Triphenylphosphine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Under the conditions of a 90-day Neurotoxicity study with extended examinations for systemic toxicity and fertility (OECD TG 408), the oral administration of the test item in an aqueous vehicle by gavage to Wistar rats revealed signs of systemic toxicity at a dose level of 60 mg/kg bw/d and 120 mg/kg bw/d. Thus, the NOAEL for general systemic toxicity was 6 mg/kg bw/d for male and female Wistar rats. The NOAEL for fertility was set to 120 mg/kg bw/d for male and female Wistar rats since no adverse effects were observed up to the highest tested concentration [BASF, 2002].

No data are available for the dermal or inhalation route.

Link to relevant study records

Reference

Endpoint:

fertility, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2000 - November 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD testing guideline No. 408

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesanstalt für Pflanzenbau und Pflanzenschutz, Essenheimer Straße 144, D-55128 Mainz.

Limit test:

no

Specific details on test material used for the study:

- Purity: 99.82%.

SOURCE OF TEST MATERIAL
- Batch No.of test material: "Ablieferungsnummer: 01-0140".
- Date of production: May 18,1999.
- Physical state/color: Solid/white powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature.
- Stability under test conditions: The stability was given for 2 years (information of the sponsor).
- Solubility and stability of the test substance in the solvent/vehicle: The stability of the test substance in aqua bidest with 0.5% carboxymethylcellulose was demonstrated over a period of up to 24 hours at room temperature. Ac the mixtures were administered directly after preparation, the stability was guaranteed.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was pulverised, weighed depending on the dose group. Then the vehicle was filled up to the desired volume and mixed with a magnetic stirrer or highspeed sonicator (ultra turrax). During the administration the test substance peparations were kept homogenous by a magnetic stirrer.

FORM AS APPLIED IN THE TEST (if different from that of starting material): The test substance was administered in aqua bidest with 0.5% carboxymethylcellulose.

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Male and female Wistar rats were supplied by Charles River GmbH, Sulzfeld, Germany.
- Animal identification: The rats were identified clearly by ear tattoo. The animals were tattooed in consecutive order after arrival and before randomization.
- Females (if applicable) nulliparous and non-pregnant: not specified.
- Age at study initiation: supplied at an age of 33-37 days (males) and 32-36 days (females).
- Weight at study initiation: 120-150g at day -7, 155-192 g at day 0.
- Housing: The rats were housed singly in type DK III stainless steel wire cages from Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm2). Underneath the cages, waste trays were fixed containing absorbent material (type 314 dustfree embedding, supplied by SSNIFF, Soest, Germany). The motor activity measurements were conducted in Polycarbonate cages with wire Covers from Ehret, Emmendingen, Germany (floor area about 800 cm2 and small amounts of absorbent material (see above). The animals were housed in a completely air-conditioned room in which a central air-conditioner ensured temperatures and relative humidity values. The room was completely disinfected using a disinfector ("AUTEX", fully automatic, formalinammonia-based terminal disinfector) before the start of the study. The walls and the floor were cleaned once a week, in each case using water containing 0.1 % lncidin perfect (supplied by Henkel, Düsseldorf, Germany).
- Diet: The food used was basic maintenance diet for mouselrat, 9433 LL Meal, supplied by Eberle Nafag AG Gossau, Switzerland. Food was available ad libitum (except during motor activity measurements, functional observational batteries and during fasting periods).
- Water (e.g. ad libitum): Drinking water was available ad libitum from water bottles (except during motor activity measurements and functional observational batteries).
- Acclimation period: Only animals free from clinical signs of disease were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C.
- Humidity (%): 30 - 70%.
- Photoperiod (hrs dark / hrs light): 12 hours dark from 6:00 p.m. - 6:00 a.m.
IN-LIFE DATES: From: 28.07.2000 To: 09. and 10.11.2000

Route of administration:

oral: gavage

Vehicle:

other: aqua bidest. with 0.5% CMC

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was pulverised, weighed depending on the dose group. Then the vehicle was filled up to the desired volume and mixed with a magnetic stirrer or highspeed sonicator (ultra turrax). During the administration the test substance preparations were kept homogenous by a magnetic stirrer. The test substance preparations were made daily.

VEHICLE
- Concentration in vehicle: 6, 60 and 120 mg/kg bw/d.
- Amount of vehicle (if gavage): 5 mL/kg bw.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in aqua bidest with 0.5% carboxymethylcellulose was demonstrated over a period of up to 24 hours at room temperature. All the mixtures were administered directly after preparation, the stability was guaranteed.

Duration of treatment / exposure:

91 days

Frequency of treatment:

once daily, 7 days per week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The following dose levels were selected: 120 mg/kg bw/day as high dose level with expected toxic effects; 60 mg/kg bwlday as mid dose level; 6 mg/kg bw/day as low dose level. A factor of 10 between mid and low dose level was selected in order to assure a safe no observed adverse effect level. The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.

Positive control:

yes

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: twice a day.
- The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays. Additionally, further general clinical examinations were carried out daily.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: twice a day.

BODY WEIGHT: Yes.
- Time schedule for examinations: The body weight was determined before the first neurofunctional test in order to randomize the animals. During the conduct of the study, the body weight was determined on day 0 (start of administration period) and thereafter in weekly intervals. Body weight was also determined on the days when functional observational batteries were carried out. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
- Food consumption was determined weekly. The values were calculated as grams per animal per day.

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: Yes.
- Food efficiency (group means) was calculated based upon individual values for body weight and food consumption.

WATER CONSUMPTION: Yes.
- Time schedule for examinations: Water consumption was observed daily by visual inspection of the water bottles for any overt changes in volume.

OPHTHALMOSCOPIC EXAMINATION: No.

HAEMATOLOGY: Yes.
- Time schedule for collection of blood: at the end of the application period (Study day 92).
- Anaesthetic used for blood collection: No.
- Animals fasted: Yes.
- How many animals: 10 animals per test group and Sex.

CLINICAL CHEMISTRY: Yes.
- Time schedule for collection of blood: at the end of the application period (Study day 92).
- Animals fasted: Yes.
- How many animals: How many animals: 10 animals per test group and Sex.

URINALYSIS: No.

NEUROBEHAVIOURAL EXAMINATION: Yes.
- Time schedule for examinations: on study days -7, 22, 50 and 85.
- Dose groups that were examined: all dose groups.
- Battery of functions tested: Functional observational battery (Home cage observations, Open field observiations, Sensorimotor Tests/Reflexes), Motor activity assessment
- FOBs were performed in all animals as given in the time table, each time starting at 10 a.m.. The FOB started with passive observations, without disturbing the animals, followed by removal from the home cage and Open field observations in a standard arena. Thereafter, sensorimotor tests and reflex tests were conducted. The examinations were carried out by trained technicians which performed positive control studies as part of their training. The findings were ranked according to the degree of severity, if applicable. In order to guarantee the blind status of the observer, the cages were randomly distributed in the racks at least 30 minutes prior to the examinations, and the cage labels (indicating the dose group) were turned. Thus only the animal number, but not the allocation of the animals to the different dose groups could be identified by the observer. Moreover, the examinations were carried out in randomized order. The findings and values obtained were documented by another technician knowing the identification of the animals.
- Home cage observations: The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals.
- Open field observations: The animals were transferred to a standard arena (50 X 50 cm with sides of 25 cm height) and observed for at least 2 minutes.
- The animals were removed from the Open field and subjected to following sensorimotor or reflex tests.
- Motor activity was measured on the same day as FOB was performed. The measurement was performed in the dark using the Multi-Varimex-System (Columbus Instruments Int. Corp., Ohio, USA) with 4 infrared beams per cage. During the measurement the animals were kept in Polycarbonate cages with absorbent material. The cages were cleaned prior to each use. The animals were put into the cages in a randomized order. The measurements started at about 2 p.m. and the number of beam interrupts were counted over 12 intervals, each lasting 5 minutes. Measurement did not commence at the same instant for all cages; the period of assessment for each animal started when the first beam was interrupted by pushing the cage into the rack (staggered start). Measurements ended exactly 60 minutes thereafter. During the measurements the animals received no food and no water.

IMMUNOLOGY: No.

OTHER: Sperm examination.

Sperm parameters (parental animals):

The following parameters were determined:
Sperm motility
Sperm morphology
Sperm head count (cauda epididymis)
Sperm head count (testis)
Sperm motility examinations were carried out in a randomized sequence.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes.

HISTOPATHOLOGY: Yes.

Statistics:

Statistics of clinical examinations, statistics of clinical pathology and statistics of pathology

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Two high dose female animals showed anogenital region smeared with urine (slight) during the later phase of the study. A relationship to treatment cannot be excluded with certainty.
One low dose female animal showed loss of tail end. This was clearly incidental in nature.

Mortality:

no mortality observed

Description (incidence):

No animal died during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight data were similar for all groups, including the controls. No substance-related findings were observed.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was similar for all groups, including the controls. No substance-related findings were observed.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Food efficiency was statistically significantly decreased in mid dose males on day 84. Due to the isolated occurrence and the lack of a dose-response relationship, this was clearly incidental.

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

no effects observed

Description (incidence and severity):

Water consumption was similar for all groups, including the controls. No substance-related findings were observed.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the administration period prothrombin times were significantly shortened in the plasma of the mid and high dose females. No effects related to treatment were seen in the other hematology parameters measured.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Enzyme examinations revealed reduced alanine aminotransferase and aspartate aminotransferase activities in the serum of the mid and high dose females after 3 months of test substance administration. In the serum of the low dose females the activity of aspartate aminotransferase was also decreased at the end of the study. The decrease in aspartate aminotransferase activities was dose dependent (-29%, -33%, -38% at 6, 60 and 120 mg/kg bw), but not considered as of toxicological relevance by the study authors (increase in activities advers but decrease questionable). No treatment related changes were found in the other enzyme measurements.
Blood chemistry investigations showed decreased glucose levels in the serum of the high-dose males and increased calcium, total protein, globulin and triglyceride concentrations in the peripheral blood of the high dose females at the 3-month interval. No toxicologically relevant changes were seen in the other blood chemistry parameters.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The treatment with the substance did not influence findings in the functional observation battery.
No significant or biologically relevant changes were observed in the motor activity assessment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Axonal degeneration was found in the proximal sciatic nerve in one top dose female and axonal degeneration was also seen in the sural nerve of another top dose female out of five examined female animals.
These single findings were assessed as being of spontaneous or incidental nature and not related to treatment. No other significant substance-related effects were seen after detailed examination of the central and peripheral nervous system.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In both sexes, centrilobular hepatocyte hypertrophy was observed at 60 and 120 mg/kg bw/d in both sexes. The extent and higher incidence of centrilobular hypertrophies in the dose groups is regarded to be treatment-related.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Sperm analysis: Slightly decreased spermatid counts were found in the testes of the high dose males at the end of the study. In the other sperm parameters no treatment-related effects were seen.

Food consumption, water consumption, and body weight data were similar for all groups, including the controls. The treatment with the substance did not influence findings in the functional observation battery. No significant or biologically relevant changes were observed in the motor activity assessment. Absolute adrenal weight of male animals in the low- and mid-dose groups was increased, but without dose-dependency, and no increase was seen in high-dose animals. The effect was therefore considered as of no biological relevance. The only finding at 6 mg/kg bw/day was a significantly decrease in aspartate aminotransferase activity in females. The decrease in aspartate aminotransferase activities was dose dependent (-29%, -33%, -38% at 6, 60 and 120 mg/kg bw), but not considered as of toxicological relevance by the study authors (increase in activities advers but decrease questionable). In females 60 mg/kg bw/day induced a decrease in plasma prothrombin time, a statistically significant increase in absolute liver weights (+16%) and a slight increase in relative liver weights (+11%, not significant), and, in both sexes, centrilobular hepatocyte hypertrophy. At 120 mg/kg bw/day, increased levels of calcium, total protein, globulin and triglycerides were found in peripheral blood of females, decreased serum glucose levels in males and a shortened plasma prothrombin time in females. Liver weights were statistically significant increased in males (+14% absolute weight, +18% relative weight) and in females (+31%, absolute weight, s.s., +30% relative weight). The observed changes concerning the liver and in clinical chemistry are indicative of liver enzyme induction. Kidney weights were statistically significant increased in females (+15% absolute weight, +13% relative weight), and centrilobular hypertrophy of hepatocytes was found in both sexes. Axonal degeneration was found in the proximal sciatic nerve in one top dose female and axonal degeneration was also seen in the sural nerve of another top dose female out of five examined female animals. These single findings were assessed as being of spontaneous or incidental nature and not related to treatment. No other significant substance-related effects were seen after detailed examination of the central and peripheral nervous system. The NOAEL of this study was at 6 mg/kg bw/day, critical effects at the LOAEL (60 mg/kg bw) were a decrease in plasma prothrombin time and a slight increase in liver weights in females, and, in both sexes, centrilobular hepatocyte hypertrophy.

No indications for an adverse effect on reproductive organs were found, as there were no changes in the organ weights for adrenal glands, testes, epididymides, ovaries, uterus, and prostate, and because no histopathological changes were seen in testes, ovaries, oviducts/uterus/vagina, epididymides/prostate/seminal vesicle, and the female mammary glands up to and including the highest dose tested (120 mg/kg bw/day).
At 120 mg/kg bw/day the spermatid count was slightly decreased (107 versus 130 million per g testis in control), however, the number of spermatids/g testis in the high dose males is within the limits of the historical control data (85-129 million/g) and no dose response-relationship was seen. No effects on other sperm parameter were detected.

Dose descriptor:

NOAEL

Effect level:

6 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: general toxicity

Remarks on result:

other: no adverse effects at this dose

Dose descriptor:

LOAEL

Effect level:

60 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: general toxicity (organ weights, clinical biochemistry, food consumption)

Dose descriptor:

NOAEL

Effect level:

>= 120 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: reproduction toxicity

Remarks on result:

other: no reproduction toxicity up to the highest tested dose

Critical effects observed:

no

Remarks on result:

other: not applicable

Remarks on result:

other: not applicable

Remarks on result:

other: not applicable

Reproductive effects observed:

no

Executive summary:

The test substance was administered to groups of 10 male and 10 female Wistar rats by gavage at dose levels of 0 (vehicle control), 6, 60 and 120 mg/kg body weight/day (mg/kg bw/day) for 3 months. The vehicle used was aqua bidest with 0.5% carboxymethylcellulose, and the administration volume was 5 ml/kg bw.

 

Food consumption was determined once a week. Body weight was determined once a week and on the days when functional observational batteries were performed. A check of the general state of health was made at least daily. The animals were observed at least twice a day and palpated once a week. Functional observational batteries and motor activity measurements were carried out in 10 animals per sex and group on days -7, 22, 50 and 85. Clinicochemical, hematological examinations and sperm analyses were carried out towards the end of the administration period. Five animals per sex and dose were fixed by in situ perfusion and subjected to neuropathological examinations. The remaining animals were subjected to gross-pathological assessment, followed by histopathological examinations.

Following the oral administration of changes associated with induction of the microsomal enzyme system in the liver, increased liver weights and centrilobular hypertrophy of hepatocytes were observed. Decreases in alanine and aspartate aminotransferase activities were also seen, but were considered not to be an adverse toxic effect. At the end of the administration period prothrombin times were significantly shortened in the plasma of the mid and high dose females. No effects related to treatment were seen in the other hematology parameters measured. Furthermore, the treatment with the substance did not influence findings in the functional observation battery. No significant or biologically relevant changes were observed in the motor activity assessment.

Moreover, no indications for an adverse effect on reproductive organs were found, as there were no changes in the organ weights for adrenal glands, testes, epididymides, ovaries, uterus, and prostate, and because no histopathological changes were seen in testes, ovaries, oviducts/uterus/vagina, epididymides/prostate/seminal vesicle, and the female mammary glands up to and including the highest dose tested (120 mg/kg bw/day). At 120 mg/kg bw/day the spermatid count was slightly decreased (107 versus 130 million per g testis in control), however, the number of spermatids/g testis in the high dose males is within the limits of the historical control data (85-129 million/g) and no dose response-relationship was seen. No effects on other sperm parameter were detected.

The NOAEL of this study for general toxicity was 6 mg/kg, the NOAEL for reproductive toxicity was 120 mg/kg.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

120 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Additional information

Male and female rats were given doses of 6, 60, or 120 mg/kg bw in the course of a 3-month neurotoxicity gavage study according to OECD TG 408 and GLP [BASF, 2002]. Following the oral administration of changes associated with induction of the microsomal enzyme system in the liver, increased liver weights and centrilobular hypertrophy of hepatocytes were observed. Decreases in alanine and aspartate aminotransferase activities were also seen, but were considered not to be an adverse toxic effect.At the end of the administration period prothrombin times were significantly shortened in the plasma of the mid and high dose females. No effects related to treatment were seen in the other hematology parameters measured.Furthermore, the treatment with the substance did not influence findings in the functional observation battery. No significant or biologically relevant changes were observed in the motor activity assessment. Moreover, no indications for an adverse effect on reproductive organs were found, as there were no changes in the organ weights for adrenal glands, testes, epididymides, ovaries, uterus, and prostate, and because no histopathological changes were seen in testes, ovaries, oviducts/uterus/vagina, epididymides/prostate/seminal vesicle, and the female mammary glands up to and including the highest dose tested (120 mg/kg bw/day). At 120 mg/kg bw/day the spermatid count was slightly decreased (107 versus 130 million per g testis in control), however, the number of spermatids/g testis in the high dose males is within the limits of the historical control data (85-129 million/g) and no dose response-relationship was seen. No effects on other sperm parameter were detected. The NOAEL of this studyfor general toxicity was 6 mg/kg, the NOAEL for reproductive toxicity was 120 mg/kg. Therefore,with an extended examination of the reproductive organs was found to be 6 mg/kg and the LOAEL was found to be 60 mg/kg for male and female rats.

Effects on developmental toxicity

Description of key information

Under the conditions of a Prenatal Development Toxicity Study (OECD 414), the oral administration by gavage of the test item to Wistar rats had no influence on gestational parameters and induced no adverse signs of developmental toxicity up to and including the high dose level of 90 mg/kg body weight/day; especially, no indications of teratogenic effects occurred which could be causally related to the test substance administration. Based on these results, the no observed adverse effect level (NOAEL) for maternal toxicity is 30 mg/kg body weight/day. The NOAEL for prenatal developmental toxicity is 90 mg/kg body weight/day.

No data are available for the dermal or inhalation route.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October - November 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesanstalt für Pflanzenbau und Pflanzenschutz

Limit test:

no

Specific details on test material used for the study:

- Purity: 99.82%

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: Ablieferungs-Nr. 01-0140
- Purity test date: May 1999

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature.
- Stability under test conditions: The stability under storage conditions over the exposure period was guaranteed by the Sponsor.
- Solubility and stability of the test substance in the solvent/vehicle: Analytical verifications of the stability of the test substance in doubly distilled water for a period of at least 24 hours at room temperature were carried out before the study was initiated. Samples of the test substance suspensions were sent to the analytical laboratory twice during the study period (at the beginning and towards the end) for verification of the concentrations

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: For the preparation of the suspensions, an appropriate amount of the test substance was weighed depending on the dose group, in calibrated beakers and subsequently suspended in 0.5% Carboxymethylcellulose in doubly distilled water using a high-speed homogenizer (Ultra Turrax, JANKE & KUNKEL KG, Germany). A magnetic stirrer was used to keep the suspensions homogeneous during treatment of the animals
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material): aqueous suspension

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Germany.
- Age at study initiation: The female animals were supplied at an age of about 70 - 84 days. The animals were mated by the breeder and supplied on day 0 post coitum (= detection of vaginal plug / sperm).
- Weight at study initiation: Based on the pregnant animals the body weight on day 0 varied between 145.0 - 183.4 g. The mean maternal body weight at the day of first administration (gestation day 6 p.c.) was 188 - 190 g.
- Housing: The rats were housed singly from day 0 - 20 p.c. in type DK III stainless steel wire mesh cages supplied by BECKER & CO., Castrop-Rauxel, FRG (height: 15 cm, length: 37,5 cm, width: 21 Cm; floor area about 800 cm2). The animals were accommodated in fully air-conditioned rooms in which central air conditioning guaranteed the range of temperature and relative humidity. Before the study started, the animal room was completely disinfected using a disinfector ("AUTEX", fully automatic, formalin-ammonia-based terminal disinfection). In general, each week the walls and the floor were cleaned with water containing about 0.5% Mikro-Quat (supplied by ECOSAN GmbH, FRG).
- Diet: The food used was ground Kliba maintenance diet ratlmouselhamster meal, supplied by PROVlMl KLlBA SA, Kaiseraugst, Switzerland. Food was available to the animals ad libitum throughout the study (from the day of supply to the day of necropsy).
- Water drinking water of tap water quality from water bottles ad libitum.
- Acclimation period: Between start of the study (beginning of the experimental phase) and first administration (day 6 p.c.) the animals were acclimated to the laboratory conditions.
- Other specifics: Time-mated Wistar rats (CrlGlxBrlHan:WI) which were free from clinical signs of disease.
- Randomization: The animals were assigned to the test groups by taken random selection from the transport box.
- Identification: After randomization the rats were identified uniquely by ear tattoo.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C.
- Humidity (%): 30 - 70%.
- Photoperiod (hrs dark / hrs light): The day/night rhythm was 12 hours (12 hours light from 6.00 a.m. to 6.00 p.m. and 12 hours darkness from 6.00 p.m. to 6.00 a.m.).


IN-LIFE DATES: From: October 23, 2001 To: November 14, 2001.

Route of administration:

oral: gavage

Vehicle:

other: 0.5% Carboxymethylcellulose in doubly distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The aqueous test substance suspensions were prepared at the beginning of the administration period and thereafter at intervals which took into account the analytical results of the stability verification. For the preparation of the suspensions, an appropriate amount of the test substance was weighed depending on the dose group, in calibrated beakers and subsequently suspended in 0.5% carboxymethylcellulose in doubly distilled water using a high-speed homogenizer (Ultra Turrax, JANKE & KUNKEL KG, Germany). A magnetic stirrer was used to keep the suspensions homogeneous during treatment of the animals.

VEHICLE
- Concentration in vehicle: nominal concentrations of 0.1, 0.3 and 0.9 g/100mL. Test substance concentrations in Carboxymethylcellulose 0.5% in doubly distilled water were found to be in the range of 88.7-124.0 % of the nominal concentration.
- Amount of vehicle (if gavage): A standard dose volume of 10 mL/kg body weight was used for each group .

EXPOSURE
- The test substance suspensions were administered to the animals orally (by gavage) once a day from implantation to one day prior to the expected day of parturition (day 6 to day 19 p.c.) always at approx. the same time of day (in the morning). The animals of the control group were treated in the same way with the vehicle (0.5% carboxyrnethylcellulose in doubly distilled water). The volume administered each day was 10 ml/kg body weight. The calculation of the volume administered was based on the last individual body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance in doubly distilled water for a period of at least 24 hours at room temperature were carried out before the study was initiated.
Samples of the test substance suspensions were sent to the analytical laboratory twice during the study period (at the beginning and towards the end) for verification of the concentrations. The samples which were taken for the first concentration control analyses at the beginning of the administration period were also used to verify the hornogeneity for the samples of the low and the high concentrations (10 and 90 mglkg body weightlday). 3 samples (one from the top, middle and bottom in each case) were taken for each of these concentrations from the beaker with a magnetic stirrer running.

Details on mating procedure:

The female animals were supplied at an age of about 70 - 84 days. The animals were mated by the breeder ("time-mated") and supplied on day 0 post
coitum (= detection of vaginal plug / sperm). The animals arrived on the same day (i.e. day 0 p.c.) at the experimental laboratory. The following day was designed "day 1 " post coitum (p.c.). Between start of the study (beginning of the experimental phase) and first administration (day 6 p.c.) the animals were acclimated to the laboratory conditions.

Duration of treatment / exposure:

day 6 - 19 p.c.

Frequency of treatment:

daily

Duration of test:

until day 20 p.c.

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

90 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

The following doses were chosen for the present full-scale toxicity study in Wistar rats:
10 mg/kg body weight/day: as the expected no observed adverse effect level
30 mg/kg body weight/day: as intermediate dose level
90 mg/kg body weight/day: as the dose level which should induce some developmental andlor maternal toxicity but not death or severe suffering
The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.

Maternal examinations:

A check was made twice a day on working days or once a day (Saturday, Sunday or on public holidays) (days 0 - 20 p.c.).
The animals were examined for clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited (days 0 - 20 p.c.).
With the exception of day 0, the consumption of food was determined on the same days as was body weight.
All animals were weighed on days 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20 p.c.. The body weight change of the animals was calculated from these results.
Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on day 20 p.c. minus weight of the unopened uterus minus body weight on day 6 p.c.).
Blood was taken from the retroorbital venous plexus in the morning from non-fasted animals without anesthesia. The blood sampling procedure and the subsequent analysis of the serum samples were carried out in a randomized sequence. The list of randomization instructions was compiled with a computer using a random number generator. The assays of serum parameters were performed under internal laboratory quality control conditions with commercial reference controls to assure reliable test results. The results of the clinical pathology examinations are expressed in units of the International System (SI).
An automatic analyzer (Hitachi 91 7; Roche, Mannheim, Gerrnany) was used to examine the clinicochemical parameters.
The following examinations were carried out in 12 animals per test group before necropsy.
On day 20 p.c. immediately after blood was taken from the retroorbital venous plexus of 12 females/group in a randomized sequence, all dams were sacrificed in randomized order by cervical dislocation and the fetuses removed from the uterus.

Ovaries and uterine content:

After the dams had been sacrificed, they were necropsied and assessed by gross pathology. The liver, the uterus and the ovaries were removed and the following data were recorded:
- Weight of the liver
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
live fetuses
dead implantations:
a) early resorptions (only decidual or placental tissues visible or according to SALEWSKI (Salewski, 1964) from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy)
b) late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c) dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)

Fetal examinations:

Examination of the fetuses after dissection from the uterus:
At necropsy each fetus was weighed, sexed and examined macroscopically for any external findings. The sex was determined by observing the distance between the anus and the base of the genital tubercle and was later confirmed in all fetuses fixed in BOUIN'S solution by internal examination. If there were discrepancies between the "external" and the "internal" sex of a fetus, the fetus was finally sexed according to the Y appearance of its gonads.
Furthermore, the viability of the fetuses and the condition of the placentae, the umbilical cords, the fetal membranes and fluids were examined. Individual placental weights were recorded.

Soft tissue examination of the fetuses:
Thereafter, the fetuses were sacrificed by subcutaneous injection of a pentobarbital. After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and placed in ethyl alcohol, the other half was placed in BOUIN's solution for fixation and further evaluation.
The fetuses fixed in BOUIN's solution were examined for any visceral findings according to the method of BARROW and TAYLOR (Barrow and Taylor, 1969). After this examination these fetuses were discarded.

Skeletal examination of the fetuses:
The skeletons of the fetuses fixed in ethyl alcohol were stained according to a modified method of KIMMEL and TRAMMELL (Kimmel, C.A. and Trammell C., 1981). Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After this examination the stained fetal skeletons were retained individually.

Statistics:

Statistics of clinical, necropsy and fetal examinations; statistics of clinical pathology

Historical control data:

yes

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical findings occurred in any of the dams during the in-life phase of this study.

Mortality:

no mortality observed

Description (incidence):

There were no substance-related or spontaneous mortalities in any of the groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no statistically significant or biologically relevant differences between the controls and the substance-treated dams concerning mean body weights during the administration period (days 6 - 19 p.c.) or during the entire study phase (days 0 - 20 p.c.).

The mean body weight gain of the high dose rats (90 mglkg body weightlday) showed a statistically significant impairment (about 54% below the concurrent control value) at initiation of dosing (days 6 - 8 p.c.). On the following study days (8 - 20 p.c.), weight gains of the 90 mglkg females reached or even exceeded the weight gains of the concurrent control group. The weight gains of the high dose rats were even statistically significantly increased on days 8 - 10 and 19 - 20 p.c.). As the food consumption of these rats was also transiently diminished at initiation of dosing (see 4.2.1.3.), this is considered to be a substance-related sign of maternal toxicity.

Body weight gains of the dams of test groups 1 and 2 (10 and 30 mglkg body weightlday) were similar to those of the concurrent controls. All observable differences in these 2 groups in comparison to the controls during the treatment period are without any biological relevance.

The corrected body weight gain (terminal body weight on day 20 p.c. minus weight of the unopened uterus minus body weight on day 6 p.c.) of the dams of test group 1, 2 and 3 (10; 30 and 90 mglkg body weightlday) was not statistically significantly different from the corresponding control value and did not show a clear relation to dosing. Moreover, the mean carcass weights were unaffected by treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The high dose dams showed transient reductions in food consumption (about 21 % below controls) in association with temporary impairments in absolute body weight gain (about 54% below controls) at initiation of treatment (days 6 - 8 p.c.). This finding is considered to be substance-induced.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Enzyme examinations revealed reduced alanine aminotransferase activities in the serum of all treatment groups. No test substance-related changes were found in the other serum enzyme measurements. The decreased enzyme activities are considered to be test substance-related, but have no pathognomonic relevance.
In general, increases in serum alanine aminotransferase activities correlate well with hepatic diseases involving liver cell injury and, thus, are useful tools in the assessment of hepatotoxicity. The clinical implications of elevated aminotransferase activities are well known. Decreases of serum aminotransferase activities, however, are poorly understood and the assessment of enzyme reduction as an adverse toxic effect is debatable. Since no adverse effects were observed in this study which could be associated with the fall in alanine aminotransferase activities, a pathognomonic relevance is not assigned to the decreased enzyme activities. Therefore, the decreases in alanine arninotransferase are not considered to be of toxicological importance.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative mean liver weights of the high dose dams (90 mg/kg body weight/day) were statistically significantly increased and about 14% above the corresponding control values. It is very likely that the weight increases of the liver represent an adaptive response of this Organ to treatment (enzyme induction). Mean liver weights (absolute and relative) of the rats of test groups 1 and 2 (10 and 30 mglkg body weighuday) were similar to the corresponding control values and did not show any relation to treatment.

The gravid uterus weight of the animals of test groups 1, 2 and 3 (10; 30 and 90 mglkg body weightlday) was not influenced by the administration of the test substance. The differences between these groups and the control group are considered to be without any biological relevance and represent the usual fluctuations in the strain of rats used for this study.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no substance-related observations at necropsy in any of the dams.

A hydrometra was detected in one low dose dam (No. 44), one mid dose dam (No. 73) and one high dose dam (No. 86) each. Consequently none of these dams became pregnant. The scattered occurrence of this finding does not suggest any relation to treatment and is spontaneous in nature

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no substance-related and/or biologically relevant differences between the test groups in the pre- and the postimplantation losses.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no substance-related and/or biologically relevant differences between the test groups in the number of resorptions
Two dams (Nos. 52 and 70) of test group 2 (30 mglkg body weight/day) had only early resorptions, but no viable fetuses in the uterus. This is not an uncommon finding in untreated rats and thus is considered to be spontaneous in nature.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no substance-related and/or biologically relevant differences between the number of viable fetuses.
This statement includes the statistically significantly increased rate of live fetuses in test group I (10 mglkg body weight/day; 97.8% vs. 90.8% in the control group) and the consequently statistically significantly increased rate of live male fetuses in this test group.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The conception rate reached 92% in test groups 0 and 3 (0 and 90 mglkg body weight/day) and 88% in test groups 1 and 2 (10 and 30 mg/kg body weight/day).
There were no substance-related and/or biologically relevant differences between the test groups in the conception rate, in the mean number of Corpora lutea and implantation sites.

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Basis for effect level:

other: maternal toxicity

Remarks on result:

other: no maternal toxicity at this dose

Dose descriptor:

LOAEL

Effect level:

90 mg/kg bw/day (nominal)

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

>= 90 mg/kg bw/day (nominal)

Basis for effect level:

other: maternal developmental toxicity

Remarks on result:

other: no influence on gestational parametersand no adverse signs of maternal developmental toxicity up to the highest tested dose

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal body weights in test groups 1, 2 and 3 (10; 30 and 90 mg/kg body weight/day) were close to or even identical with the corresponding control values. They were not influenced by the test substance administration to the dams.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The Sex distribution of the fetuses in test groups 1 - 3 (10; 30 and 90 mg/kg body weight/day) was comparable with that of the control fetuses. The differences observed in comparison to the control were without any biological relevance. This includes the by chance statistically significantly increased rate of male fetuses at 10 mg/kg body weight/day.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External malformations occurred in one control and one mid dose fetus each, but not in the low or the high dose fetuses.
Anophthalmia of left eye was recorded for female mid dose fetus No. 8 of dam No. 68. Moreover, female fetus No. 12 of dam No. 4 of the control group had multiple external malformations substantiated by mandibular micrognathia, astomia and malpositioned pinna.
In total, one out of 194 examined control fetuses [= 0.5%] (in one out of 23 litters [= 4.3%]), none out of 212 low dose fetuses (from 22 litters), one out of 172 mid dose fetuses [= 0.6%] (in one out of 20 Iitters [= 5.0%]) and none out of 208 high dose fetuses (from 23 litters) showed external malformations. The mean percentages of affected fetuses/litter with external malformations amounted to 0.4, 0.0, 0.5 and 0.0%, respectively without attaining statistical significance in any of the test groups (0; 10; 30 or 90 mg/kg body weight/day).
The isolated and scattered occurrence of these 2 external malformations without any dose-response relationship does not suggest a substance-related background. No external variations were seen in any fetuses of any group.
There appeared one unclassified observation. Blood coagulum around the placenta was observed for control fetus No. 12 of dam No. 4.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Malformations of the skeletons occurred in three low dose, one mid dose and 2 high dose fetus each, but not in the lcontrol group fetuses.
- 10 mg/kg: Lumbar hemivertebra, misshapen lumbar vertebra was recorded for female low dose fetus No. 7 of dam No. 26. Malpositioned and bipartite sternebra (unchanged cartilage) were recorded for male low dose fetus No. 5 of dam No. 34 and female low dose fetus No. 1 of dam No. 40.
- 30 mg/kg: Misshapen humerus was recorded for male mid dose fetus No. 5 of dam No. 69.
- 90 mg/kg: Misshapen sacral vertebra was recorded for female high dose fetus No. 9 of dam No. 87. Deformed clavicle was recorded for female high dose fetus No. 9 of dam No. 96.

In total, none of the 102 examined control fetuses (from 23 litters), 3 out of 111 low dose fetuses [= 2.7%] in 3 out of 22 litters [= 14%], one out of 93 mid dose fetuses [= 1.1 %] in one out of 20 litters [= 5.0%] and 2 out of 108 high dose fetuses [= 1.9%] in 2 out of 23 litters [= 8.7%] showed skeletal malformations. The mean percentages of affected fetuses/litter with skeletal malformations amounted to 0.0, 2.2*, 0.8 and 2.0% (* =p ≤ 0.05). All of the noted skeletal malformations appeared without a clear relation to dosing, without biologically relevant differences between the groups and/or can be found at a comparable frequency in the historical control data.
These statements include the statistically significantly increased mean percentage of affected fetuses/litter with total skeletal malformations at the low dose group. The respective value (2.2*% (* = p ≤ 0.05)) is fully within the range of the corresponding
historical control data (mean value: 1.8%; range: 0.7 - 5.3), a dose-response relationship is not present.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The examination of the organs of the fetuses revealed only one soft tissue malformation in one mid dose fetus, but not in any of the fetuses of test groups 0, 1 or 3 (0; 10 or 90 mg/kg body weight/day).
Female fetus No. 8 of dam No. 68 (which showed already unilateral anophthalmia during its external examination) had a globular shaped heart.
In total, none out of 92 control fetuses (from 23 litters), none out of 101 low dose fetuses (from 22 litters), one out of 79 mid dose fetuses [= 1.3%] (in one out of 20 litters [= 5.0%], and none out of 100 high dose fetuses (from 23 litters) showed visceral malformations. The mean percentages of affected fetuses/litter with soft tissue malformations amounted to 0.0, 0.0, 1.0 and 0.0%, respectively without attaining statistical significance in any of the test groups (0; 10; 30 or 90 mg/kg body weight/day).
The isolated occurrence of the observed soft tissue malformation in one mid dose fetus does not suggest any treatment-relationship.
Two soft tissue variations (uni- or bilateral dilation of renal pelvis and/or ureter) were detected in each group including the controls in 3 - 12 fetuses from 3 - 8 litters. Dilatations of these parts of the urinary tract are very common fetal soft tissue findings in
the strain of used for this study.
The mean percentages of affected fetuses/litter with total soft tissue variations amounted to 7.0% (control), 6.5% (10 mg/kg body weight/day), 4.6% (30 mg/kg body weight/day) and 11.9% (90 mg/kg body weight/day), respectively. Thus, a clear relation to dosing is not present. Moreover, all of these values are fully within the historical control data range (mean value: 11.6%; range: 4.4 - 22.2%). Therefore a substance-induced effect concerning the occurrence of soft tissue variations can be excluded with certainty.
No unclassified soft tissue observation (like blood imbibition of kidney(s)) was recorded in any of the fetuses.

Other effects:

no effects observed

Description (incidence and severity):

The mean placental weights in test groups 1,2 and 3 (10; 30 and 90 mglkg body weight/day) were comparable to the control values and not influenced by the test substance administration. This statement includes the statistically significantly lower
mean placental weights of the low dose female fetuses. As no relation to dosing is given and all respective values are fully within the historical control range

Dose descriptor:

NOAEL

Effect level:

>= 90 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: no adverse effects at the highest tested dose

Abnormalities:

effects observed, non-treatment-related

Developmental effects observed:

no

In summary, the following substance related findings were obtained:

Test group 3 (90 mg/kg body weight/day):

· statistically significantly reduced food consumption at initiation of dosing, i.e. on days 6 - 8 p.c. (about 21% below controls); food uptake reached or exceeded control values on the subsequent days

· statistically significantly impaired absolute body weight gain at initiation of dosing, i.e. on days 6 - 8 p.c. (about 54% below controls); weight gains reached or exceeded control values on the subsequent days

· decreased serum alanine aminotransferase activities

· statistically significantly increased absolute and relative liver weights (about 14% above controls)

· no substance-related adverse effects on gestational parameters or fetuses

Test groups 2 (30 mg/kg body weight/day):

· decreased serum alanine aminotransferase activities

· no substance-related adverse effects on gestational parameters or fetuses

Test group 1 (10 mg/kg body weight/day):

· decreased serum alanine aminotransferase activities

· no substance-related adverse effects on gestational parameters or fetuses

Conclusions:

Under the conditions of this full-scale study, the administration of 90 mg Test substance/kg body weight/day to pregnant female Wistar rats elicited substance-induced effects on the dams including signs of maternal toxicity. At the low and mid dose the only effect on the dams was a decrease in alanine aminotransferase activity, which does not represent an adverse toxic effect. The test substance had no influence on gestational parameters and induced no adverse signs of developmental toxicity up to and including the high dose level (90 mg/kg body weight/day); especially, no indications of teratogenic effects occurred which could be causally related to the test substance administration.

Executive summary:

The test substance was tested for its prenatal developmental toxicity in Wistar rats according to OECD TG 414 and GLP.

The test substance was administered as an aqueous suspension to 25 presumably pregnant female Wistar rats/group by stomach tube at doses of 10; 30 and 90 mg/kg body weight on day 6 through day 19 post coitum (p.c.). A standard dose volume of 10 ml/kg body weight was used for each group. The control group, consisting of 25 females, was dosed with the vehicle only (0.5% Carboxymethylcellulose in doubly distilled water). Between 22 - 23 females/group had implantation sites at terminal sacrifice.

 

Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On day 20 post coitum, blood was taken from 12 females/group, which were subsequently sacrificed like the remaining rats and assessed by gross pathology (including weight determinations of the unopened uterus, the liver and the placentae). For each dam, Corpora lutea were counted and number and distribution of implantation sites (differentiated as resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for any external findings. Thereafter, nearly one half of the fetuses of each litter was examined for soft tissue findings and the remaining fetuses for skeletal (incl. cartilage) findings.

 

The following substance-related findings were obtained:

Test group 3 (90 mg/kg body weight/day):

• statistically significantly reduced food consumption at initiation of dosing, i.e. on days 6 - 8 p.c. (about 21 % below controls); food uptake reached or exceeded control values on the subsequent days
• statistically significantly impaired absolute body weight gain at initiation of dosing, i.e. on days 6 - 8 p.c. (about 54% below controls); weight gains reached or exceeded control values on the subsequent days
• decreased alanine aminotransferase activities
• statistically significantly increased absolute and relative liver weights (about 14% above controls)
• no substance-related adverse effects on gestational parameters or fetuses

 

Test groups 2 (30 mg/kg body weight/day):

• decreased alanine aminotransferase activities
• no substance-related adverse effects on gestational parameters or fetuses

 

Test group 1 (10 mg/kg body weight/day):

• decreased alanine aminotransferase activities
• no substance-related adverse effects on gestational parameters or fetuses

 

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (days 6 - 19 p.c.) elicited substance-induced effects on the dams including signs of maternal toxicity at 90 mg/kg body weight/day. At the low and mid dose (10 and 30 mg/kg body weight/day) the only effect on the dams was a decrease in alanine aminotransferase activity, which does not represent an adverse toxic effect. The test substance had no influence on gestational parameters and induced no adverse signs of developmental toxicity up to and including the high dose level (90 mg/kg body weight/day); especially, no indications of teratogenic effects occurred which could be causally related to the test substance administration.

Based on these results, the no observed adverse effect level (NOAEL) for maternal toxicity is 30 mg/kg body weight/day. The NOAEL for prenatal developmental toxicity is 90 mg/kg body weight/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

90 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

The test substance was tested for its prenatal developmental toxicity in Wistar rats according to OECD TG 414 and GLP [BASF, 2002].

The test substance was administered as an aqueous suspension to 25 presumably pregnant female Wistar rats/group by stomach tube at doses of 10; 30 and 90 mg/kg body weight on day 6 through day 19 post coitum (p.c.). A standard dose volume of 10 ml/kg body weight was used for each group. The control group, consisting of 25 females, was dosed with the vehicle only (0.5% Carboxymethylcellulose in doubly distilled water). Between 22 - 23 females/group had implantation sites at terminal sacrifice.

 

Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On day 20 post coitum, blood was taken from 12 females/group, which were subsequently sacrificed like the remaining rats and assessed by gross pathology (including weight determinations of the unopened uterus, the liver and the placentae). For each dam, Corpora lutea were counted and number and distribution of implantation sites (differentiated as resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for any external findings. Thereafter, nearly one half of the fetuses of each litter was examined for soft tissue findings and the remaining fetuses for skeletal (incl. cartilage) findings.

 

The following substance-related findings were obtained:

Test group 3 (90 mg/kg body weight/day):

• statistically significantly reduced food consumption at initiation of dosing, i.e. on days 6 - 8 p.c. (about 21 % below controls); food uptake reached or exceeded control values on the subsequent days
• statistically significantly impaired absolute body weight gain at initiation of dosing, i.e. on days 6 - 8 p.c. (about 54% below controls); weight gains reached or exceeded control values on the subsequent days
• decreased alanine aminotransferase activities
• statistically significantly increased absolute and relative liver weights (about 14% above controls)
• no substance-related adverse effects on gestational parameters or fetuses

 

Test groups 2 (30 mg/kg body weight/day):

• decreased alanine aminotransferase activities
• no substance-related adverse effects on gestational parameters or fetuses

 

Test group 1 (10 mg/kg body weight/day):

• decreased alanine aminotransferase activities
• no substance-related adverse effects on gestational parameters or fetuses

 

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (days 6 - 19 p.c.) elicited substance-induced effects on the dams including signs of maternal toxicity at 90 mg/kg body weight/day. At the low and mid dose (10 and 30 mg/kg body weight/day) the only effect on the dams was a decrease in alanine aminotransferase activity, which does not represent an adverse toxic effect. The test substance had no influence on gestational parameters and induced no adverse signs of developmental toxicity up to and including the high dose level (90 mg/kg body weight/day); especially, no indications of teratogenic effects occurred which could be causally related to the test substance administration.

Based on these results, the no observed adverse effect level (NOAEL) for maternal toxicity is 30 mg/kg body weight/day. The NOAELfor prenatal developmental toxicity is 90 mg/kg body weight/day.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for reproductive toxicity under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.