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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Translation of a japanes study report
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Phosphoric acid, dodecyl ester, sodium salt
EC Number:
256-865-1
EC Name:
Phosphoric acid, dodecyl ester, sodium salt
Cas Number:
50957-96-5
IUPAC Name:
sodium dodecyl hydrogen phosphate
Test material form:
solid
Details on test material:
- Physical state: solid
- Analytical purity: not given in test report, but according to supplier 100%

Test animals

Species:
rat
Strain:
other: Sprague-Dawley SPF (Crj;CD (SD) IGS
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 10 weeks
- Weight at study initiation: 338 to 395 g (males), 219 to 256 g (females)
- Housing:
- individually in bracket type metal wire cages (250 mm [W] × 350 mm [D] × 200 mm [H];
- during mating: one pair of male and female animals per cage
- from gestation day 17 to day 5 of lactation: individual housing in a plastic Econ cage (340 mm [W] × 400 mm [D] × 185 mm [H]) with bedding
- Diet (e.g. ad libitum): Solid chow NMF, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C to 26°C
- Humidity (%):37% to 77%
- Air changes (per hr): 10 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
the test substance was suspended in olive oil in an agate mortar;
prepared at a frequency of at least once every 7 days and stored in refrigerator (observed temperature: 3°C-5°C) in brown glass bottles until use

VEHICLE
- Justification for use and choice of vehicle (if other than water): olive oil
- Concentration in vehicle: 50, 100, and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight, exact amount based on latest body weight
- Lot/batch no. (if required): Maruishi Pharmaceutical Co., Ltd., batch No. 4604, 4720
- Purity: no data

PREPARATION OF DOSING SOLUTIONS:
the test substance was suspended in olive oil in an agate mortar;
prepared at a frequency of at least once every 7 days and stored in refrigerator (observed temperature: 3°C-5°C) in brown glass bottles until use

VEHICLE
- Justification for use and choice of vehicle (if other than water): olive oil; no justification given on choice
- Concentration in vehicle: 50, 100, and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight, exact amount based on latest body weight
- Lot/batch no. (if required): Maruishi Pharmaceutical Co., Ltd., batch No. 4604, 4720
- Purity: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- suspensions of each concentration used for administration at week 1 and on the last week of administration were analyzed by HPLC
- for all suspensions tested, the percentage of the test substance relative to the nominal value was in the range of 96.5% to 105.0%, with a C.V. in the range of 1.0% to 5.3%, which were within the acceptable range
Duration of treatment / exposure:
Males: 42 days total; 14 days before mating, through the mating period, up to 1 day before necropsy
Females: 42 to 45 days total; 14 days before mating, through the mating and gestation period, up to day 4 of lactation
Recovery groups: in the 0 and 1000 mg/kg groups, a 14-day recovery period was allowed after the 42-day administration period to investigate the reversibility of the toxic changes

Supplementary study:
males and females: 45 days
Frequency of treatment:
Once daily for 7 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 250, 500, 1000 mg/kg bw/day + 62.5, 125 mg/kg bw/day in supplementary study
Basis:
actual ingested
No. of animals per sex per dose:
12 males, 12 females;
recovery group: 5 males, 5 females (0 and 1000 mg/kg bw/day)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In a preliminary 14-day repeated-dose oral toxicity study (doses: 125, 250, 500, 1000 mg/kg, administration of the test substance did not produce any effect up to 1000 mg/kg. Therefore, 1000 mg/kg was set as the highest dose, and doses of 500 and 250 mg/kg were derived by dividing by a common factor of 2.
A supplementary study was conducted with 62.5 and 125 mg/kg bw/day to determine NOEL, as with the lowest dose of the main test (125 mg/kg) effects have been observed.

- Rationale for animal assignment (if not random):
Animals were stratified according to the body weight on the day of group assignment (1 day before the start of administration), and assigned to each group in such a way that the mean body weight was as close as possible among the groups. The assignment of individual animals was performed by a
combination of the block placement method and random sampling.

- Rationale for selecting satellite groups: not given
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): not given
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
external appearance, nutritional condition, posture, behavior, and excrements, 3 times daily
(before, immediately after, and 2 hours after the administration) during the administration period and
once every morning during the recovery period


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
before the start of administration
males: once every week during administration
females: once every week during pre-mating and mating period, days 1, 7, 14 and 20 of gestation, day 4 of lactation
recovery groups: once every week during administration and recovery
BODY WEIGHT: Yes
- Time schedule for examinations:
males, main group: days 1, 4, 8, 11, 15, 18, 22, 25, 29, 32, 36, 39 and 42 of administration, day of necropsy
females, main group: days 1, 4, 8, 11 and 15 of administration, days 0, 4, 7, 11, 14, 17 and 20 of gestation, days 0 and 4 of lactation
males + females, recovery group: additional days 1, 4, 8, 11 and 14 of the recovery period, day of necropsy


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
males, main group: days 1, 4, 8, 11, 15, 32, 36, 39 and 42 of administration
females, main group: days 1, 4, 8, 11 and 15 of administration, days 1, 4, 7, 11, 14, 17 and 20 of gestation, days 2 and 4 of lactation
males+females, recovery group: additional on days 1, 4, 8, 11 and 14 of the recovery period


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data


WATER CONSUMPTION: Yes
- Time schedule for examinations: week 6 of administration


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 6 of administration
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes (overnight)
- How many animals: 5 males, 5 females of each group
- Parameters checked in table 2 were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 6 of administration
- Animals fasted: Yes (overnight)
- How many animals: 5 males, 5 females of each group
- Parameters checked in table 2 were examined.


URINALYSIS: Yes
- Time schedule for collection of urine: week 6 of administration, week 2 of recovery, only male rats
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes - for 4 hours urine specimen collection, 20 h urine specimen collection with free access to food and water
- Parameters checked in table 2 were examined.


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
males, main group: week 6 of administration
males, recovery group: week 6 of administration, week 2 of recovery
females, main group: day 4 of lactation
females, recovery group: week 6 of administration, week 2 of recovery
- Dose groups that were examined: 0, 250, 500, 1000 mg/kg / 5 animals
- Battery of functions tested: sensory activity (auditory response, approach response, touch response, tail pinch response, papillary reflex, aerial righting reflex, landing foot splay) / grip strength – fore limb + hind limb / motor activity / other:


OTHER:
vaginal smear test in females of the main group, during pre-mating period
- Time schedule for examinations: day 1 of administration until copulation was confirmed
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 3)
5 animals (testis, epididymis all animals)

HISTOPATHOLOGY: Yes (see table 3)
specimens from 5 male and female animals subjected to microscopic examination
Other examinations:
Copulation rate
Conception rate
Fertility rate
Delivery rate
in females (mother animals): corpora lutea and number of implantation sites counted
Statistics:
Bartlett test for homoscedasticity (2-sided significance level, 0.01), Dunnett’s test if homoscedastic, otherwise Dunnett-type test (2-sided significance level 0.05 and 0.01):
body weight, food consumption, water intake, number of occurrences of the estrus profile, estrous cycle, days to copulation, gestation period, number of corpora lutea, number of implantation sites, number of live-born pups, open field observations (defecating frequency, standing frequency), function tests (landing foot splay), grip strength and spontaneous motor activity, quantitative urinalysis parameters, hematological tests, blood chemistry tests, organ weight;
mean body weight of the pups, implantation rate, stillbirth rate, live birth rate, external anomaly rate, and viability rate of the pups for each mother animal,

χ2 test with Yates’ continuity correction (2-sided significance level, 0.05 and 0.01), or if there were cells with an expected frequency of 5 or less, Fisher’s exact test (2-sided significance level, 0.05 and 0.01):
copulation rate, fertility rate, conception rate, delivery rate, sex ratio of the pups, auditory response, approach reaction, contact reaction, pain reaction, pupillary reflex, and air righting reflex, the number of animals that copulated, the number of pregnant animals, the number of females that gave birth to live pups, the number of live male pups, the number of live female pups, and the number of animals that showed normal reflexes

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see details on results
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
see details on results
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Urinalysis findings:
no effects observed
Description (incidence and severity):
see details on results
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
see details on results
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
- no mortality occurred during study period
- no abnormalities in home cage observations

- one female (500 mg/kg) showed opacity of an eyeball (unilateral) from gestation day 5, which was not related to the dose and was therefore considered to be an incidental change
-one male (1000 mg/kg, recovery group) showed decreased body weight gain during the administration period and decreased body weight during the recovery period; decreased spontaneous activity; wheezing in the observation of the general condition (body weight of 466 g before manifestation of the symptom and 261 g on day 14 of the recovery period).
- males of the 1000 mg/kg group (main group) showed a significant increase of the defecation frequency during weeks 1 and 2 of administration, which was a very mild transient change and considered to be within normal range

BODY WEIGHT AND WEIGHT GAIN
- a significantly greater increase in body weight in the females of the 250 and 1000 mg/kg groups during the lactation period, but not dose-related
- one male (1000 mg/kg, recovery group): decreased body weight gain during the administration period and decreased body weight during the recovery period; decreased spontaneous activity; wheezing in the observation of the general condition (body weight of 466 g before manifestation of the symptom and 261 g on day 14 of the recovery period).
- the body weights of the other 4 males and 5 females in the same group were similar to those of the animals in the control group, showing no statistically significant differences

FOOD CONSUMPTION
- a significant increase was observed on days 2 and 4 of lactation in the females of the 250 mg/kg dose group (main group), but this was not dose-related.

WATER CONSUMPTION
- no significant differences between treated and control animals

HAEMATOLOGY
- at 250 mg/kg females: significant increase in the serum level of fibrinogen
- at 500 mg/kg females: significant decrease in the percentage of lymphocytes + significant increase in the percentage of segmented neutrophils
- none of these changes were observed in the 1000 mg/kg group, suggesting that they were within the range of physiological variations
- at 1000 mg/kg females (recovery group) showed a significant increase in the mean corpuscular volume of red blood cells, significant decrease of the platelet count, significant increase in the percentage of lymphocytes, and significant decrease in the percentage of segmented neutrophils; changes were not observed at the end of the administration period, which suggested that they were within the range of physiological variations
- no significant differences in the male animals between the control and any of the treatment groups

CLINICAL CHEMISTRY
- at 250 mg/kg significant decrease of the serum level of inorganic phosphorus in males; not dose related, therefore considered to be within the range of physiological variations
- at 1000 mg/kg males showed a significant increase in the serum level of ALT; as no histopathological correlation was observed, this is considered to be of no toxicological relevance
- at 1000 mg/kg (recovery group) females showed a significant increase in the serum level of total protein; no such change observed at the end of the administration period; the increase was considered to be within the range of physiological variations

URINALYSIS
- no abnormalities observed in the qualitative + quantitative parameter values in any of animals in either the main group or in the recovery group

NEUROBEHAVIOUR
- no significant differences in auditory response, approach response, touch response, tail pinch response, pupillary reflex, grip strength, spontaneous motor activity, air righting reflex or landing foot splay between the control group and any of the treatment groups

ORGAN WEIGHTS
- at 500 mg/kg males showed a significant decrease in absolute and relative thymus gland weight
- at 500 mg/kg males showed a significant increase in relative testis weight
- at 500 mg/kg females showed a significant increase in absolute heart weight
- those changes were not dose-related and are therefore considered to be of no toxicological relevance

- at 1000 mg/kg (recovery group) females showed a significant decrease in absolute and relative thyroid gland weight
- no such change was observed at the end of the administration period

GROSS PATHOLOGY
- indentation of the anterior stomach was observed in 0 (250 mg/kg), 5 (500 mg/kg) and 7 (1000 mg/kg) males, and 1 (250 mg/kg), 1 (500 mg/kg) and 3 (1000 mg/kg)females
- white foci in the anterior stomach were observed in 1 male of the 500 mg/kg group
- rough mucosa in the anterior stomach was observed in 5 (500 mg/kg) and 9 (1000 mg/kg) males, and 5 (500 mg/kg) and 6 (1000 mg/kg) females
- indentation of the glandular stomach was observed in 1 female of the 500 mg/kg group
- dark red foci in the glandular stomach were observed in 1 (250 mg/kg), 2 (500 mg/kg) and 1 (1000 mg/kg) males and 4 (control), 2 (250 mg/kg), 1 (500 mg/kg) and 1 (1000 mg/kg) females

- renal pelvis dilation was observed in 2 (250 mg/kg) and 1 (1000 mg/kg) males; no dose-related effect and therefore considered to be of no toxicologocal relevence
- unilateral corneal opacity was observed in 1 female of the 500 mg/kg group

- rough mucosa in the anterior stomach was observed in 1 male of the 1000 mg/kg group at the end of the recovery period. This animal also showed expansion of the digestive tract from the stomach to the colon due to gas accumulation, and a mild decrease in the size of the testis.
- dark red foci were observed in the lung of 1 female of the 1000 mg/kg group at the end of the recovery period

HISTOPATHOLOGY: NON-NEOPLASTIC
Administration of the test substance had effects on the anterior stomach of the animals in the 250 mg/kg and higher dose groups:
- mild to moderate erosions or ulcers of the anterior stomach were observed in 4 (500 mg/kg) and 4 (1000 mg/kg) males and 1 (250 mg/kg), 1 (500 mg/kg) and 1 (1000 mg/kg) females
- very mild to moderate thickening of the anterior stomach mucosa was observed in 1 (250 mg/kg), 4 (500 mg/kg) and 5 (1000 mg/kg) males and 1 (250 mg/kg), 4 (500 mg/kg) and 3 (1000 mg/kg) females
- very mild to mild edema of the submucosal tissue in the anterior stomach was observed in 1 (250 mg/kg), 5 (500 mg/kg) and 5 (1000 mg/kg) males and 4 (500 mg/kg) and 3 (1000 mg/kg) females
Most of these changes in the anterior stomach were localized findings.

- At the end of the recovery period, moderate thickening of the anterior stomach mucosa was observed in 1 male of the 1000 mg/kg group.

All other findings observed were considered to be incidental changes as judged from the frequency of their occurrence and therefore considered to be of no toxicological relevance:
- Epididymis: very mild infiltration by stromal cells was observed in 1 male of the control group.
- Heart: very mild localized myocarditis was observed in 4 males of the control group and 1 male of the 1000 mg/kg group.
- Kidney: very mild basophilic tubules were observed in 3 males of the control group and 1 male and 1 female in the 1000 mg/kg group.
- Liver: very mild, minute granulomas were observed in 3 males of the control group and 1 male of the 1000 mg/kg group.
- Lung (including bronchi): very mild mineral deposits in the arterial walls were observed in 1 male of the control group and 1 female of the 1000 mg group. Very mild accumulation of foam cells was observed in 2 males and 1 female of the control group, and 1 male and 3 females of the 1000 mg/kg group.
- Spleen: very mild to mild extramedullary hematopoiesis was observed in 5 females each in the control group and 1000 mg/kg group.
- Stomach: inclusion cysts were observed in 1 male of the 500 mg/kg group. Very mild to mild erosions in the glandular stomach were observed in 1 male (1000 mg/kg group) and 3 (control), 1 (250 mg/kg) and 1 (500 mg/kg) females

OTHER FINDINGS

- ESTRUS CYCLE
-no significant differences in the mean length of the estrous cycle between the control group and any of the treatment groups

- MATING RESULTS
- one pair in the 500 mg/kg group did not copulate
- copulation occurred in all of the other pairs by day 4 after the start of mating, resulting in pregnancy in all of the females that copulated.
- no significant differences in the days to copulation, copulation rate, fertility rate, or conception rate between the control group and any of the treatment groups

- DELIVERY RESULTS, DELIVERY/LACTATING STATE
- significant increases in the number of corpora lutea and live-born pups were observed in the 250 mg/kg group, but not dose-related
- no significant difference in the delivery rate, gestation period, number of corpora lutea, number of implantation sites, implantation rate, stillbirth rate, number of live-born pups, and live birth rate
- no abnormalities observed in nest building, pup gathering, or lactating behavior in any of the mother animals

OBSERVATION OF PUPS
- no significant differences were observed in the sex ratio, body weight at birth, or external anomaly rate between the control group and any of the treatment groups
- observation for external anomalies showed kinking of the tail in 1 animal of the 500 mg/kg group, which was considered to be a spontaneous occurrence as judged from the frequency of occurrence and the type of the anomaly

VIABILITY OF THE PUPS
- during the lactation period, death occurred in only 4 pups in the control group and 2 pups in the 1000 mg/kg group.
- no significant differences in the viability rate on day 4 of lactation between the control group and treatment groups.

BODY WEIGHT OF THE PUPS
- no significant differences at birth or on day 4 of lactation between the control group and any of the treatment groups

NECROPSY FINDINGS OF PUPS ON DAY 4 OF LACTATION
- thymic cervical residue was observed in 1 (control), 1 (250 mg/kg), 2 (1000 mg/kg) males and 3 (control), 1 (250 mg/kg), 3 (1000 mg/kg) females
- these findings were considered not to be a sign of toxicity as no dose response relationship was noted

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No substance related systemic effects
Dose descriptor:
LOEL
Remarks:
local effects
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: at 250 mg/kg bw/day: intendation of the anterior stomach, at 500 and 1000 mg/kg bw/day: rough mucosa and/or white foci in the anterior stomach, histologically: erosion/ulcers, thickening of the anterior stomach mucosa, edema in submucosal tissue
Dose descriptor:
NOEL
Remarks:
local effcts
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: edema in the submucosal tissue of the anterior stomach
Dose descriptor:
NOEL
Remarks:
local effects
Effect level:
62.5 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
female
Basis for effect level:
other: edema in the submucosal tissue of the anterior stomach

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

In one male of the 1000 mg/kg group (recovery group), decreased spontaneous motility, wheezing, and decreased body weight were observed. Gross examination showed rough mucosa in the forestomach and expansion of the digestive tract from the stomach to the colon due to gas accumulation, and histological examination showed thickening of the gastric mucosa. Body weight in this animal showed a similar increase as that observed in the control group until the above symptoms manifested themselves on day 37 of administration, but continuously decreased after the manifestation of these symptoms, from which these symptoms were judged to be due to inadvertent administration of the test substance into the upper respiratory tract.

A significant increase of the serum ALT was observed in the males of the 1000 mg/kg group at the end of the administration period. Since the findings were very mild and within the range of physiological variations and not accompanied by histopathological abnormalities, it was judged that these findings were not of toxicological relevance.

SUPPLEMENTARY STUDY

GROSS PATHOLOGY

- at 125 mg/kg females showed edema in the submucosal tissue of the anterior stomach

Applicant's summary and conclusion

Conclusions:
Administration of Phosphoric acid, dodecyl ester, sodium salt had no effect on any of the following: general condition, results of function tests, grip strength, spontaneous motor activity, body weight, food consumption, or the results of urinalysis (including water intake), or haematological tests.

In the pathological examination performed at the end of the administration period, gross observation showed indentation of the forestomach in the animals of the 250 mg/kg and higher dose groups, and rough mucosa and/or white foci in the anterior stomach in the animals of the 500 mg/kg and higher dose groups. Histological observation showed erosions/ulcers of the forestomach, thickening of the forestomach mucosa, and edema in the submucosal tissue.
Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD guideline 422 Phosphoric acid, dodecyl ester, sodium salt was administered 12 Sprague-Dawley rats/sex/dose daily by oral gavage at dose levels of 0 (control), 250, 500, 1000 mg/kg bw/day.

The males were exposed 14 days before mating, through the mating period, up to 1 day before termination (42 days in total). The females were exposed 14 days before mating, through the mating and gestation period, up to day 4 of lactation (42 to 45 days in total). Additional 5 males and 5 females were added to the control group and 1000 mg/kg group as a recovery group to examine the reversibility of the effects. Those animals were observed for another 14 days.

Administration of Phosphoric acid, dodecyl ester, sodium salt had no effect on any of the following: general condition, results of function tests, grip strength, spontaneous motor activity, body weight, food consumption, or the results of urinalysis (including water intake), or haematological tests.

A mild but significant increase of the serum ALT in males of the highest dose group at the end of the administration period was not accompanied by histopathological findings and was within the range of physiological variations in historical controls. Therefore it was judged, that these findings were not of toxicological relevance.

In the pathological examination performed at the end of the administration period, gross observation showed indentation of the forestomach in the animals of the 250 mg/kg and higher dose groups, and rough mucosa and/or white foci in the anterior stomach in the animals of the 500 mg/kg and higher dose groups. Histological observation showed erosions/ulcers of the forestomach, thickening of the forestomach mucosa, and edema in the submucosal tissue. In the pathological examination performed at the end of the recovery period, only one male animal of the highest dose group showed moderate thickening of the anterior stomach mucosa.

Forestomach findings related to an irritant activity of the test item are common findings in rat gavage studies. The stomach of rats is anatomically different from the human stomach. The rat stomach consists of two anatomically distinct parts of approximately equal size: a non-glandular forestomach and a glandular stomach. The forestomach is connected to the oesophagus at the gastro-oesophageal junction, and is clearly separated from the glandular stomach by a distinct border called the limiting ridge. The forestomach is not present in humans. The main function of the rat forestomach is storage and trituration of ingested food prior to digestion in the glandular stomach. The forestomach is a holding compartment and due to this function a long exposure time to orally - especially by gavage as bolus dose - administered doses in this organ occurs. Thus, an irritant test item can easily take effect at the forestomach squamous epithel.

A functional equivalent to the rat forestomach is missing in humans. A histological similar in humans is the oesophagus, because the epithelium of the human oesophagus is morphologically of the same type as the forestomach epithelium of rats. However, tissue exposure in the human oesophagus is likely to be minimal compared to tissue exposure in the rat forestomach since the epithelial contact is much shorter in the human oesophagus compared to the rat forestomach and it seems very unlikely that exposure to concentrations far below those having an irritating potential is hazardous to man. Further on, a constantly repeated oral bolus ingestion of substances like the PAEs, is very unlikely. When test items are administered with the feed or the drinking water – exposure procedures more realistic to the exposure situation of humans –, forestomach effects do not occur.

The NOAEL for systemic toxicity is 1000 mg/kg bw/day

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirements of OECD TG 422.