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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
Study period:
From 15 April 2013 to 09 September 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to international test guidelines and to GLP.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2014

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)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Reaction Mass of Diisobutyl hydrogen phosphate and Isobutyl dihydrogen phosphate
IUPAC Name:
Reaction Mass of Diisobutyl hydrogen phosphate and Isobutyl dihydrogen phosphate
Constituent 2
Chemical structure
Reference substance name:
Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate
EC Number:
911-351-2
Molecular formula:
Not applicable
IUPAC Name:
Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate
Test material form:
other: brownish liquid stored at room temperature
Details on test material:
- Name of test material (as cited in study report): Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate. ISOBUTYL ACID PHOSPHATE, IBAP
- Physical state: brownish liquid.
- Stability under test conditions: the test item is assumed to be stable by the sponsor.
- Storage condition of test material: room temperature.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Italia, Calco, Italy
- Age at study initiation: males were approximately 10 weeks old and the females were approximately 9 weeks old.
- Weight at study initiation: males had a mean body weight of 373 g (range: 324 g to 431 g) and the females had a mean body weight of 214 g (range: 196 g to 245 g).
- Fasting period before study: no
- Housing: the animals were individually housed, except during mating (males and females together during nights) and lactation (females with their litter), in polycarbonate cages (Tecniplast 2154, 940 cm²) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing was chosen for mating trial purposes and since it is preferable for pregnant animals.
Toward the end of gestation and during lactation, autoclaved wood shavings (SICSA, Alfortville, France) were provided to females and their litter as nesting material.
- Diet (e.g. ad libitum): SSNIFF R/M-H pelleted maintenance diet, batch Nos. 4898480 (SSNIFF Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): tap water (filtered with a 0.22 µm filter).
- Acclimation period: 8 days before the beginning of the treatment period. One supplementary animal per sex was acclimated to permit the selection and/or replacement of individuals.

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: 16 April 2013 to 09 June 2013.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
(PEG 400)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as an emulsion in the vehicle. It was mixed with the required quantity of vehicle. No correction factor was applied for the dose calculations.
VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw/day
- Lot/batch no. (if required): MKBG6036V
- Purity: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of the test item in the dose formulations have been quantified by a validated Ion Chromatography (IC) with conductivity detection analytical method.
The validation of the analytical method was conducted in CiToxLAB France/Study No. 40028 VAA and precise details concerning the checked parameters, acceptance criteria and obtained results are documented in the corresponding validation report.
Duration of treatment / exposure:
In the males:
- 2 weeks before mating (from study day 1 to 14)
- during the mating period (from study day 15 until study day 16 to 19),
- until sacrifice (at least 5 weeks in total) (from study day 17 to 20 until study day 36).

In the females:
- 2 weeks before mating (from study day 1 to 14),
- during the mating period (from study day 15 until study day 16 to 19),
- during gestation (from study day 16 to 19 until study day 36 to 40),
- during lactation until day 5 p.p. inclusive (from study day 37 to 41 until study day 42 to 46),
- until sacrifice for the non-pregnant female (until study day 42).

Day 1 corresponds to the first day of the treatment period.
Frequency of treatment:
Once daily, at approximately the same time (7 days/week)
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 400 and 1000 mg/kg bw/day.
Basis:
other: Nominal concentration
No. of animals per sex per dose:
10 animals/sex/dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose-levels were set based on the results of a previous 2-week dose-range finding study performed in the same species and strain (CiToxLAB France/Study No. 40031 TSR). In this study, four groups of three males and three females received 0 (vehicle: PEG 400), 100, 400 or 1000 mg/kg/day daily for 2 weeks by gavage with a constant dose-volume of 5 mL/kg/day. There was no mortality and clinical signs consisting in piloerection, hunched posture, soft feces and ptyalism have been observed sporadically at 400 (in one male and all females) and 1000 (in all animals) mg/kg/day in the second week of treatment. There were no obvious effects on mean body weight, mean food consumption and mean organ weights. There were no adverse macroscopic findings at necropsy (distended intestinal track was present in 1/3 male from 100 mg/kg/day). Therefore, the same dose-levels were used for this main study.

- Rationale for animal assignment (if not random): during the acclimation period, the required number of animals (40 males and 40 females) was selected according to body weight and clinical condition. The animals were allocated to groups (by sex) using a computerized stratification procedure based on body weight, so that the average body weight of each group was similar.
Positive control:
Not used.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: each animal was checked for mortality or signs of morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays. From arrival, each animal was observed once a day as part of routine examinations. From the start of the treatment period, each animal was observed once a day, at approximately the same time, for the recording of clinical signs. Additionally, the females were observed for signs of difficult or prolonged parturition.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: detailed clinical examinations were performed on all animals once before the beginning of the treatment period and then once a week until the end of the study.

BODY WEIGHT: Yes
- Time schedule for examinations: the body weight of each male was recorded on the first day of treatment (day 1), then once a week until sacrifice.
The body weight of each female was recorded on the first day of treatment (day 1), then once a week until mated and on days 0, 7, 14 and 20 post-coitum (p.c.) and days 1 and 5 p.p..


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
The quantity of food consumed by each male was measured once a week, over a 7 day period, from the first day of treatment until the start of the mating period.
The quantity of food consumed by each female was measured once a week, over a 7 day period, from the first day of treatment until the start of the mating period, during pregnancy at the intervals days 0-7, 7 14 and 14-20 p.c. and during lactation for interval days 1 5 p.p..

During the mating period, food consumption was not measured for males or females.

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study.
- Anaesthetic used for blood collection: Yes (light isoflurane)
- Animals fasted: Yes
- How many animals: the first five males and the first five females to be sacrificed on Day 6 p.p. from each group on the day of scheduled sacrifice.
- The following parameters were determined: Erythrocytes (RBC), Mean cell volume (MCV), Packed cell volume (PCV), Hemoglobin (HB), Mean cell hemoglobin concentration (MCHC),Thrombocytes (PLT), Leucocytes (WBC), Differential white cell count with cell morphology (Neutrophils (N), eosinophils (E), basophils (B), lymphocytes and large unstained cells (L+LUC) and monocytes (M)) and Reticulocytes (RTC).
Coagulation: Prothrombin time (PT) + Fibrinogen (FIB) + Activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study.
- Animals fasted: Yes
- How many animals: the first five males and the first five females to be sacrificed on Day 6 p.p. from each group on the day of scheduled sacrifice.
- Parameters (examined): Sodium (Na+), Potassium (K+), Chloride (Cl-), Calcium (Ca++), Inorganic phosphorus (I.PHOS), Glucose (GLUC), Urea (UREA), Creatinine (CREAT), Total bilirubin (TOT.BIL.), Total cholesterol (CHOL), Triglycerides (TRIG), Alkaline phosphatase (ALP), Alanine
aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Total proteins (PROT), Albumin (ALB) and Albumin/globulin ratio (A/G).


URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the study.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters (examined):
1- Quantitative parameters: volume (Volume), pH (pH) and Specific gravity (SP.GRAV).
2- Semi-quantitative parameters: Proteins (PROT), Glucose (GLUC), Ketones (CETO), Bilirubin (BILI), Nitrites (NITR), Blood (hemoglobin) (BLOOD), Urobilinogen (UROB) and Cytology of sediment (leucocytes (WBC), erythrocytes (RBC), cylinders (CYLIN), magnesium ammonium phosphate crystals (AMM.PH), calcium phosphate crystals (CAL.PH), calcium oxalate crystals (CAL.OX.), epithelial cells (CELLS))
3- Qualitative parameters: Appearance (APP) and Color (COLOR).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the treatment period. For females, this was performed on day 5 p.p. after sacrifice of the pups.
- Dose groups that were examined: the first five males and the first five females to be sacrificed on day 6 p.p. from each group were evaluated
- Battery of functions tested: touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay and at the end of observation: rectal temperature.


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
A complete macroscopic post-mortem examination was performed on all animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues, including the urinary bladder and the internal surface of the esophagus and stomach, and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs.
The numbers of corpora lutea and implantation sites were recorded for females sacrificed as scheduled on day 6 p.p. and one female (group 3) sacrificed on day 25 p.c. for no delivery.

HISTOPATHOLOGY: Yes
A microscopic examination was performed on:
- all tissues listed in the Tissue Procedure Table (Table 7.5.1/1) from the first five sacrificed as scheduled males and the first five females sacrificed on day 6 p.p. of the control and high-dose groups (groups 1 and 4),
- forestomach, stomach, duodenum, jejunum and ileum from the first five sacrificed as scheduled males and the first five females sacrificed on Day 6 p.p. of the low- and intermediate-dose groups (groups 2 and 3) following the results obtained on those organs at macroscopy and/or microscopy in the high-dose group,
- reproductive organs from one female and one male (group 3) that did not conceive to investigate possible causes,
- all macroscopic lesions of all groups.

Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Other examinations:
See "Tox.reproOECD 422 study V1 CiToxLAB 2013" section
Statistics:
The following statistical methods were used to analyze body weights, food consumption, reproduction and skeletal examination data:
• Means and standard deviations of various data were calculated.
• If the variables could be assumed to follow a normal distribution, the Dunnett t-test, based on a pooled variance estimate, was used for inter-group comparisons (i.e. single treatment groups against the control group).
• The Steel test (rank test) was applied when the data could not be assumed to follow a normal distribution.
• Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Observed clinical signs were considered to be related to the test item but of minor toxicological significance
Mortality:
mortality observed, treatment-related
Description (incidence):
Observed clinical signs were considered to be related to the test item but of minor toxicological significance
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Mean total bilirubin and proteinslevels were statistically significantly decreased in males treated at 1000 mg/kg/day when compared to controls.These decreases were considered to be of minor toxicological significance.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Thickening of small intestine was considered to be related to test item administration (see details on results section)
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See details on results section.
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths during the study. One female treated at 400 mg/kg/day was sacrificed on day 25 p.c. for no delivery (at necropsy, this female was not pregnant).
Ptyalism was noted in up to 6 animals per sex at 400 mg/kg/day and in all animals at 1000 mg/kg/day. This clinical sign was considered to be related to the test item (dose-relationship response and no control animals with this clinical sign) but of minor toxicological significance. There were no test item-related clinical signs at 100 mg/kg/day.

BODY WEIGHT AND WEIGHT GAIN
There were no test item-related effects on mean body weight and body weight gain.
Mean body weights were similar among groups as well as mean body weight gains in females over the pre-mating, gestation and lactation periods. Mean body weight gains in test item-treated males over the treatment period were slightly higher than in controls; however, there were no dose-relationship and no statistical level and they did not impact toxicologically the mean body weights. There were considered to be fortuitous.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were no test item-related effects on mean food consumption.
Mean food consumptions were similar between groups in both sexes. The minimally higher mean food consumption noted at 1000 mg/kg/day for females in lactation when compared to controls was considered to be fortuitous (no statistical level, individual data included in control range, amplitude of difference minimal).

OPHTHALMOSCOPIC EXAMINATION
Not examined.

HAEMATOLOGY
There were no test item-related effects and no statistically significant changes in mean hematology and coagulation data (See Table 7.5.1/2)

CLINICAL CHEMISTRY (See Table 7.5.1/3)
There were no effects on mean blood biochemistry data in females .
Mean total bilirubin and proteins (= albumin and globulins) levels were statistically significantly decreased in males treated at 1000 mg/kg/day when compared to controls. The amplitudes were not severe, one sex only was concerned and there were no adverse correlating findings (no adverse histopathology findings in liver, intestines, kidneys, no blood volume increase seen at hematology, no hemorrhages …). These decreases were therefore considered to be of minor toxicological significance.
The statistically significant decrease in mean ALP activity in 1000 mg/kg/day males was not considered to be toxicologically significant in view of the slight amplitude and direction (decrease in ALP activity is rarer than increase) of the change.

URINALYSIS
At 1000 mg/kg/day, 1/5 males and 1/5 females had several red blood cells and/or high level of blood in their urine sample. As there were no histopathological findings in the urinary bladder and kidney, an effect of the test item treatment was considered to be unlikely.
There were no test item-related effects on urine parameters at 100 and 400 mg/kg/day. The only statistically significant difference was observed at 400 mg/kg/day in males on the urine volume and was not considered to be related to the test item treatment as there was no dose-relationship and the tendency was not observed in females.

NEUROBEHAVIOUR
There were no test item-related effects on FOB tests and mean motor activity data (horizontal movements and rearing).

Slight piloerection was observed at the end of the treatment in 1/5 females at 400 mg/kg/day and in 2/5 females at 1000 mg/kg/day. In view of the slight severity and incidence and in absence of correlating clinical signs during the study, this finding was considered to be unrelated to the test item treatment.

Other findings were observed in similar incidences than in controls and there were no test item-related effects on mean motor activity data.


ORGAN WEIGHTS
There were no test item-related mean organ weight differences.

GROSS PATHOLOGY (See Table 7.5.1/4)
The dose-related thickening of small intestine was considered to be related to test item administration. It was seen in one female at 400 mg/kg/day (duodenum) and in 5 females treated at 1000 mg/kg/day at duodenum level and in one additional female treated at 1000 mg/kg/day at both jejunum and ileum levels. This correlated to the hyperplasia of crypts seen microscopically (except for the ileum for which no microscopic correlate was seen).
The other macroscopic findings had no histological correlates or correlated with common histological findings in control rats, and were thus considered to be incidental.

HISTOPATHOLOGY: NON-NEOPLASTIC (See Table 7.5.1/5)
Test item-related microscopic findings were seen in the forestomach, stomach, duodenum and jejunum of animals treated at 400 or 1000 mg/kg/day.
Minimal to moderate squamous cell hyperplasia accompanied by hyperkeratosis was noted in the forestomach from males and females treated at 400 or 1000 mg/kg/day.
In the stomach from one female treated at 1000 mg/kg/day, a slight atrophy together with glandular basophilia suggestive of regeneration was seen. This lesion was considered to be related to test item administration, while the minimal atrophy seen in one female treated at 100 mg/kg/day was considered not to be related to test item treatment since it was minimal and isolated, since no lesions were seen at 400 mg/kg/day and since there was no evidence of regeneration.
In the duodenum from males and females treated at 400 or 1000 mg/kg/day, there was minimal to slight hyperplasia of crypt, accompanied by Brunner’s gland hyperplasia in males treated at 1000 mg/kg/day only.
In the jejunum from one female treated at 1000 mg/kg/day, there was also minimal hyperplasia of crypt.
The hyperplasia of crypt correlated to the thickening of duodenum/ileum seen macroscopically.
In view of the low to moderate magnitude of these changes, of the absence of associated degenerative changes and of the absence of body weight changes, these findings were considered not to be adverse and suggested local irritant properties of the compound.
There was minimal infiltrate of eosinophils in the rectum from two females treated at 1000 mg/kg/day and in the stomach from one male treated at 400 mg/kg/day, while not seen in controls. The relationship of this finding to test item-treatment was considered to be unlikely in view of the low incidence and severity of this change.
It is noteworthy that the urinary bladder from the animals treated at 1000mg/kg/day was within normal limits.


HISTORICAL CONTROL DATA (if applicable)

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Key result
Critical effects observed:
not specified

Any other information on results incl. tables

Table 7.5.1/2: Most clinical chemistry findings

Sex

Male

Female

Dose-level (mg/kg/day)

0

100

400

1000

0

100

400

1000

Total bilirubin (µmol/L)

1.23

0.79

0.84

0.52**

1.03

1.15

0.77

0.83

Proteins (g/L)

63

60

60

57**

64

63

64

62

Albumin (g/L)

37

36

37

33*

39

39

39

37

A/G (Albumin/Globulins)

1.43

1.48

1.55

1.37

1.56

1.56

1.50

1.53

ALP (Alcaline Phosphatase; IU/L)

300

262

293

223**

174

137

119

151


Table 7.5.1/3: Incidences of macroscopic test item-related findings

Sex

Male

Female

Group

1

2

3

4

1

2

3

4

Dose-level (mg/kg/day)

0

100

400

1000

0

100

400

1000

Number of animals per group

10

10

10

10

10

10

10

10

Duodenum; thickening

0

0

0

0

0

0

1

5

Jejunum; thickening

0

0

0

0

0

0

0

1

Ileum; thickening

0

0

0

0

0

0

0

1

Table 7.5.1/4: Incidences and severities of microscopic test item-related findings

Sex

Male

Female

Group

1

2

3

4

1

2

3

4

Dose-level (mg/kg/day)

0

100

400

1000

0

100

400

1000

Forestomach/n

5

5

5

5

5

5

5

5

hyperplasia; squamous cell

0

0

3

5

0

0

2

5

. grade 1 (minimal)

0

0

3

0

0

0

2

3

. grade 2 (slight)

0

0

0

3

0

0

0

2

. grade 3 (moderate)

0

0

0

2

0

0

0

0

hyperkeratosis

0

0

3

5

0

0

2

5

. grade 1 (minimal)

0

0

3

0

0

0

2

3

. grade 2 (slight)

0

0

0

2

0

0

0

2

. grade 3 (moderate)

0

0

0

3

0

0

0

0

Stomach/n

5

5

5

5

5

5

5

5

atrophy; gland

0

0

0

0

0

1

0

1

. grade 1 (minimal)

0

0

0

0

0

1

0

0

. grade 2 (slight)

0

0

0

0

0

0

0

1

Duodenum/n

5

5

5

5

5

5

5

8

hyperplasia; crypt

0

0

1

4

0

0

2

5

. grade 1 (minimal)

0

0

1

1

0

0

2

5

. grade 2 (slight)

0

0

0

3

0

0

0

0

hyperplasia; Brunner’s glands

0

0

0

3

0

0

0

0

. grade 1 (minimal)

0

0

0

2

0

0

0

0

. grade 2 (slight)

0

0

0

1

0

0

0

0

Jejunum/n

5

5

5

5

5

5

5

5

hyperplasia; crypt

0

0

0

0

0

0

0

1

. grade 1 (minimal)

0

0

0

0

0

0

0

1

n: number of organs submitted for a microscopic examination (taking in account the organs with macroscopic findings).

Applicant's summary and conclusion

Conclusions:
Under the test conditions of this study, the No Observed Adverse Effect Level (NOAEL) for systemic and local toxicity was considered to be 1000 mg/kg/day based on ptyalism (minor toxicological significance), minor blood biochemistry findings (decrease in mean total bilirubin and protein levels in males) and non-adverse pathology findings (microscopic findings in the forestomach, stomach, duodenum and jejunum suggesting local irritant properties of the compound) noted at this dose-level.
Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (CiToxLAB, 2013) was conducted with Reaction mass of diisobutyl hydrogen phosphate and isobutyl dihydrogen phosphate according to the OECD test guideline No. 422 and in compliance with GLP.

Sprague-Dawley rats (10/sex/dose level) received the test item daily by oral (gavage) administration before mating, through mating and, for the females, through gestation until day 5 p.p.. The test item was administered at dose levels of 100, 400 and 1000 mg/kg/day as an emulsion in the vehicle (Polyethylene Glycol 400, PEG 400). Another group of ten males and ten females received the vehicle, alone, under the same experimental conditions and acted as a control group. A constant dosage volume of 5 mL/kg bw/day was used.

The animals were checked daily for clinical signs, mortality, and detailed clinical observations were conducted weekly. Body weight and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. A Functional Observation Battery (FOB), including motor activity, and laboratory investigations (hematology, blood biochemistry and urinalysis) were carried out on five males and five females from each group at the end of the study.

The males were sacrificed after at least 5 weeks of treatment and the females on day 6p.p.. Final body weights and selected organs weights (adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes, thymus and urinary bladder) were recorded and a complete macroscopic post-mortem examination was performed, with particular attention paid to the reproductive organs, the urinary bladder and the internal surface of the esophagus and stomach. A microscopic examination was performed on selected organs (including the urinary bladder) from five males and five females in the control and high-dose groups, on forestomach, stomach, duodenum, jejunum and ileum from five males and five females of the low- and intermediate-dose groups, and on all macroscopic lesions.

There were no unscheduled test item-related deaths.Ptyalism was the only recorded clinical sign,noted in up to 6 animals per sex at 400 mg/kg/day and in all animals at 1000 mg/kg/day.This finding was considered to be related to the test item but of minor toxicological importance.

There were no effects on body weight, food consumption, functional observation battery tests or motor activity data in any group and sex.

No variations from controls in mean hematology/coagulation and urine data were attributed to the test item treatment. There were 2/10 animals at 1000 mg/kg/day with blood in the urine but an effect of the test item treatment was considered to be unlikely (no histopathological findings in the urinary bladder and kidney).

There were no test item-related effects on mean blood biochemistry data in females. In males, mean total bilirubin (0.52 vs. 1.23 µmol/L, p<0.01) and protein (proteins: 57 vs. 63 g/L, p<0.01; albumin: 33 vs. 37 g/L, p<0.05; no difference in A/G ratio) levels were decreased at 1000 mg/kg/day vs. controls but considered to be of minor toxicological significance.

There were no test item-related mean organ weights differences. The dose-related thickening of duodenum, jejunum and/or ileum seen in females treated at 400 or 1000 mg/kg/day was considered to be related to test item administration.

Test item-related non-adverse microscopic findings were seen in the forestomach (squamous cell hyperplasia; hyperkeratosis), stomach (glandular atrophy), duodenum (crypt and Brunner’s gland hyperplasia) and jejunum (crypt hyperplasia) of animals treated at 400 or 1000 mg/kg/day, and suggested local irritant properties of the test item.

Based on the experimental conditions of this study:

-  the No Observed Adverse Effect Level (NOAEL) for parental toxicity (systemic and local) was considered to be 1000 mg/kg/day based on ptyalism (minor toxicological significance), minor blood biochemistry findings (decrease in mean total bilirubin and protein levels in males) and non-adverse pathology findings (microscopic findings in the forestomach, stomach, duodenum and jejunum suggesting local irritant properties of the compound)noted at this dose-level.

This study is considered as acceptable as it satisfies the main criteria of OECD guideline No.422.