days 0, 7, 14 and 20 p.c. (post-coitum) and on days 1 and 5 p.p.

FOOD CONSUMPTION:
The quantity of food consumed by each male will be measured once a week, over a 7-day period, from the first day of treatment until the start of the pairing period.
The quantity of food consumed by each female will be measured once a week, over a 7-day period, from the first day of treatment until the start of the pairing period, during gestation for the intervals days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval days 1-5 p.p.
During the pairing period, food consumption will not be measured for males or females.
Food intake per animal and per day will be calculated by noting the difference between the food given and that in the food-hopper the next time.

WATER CONSUMPTION: No

Sacrifice and pathology:

SACRIFICE
On completion of the treatment period, all F0 animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination
- Male animals: All surviving animals after the end of the pairing period (at least 5 weeks of treatment in total)
- Maternal animals: All surviving animals
+Females: on day 5 p.p.,
+Females which did not deliver on days 23 or 25 p.c.

GROSS NECROPSY
- A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all animals. Special attention was paid to the reproductive organs and to the internal surface of the esophagus and stomach.


HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.

Other examinations:

Clinical signs
-The pups will be observed daily for clinical signs, abnormal behavior and external abnormalities.
Body weight
-The body weight of each pup will be recorded on days 1 and 5 p.p.

Statistics:

See table "statistiques CIT.doc" in attached document

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See Body weight table and food comsumption table

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Kidneys and thymus

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Effect on kidney, forestomach and thymus

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths. There were no test item-related clinical signs. Incidental findings in test item-treated groups included: cutaneous lesions, areas of hair loss, and reddish vaginal discharge.


BODY WEIGHT AND WEIGHT GAIN
There were no test item-related effects on mean body weight in males. In the absence of any dose relationship, the statistically significantly higher mean body weight gain noted at 30 mg/kg/day when compared with controls was considered to be fortuitous.
There were no test item-related effects on mean body weight and mean body weight change in females during the pre-mating and gestation periods at any dose-level.
At 80 mg/kg/day, between days 1 and 5 p.p. (lactation period), half of the females lost weight (up to -8% of body weight), and the others generally had a lower body weight gain than controls. This led to statistically significantly lower mean body weight on day 5 p.p. when compared with controls. This was considered to be test item-related but non adverse.
At 12 and 30 mg/kg/day, there was a trend to a minimally dose-related lower mean body weight gain. However, in the absence of any statistical significance and effects on mean body weight on day 5 p.p., this was considered to be of no toxicologically relevance

Food consumption
There were no test item-related effects on mean food consumption in males.
There were no test item-related effects on mean food consumption in females during the pre-mating and gestation periods at any dose-level.
At 80 mg/kg/day, there was a statistically significantly lower mean food consumption from days 1 to 5 p.p. correlating with the effect seen on mean body weight. This was considered to be test item-related but non adverse.
At 30 mg/kg/day, there was a trend towards a lower mean food consumption at the same period, but in the absence of statistical significance and in view of the slight amplitude of the difference from controls, this was considered to be of no toxicologically relevance.

OPHTHALMOSCOPIC EXAMINATION
No examination performed

HAEMATOLOGY
No examination performed

CLINICAL CHEMISTRY
No examination performed

URINALYSIS
No examination performed

NEUROBEHAVIOUR
No examination performed

ORGAN WEIGHTS
The mean terminal body weight was statistically significantly lower in females given 80 mg/kg/day (p<0.05).
When compared with controls, the mean absolute and relative kidney weights were higher in females given 80 mg/kg/day, reaching a statistically significant level for the relative weight (p<0.05).
The mean absolute and relative weights of thymus were minimally lower in females given 80 mg/kg/day. This change correlated with the lymphoid atrophy seen microscopically.
The mean absolute and relative weights of testes were minimally higher in males given 80 mg/kg/day, reaching a statistically significant level for the absolute weight (p<0.05). Since no histological correlates were seen, this change was considered to be of limited toxicological importance and non adverse.

GROSS PATHOLOGY
A few treatment-related changes were seen at necropsy in kidneys, forestomach and thymus.
Kidneys
Enlargement of kidneys was seen in 2/10 females given 80 mg/kg/day. In one animal, this was associated with tan discoloration. These changes correlated histologically with tubular dilation and tubular basophilia.
Forestomach
White deposit was seen on the forestomach in 2/10 males given 80 mg/kg/day and a white mass was seen in 1/10 females. These changes correlated histologically with hyperplasia of squamous cells.
Thymus
Small thymus (reduced in size) was observed in 1/10 females given 12 mg/kg/day, 1/10 females given 30 mg/kg/day and 2/10 females given 80 mg/kg/day. This correlated histologically with lymphoid atrophy.
Red discoloration in the thymus, scabs seen on the skin and enlarged spleen were considered to be part of the normal background of changes commonly seen in the rat

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related changes were seen in kidneys, forestomach, stomach and thymus.
Kidneys
Kidney treatment-related changes were seen in both sexes at 80 mg/kg/day, but particularly in females and at 30 mg/kg/day in females. These included tubular necrosis, present in one high-dose group female but mainly basophilia and tubular dilation with peritubular mononuclear cell infiltrate (occasionally associated with a few fibroblasts). Mixed cell infiltrate was seen in papilla in females given 80 mg/kg/day. Granular casts and cellular debris (mix of desquamed and inflammatory cells including granulocytes) were seen in tubules.
At 12 mg/kg/day, no treatment-related changes were seen in kidneys. Tubular basophilia was seen at a slightly higher incidence than in the control group, however as this was seen unilaterally and focally, any relationship with the test item was considered to be unlikely at this dose-level.

Forestomach/stomach
Pronounced changes were observed in the forestomach at 80 mg/kg/day in both sexes and also at a lesser severity at 30 mg/kg/day.
These consisted of hyperplasia of squamous cells associated with hyperkeratosis, edema and inflammation. In one male and one female given 80 mg/kg/day, this was associated with erosion/ulcer. Spongiosis was seen particularly in males at the level of the limiting ridge.
These changes correlated with those seen at necropsy.
At 12 mg/kg/day, no test item-related changes were seen in the forestomach. In the stomach (glandular portion) the parietal cells appeared to be clear, particularly in males given 80 mg/kg/day. At 30 mg/kg/day, this was seen in 2/10 males only. No changes were noted at 12 mg/kg/day.
The exact nature of this change remained unclear but may correspond to degranulation of the parietal cells. This was not associated with any other treatment related changes (e.g erosion or ulceration of the gastric mucosa) and therefore was considered not to be adverse.

Thymus
Increased incidence of lymphoid atrophy was seen in the thymus of females given 80 mg/kg/day and correlated with the slightly decreased thymic weights. Hemorrhage was observed at a high incidence in all groups, including controls, particularly in males, but without dose-related trend. As this change is commonly seen in rats, this was considered to be fortuitous.

Bone marrow, femur
No treatment-related changes were seen in the bone marrow, particularly in the amount of adipose tissue. In addition, no decreased cellularity was seen in treated animals when compared with controls.

Genital organs
Careful examination of testes in males and of ovaries in females did not show any treatment related changes.
Slight focal tubular atrophy was seen unilaterally in testes of one male given 80 mg/kg/day (and a minimal atrophy involving a single tubule in one control male). This was associated with minimal focal Leydig cell hyperplasia. As these observations were seen unilaterally and in one animal only, these were considered to be unlikely related to the test item.
There were no histopathological changes which correlated with the slightly increased testicular weight noted at necropsy.

Effect levels

Dose descriptor:

NOAEL

Effect level:

12 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: For parental toxicity (Treatment-related changes were seen in kidneys and forestomach at 30 and 80 mg/kg bw/day.)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Body weight and body weight change

Mean body weights and mean body weight changes (g) are presented in the table below:

 

Sex	Male				Female
Dose-level (mg/kg/day)	0	12	30	80	0	12	30	80
Pre-mating (males and females) and whole study (males)
. Day 1	417	419	421	420	222	218	215	218
. Day 15	470	475	484	470	253	244	242	246
. Day 36	526	536	556	522	/	/	/	/
. Days 1 – 15	+52	+56	+63	+50	+30	+26	+27	+29
. Days 15 – 36	+56	+61	+72	+53	/	/	/	/
. Days 1 – 36	+109	+117	+135*	+103	/	/	/	/
Gestation
. Day 0p.c.	/	/	/	/	257	253	251	247
. Day 20p.c.	/	/	/	/	411	418	412	401
. Days 0 - 20p.c.	/	/	/	/	+154	+165	+161	+154
Lactation
. Day 1p.p.	/	/	/	/	313	309	310	302
. Day 5p.p.	/	/	/	/	326	320	317	297*
Differences from controls	/	/	/	/	/	-2%	-3%	-9%
. Days 1 - 5p.p.	/	/	/	/	+13	+11	+7	-4**

/: not applicable;Statistically significant: *: p<0.05, **: p<0.01.

 

Food consumption

Sex	Female
Dose-level (mg/kg/day)	0	12	30	80
Lactation
Days 1- 5p.p.	41	40	36	31**
Differences from controls	/	-2%	-12%	-24%

 

Organ weight

Sex	Male			Female
Group	2	3	4	2	3	4
Dose-level (mg/kg/day)	12	30	80	12	30	80
Exam. animals / Num. of animals	10/10	10/10	10/10	9/10	9/10	10/10
Body weight	+2	+6	-1	-1	-2	-8*
- Kidneys
. absolute	-2	0	-3	-3	+1	+28
. relative	-4	-5	-3	-2	+3	+40*
- Testes
. absolute	+6	+2	+9*
. relative	+4	-3	+9
- Thymus
. absolute	+2	+10	-12	-11	-8	-22
. relative	0	+5	-11	-10	-6	-16

 

Kidneys

 

Sex	Males				Females
Group	1	2	3	4	1	2	3	4
Dose-level (mg/kg/day)	0	12	30	80	0	12	30	80
Number of animals	10	10	10	10	10	10	10	10
Tubular necrosis
Total (mean severity)	-	-	-	-	-	-	-	1 (1.0)
Tubular basophilia
Total (mean severity)	7 (1.0)	4 (1.0)	10 (1.1)	8 (1.8)	2 (1.5)	5 (1.0)	6 (1.5)	10 (3.0)
Tubular dilation
Total (mean severity)	1 (1.0)	2 (1.0)	-	3 (1.3)	1 (1.0)	-	3 (1.3)	9 (2.4)
Infiltrate; mononuclear cells
Total (mean severity)	3 (1.0)	-	2 (1.0)	3 (1.0)	-	-	1 (1.0)	9 (1.1)
Cellular debris in tubules
Total (mean severity)	-	-	-	1 (1.0)	-	-	-	6 (1.3)
Cast, granular
Total (mean severity)	-	-	-	1 (1.0)	-	-	-	8 (1.4)
Infiltrate, granulocytes
Total (mean severity)	-	-	-	-	-	-	-	6 (1.0)
Desquamation, tubular lumen
Total (mean severity)	-	-	-	-	-	-	2 (1.0)	-
Edema (tip of papilla)
Total (mean severity)	-	-	-	-	-	-	1 (1.0)	1 (1.0)

-: not observed in this group.

 

Forestomach/stomach treatment-related changes

 

Sex	Males				Females
Group	1	2	3	4	1	2	3	4
Dose-level (mg/kg/day)	0	12	30	80	0	12	30	80
Number of animals	10	10	10	10	10	10	10	10
Erosion/ulcer
Total (mean severity)	-	-	-	1 (2.0)	-	-	-	1 (2.0)
Hyperplasia; squamous cell
Total (mean severity)	-	-	5 (2.2)	8 (2.5)	-	-	2 (1.0)	10 (2.2)
Hyperkeratosis
Total (mean severity)	-	-	2 (1.5)	6 (1.8)	-	-	-	6 (1.8)
Edema
Total (mean severity)	-	-	-	3 (1.0)	-	-	-	5 (1.6)
Inflammation
Total (mean severity)	-	-	-	4 (1.3)	-	-	-	4 (1.3)
Spongiosis
Total (mean severity)	-	-	1 (1.0)	9 (1.4)	-	-	-	1 (1.0)

-: not observed in this group.

Forestomach/stomach treatment-related changes

 

Sex	Males				Females
Group	1	2	3	4	1	2	3	4
Dose-level (mg/kg/day)	0	12	30	80	0	12	30	80
Number of animals	10	10	10	10	10	10	10	10
Erosion/ulcer
Total (mean severity)	-	-	-	1 (2.0)	-	-	-	1 (2.0)
Hyperplasia; squamous cell
Total (mean severity)	-	-	5 (2.2)	8 (2.5)	-	-	2 (1.0)	10 (2.2)
Hyperkeratosis
Total (mean severity)	-	-	2 (1.5)	6 (1.8)	-	-	-	6 (1.8)
Edema
Total (mean severity)	-	-	-	3 (1.0)	-	-	-	5 (1.6)
Inflammation
Total (mean severity)	-	-	-	4 (1.3)	-	-	-	4 (1.3)
Spongiosis
Total (mean severity)	-	-	1 (1.0)	9 (1.4)	-	-	-	1 (1.0)

-: not observed in this group.

 

Stomach

Sex	Males				Females
Group	1	2	3	4	1	2	3	4
Dose-level (mg/kg/day)	0	12	30	80	0	12	30	80
Number of animals	10	10	10	10	10	NA	10	10
Clear parietal cells
Total (mean severity)	1 (1.0)	-	2 (1.0)	10 (1.7)	-		-	1 (1.0)

 

Thymus

Sex	Males				Females
Group	1	2	3	4	1	2	3	4
Dose-level (mg/kg/day)	0	12	30	80	0	12	30	80
Number of animals	10	NA	10	10	10	10	10	10
Lymphoid atrophy
Minimal	-		-	1	1	3	1	4
Slight	-		-	-	-	-	1	1
Moderate	-		-	-	-	-	-	1
Total	1		-	1	1	3	2	6

 

Applicant's summary and conclusion

Conclusions:

Based on the experimental conditions of this study: the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 12 mg/kg/day, based on the effects observed on the kidney and forestomach at 30 and 80 mg/kg bw/day.

Executive summary:

In a screening reproductive toxicity (OECD 421, 1995), OPA (Octylphosphinic Acid) was administered to male and female Spargue-Dawley rats, 10/dose/sex, by oral gavage at the dose levels of 0, (vehicle), 12, 30, 80 mg/kg bw/day. The test substance was administered by gavage every day from 14 days prior to the start of mating, until day 5 post-partum for the females and at least for 5 weeks for the males.

Clinical signs, food consumption, body weight, Organ weights, macroscopic and histopathological examinations were performed.

There were no test item-related deaths or clinical signs in F0 animals. There were no toxicologically significant effects on mean body weight and mean food consumption in males at any dose-level or in females at 12 and 30 mg/kg/day. At 80 mg/kg/day, there were no test item related effects on mean body weight and mean food consumption during the pre-mating and gestation periods. However from days 1 and 5p.p., the females either lost weight (up to -8% of body weight) or gained less body weight than controls, leading to lower mean body weight on day 5p.p.when compared with controls (-9%, p<0.05). This correlated with low mean food consumption during that period (-24% from controls, p<0.01). These effects were considered to be test item-related but non adverse.

At necropsy, A few treatment-related changes were seen at necropsy in kidneys, forestomach and thymus.

Kidney treatment-related changes were seen in both sexes at 80 mg/kg/day, but particularly in females and at 30 mg/kg/day in females. These included tubular necrosis, present in one high-dose group female but mainly basophilia and tubular dilation with peritubular mononuclear cell infiltrate (occasionally associated with a few fibroblasts). Mixed cell infiltrate was seen in papilla in females given 80 mg/kg/day. Granular casts and cellular debris (mix of desquamed and inflammatory cells including granulocytes) were seen in tubules. At 12 mg/kg/day, no treatment-related changes were seen in kidneys. Tubular basophilia was seen at a slightly higher incidence than in the control group, however as this was seen unilaterally and focally, any relationship with the test item was considered to be unlikely at this dose-level.

Pronounced changes were observed in the forestomach at 80 mg/kg/day in both sexes and also at a lesser severity at 30 mg/kg/day. These consisted of hyperplasia of squamous cells associated with hyperkeratosis, edema and inflammation. In one male and one female given 80 mg/kg/day, this was associated with erosion/ulcer. Spongiosis was seen particularly in males at the level of the limiting ridge. These changes correlated with those seen at necropsy. At 12 mg/kg/day, no test item-related changes were seen in the forestomach.

In the stomach (glandular portion) the parietal cells appeared to be clear, particularly in males given 80 mg/kg/day. At 30 mg/kg/day, this was seen in 2/10 males only. No changes were noted at 12 mg/kg/day. The exact nature of this change remained unclear but may correspond to degranulation of the parietal cells. This was not associated with any other treatment related changes (e.g erosion or ulceration of the gastric mucosa) and therefore was considered not to be adverse.

Increased incidence of lymphoid atrophy was seen in the thymus of females given 80 mg/kg/day and correlated with the slightly decreased thymic weights. Hemorrhage was observed at a high incidence in all groups, including controls, particularly in males, but without dose-related trend. As this change is commonly seen in rats, this was considered to be fortuitous.

No treatment-related changes were seen in the bone marrow, particularly in the amount of adipose tissue. In addition, no decreased cellularity was seen in treated animals when compared with controls.

Based on the experimental conditions of this study, the NOAEL for parental toxicity was 12 mg/kg bw/day (kidney and forestomach effects at 30 and 80 mg/kg bw/day).