Bis(2,4,4-trimethylpentyl)phosphinic acid

Repeated dose toxicity: oral

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 March 28th, 2012 to January 9th, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

from February 16th to March 8th, 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Non GLP study. Range finding study for the OECD 422 study

Principles of method if other than guideline:

Range finding study for the OECD 422 study

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, l’Arbresle, France
- Strain and Sanitary status: Sprague-Dawley, Crl CD® (SD) IGS BR, Caesarian Obtained, Barrier Sustained-Virus Antibody Free (COBS-VAF®).
- Age/Weight: on the first day of treatment, the animals were approximately 10 weeks old. The males had a mean body weight of 393 g (range: 368 g to 416 g) and the females had a mean body weight of 227 g (range: 219 g to 242 g).
- Housing: The animals were housed by three, in polycarbonate cages with stainless steel lids(Tecniplast 2154, 940 cm2, 48 cm x 26.5 cm x 21 cm) containing autoclaved sawdust (SICSA, Alfortville, France). Each cage contained Nylabone as enrichment for the environment. The cages were placed in numerical order on the racks.
-Food and water. All animals had free access to SSNIFF R/M-H pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany), batch No. 5776558, which was distributed weekly. The animals had free access to bottles containing tap water (filtered with a 0.22 μm filter).
- Acclimation period: the animals were acclimated to the study conditions for a period of 7 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h (7:00 - 19:00)

IN-LIFE DATES: From: 2012-02-23 To: 2012-03-08

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

batch No. MKBG9425V, supplied by Sigma (Saint-Quentin-Fallavier, France).

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: the test item dose formulations were prepared daily and were stored at room temperature prior to use.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the vehicle used in this study was selected from the results of solubility assays performed by the CIT Pharmacy
- Concentration in vehicle: 100 mg/kg, 300 mg/kg and 900 mg/kg
- Amount of vehicle (if gavage): 5 ml/kg/d

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

not applicable

Duration of treatment / exposure:

2 weeks

Frequency of treatment:

Once daily

Dose / conc.:

100 other: active ingredient (mg/kg bw/d)

Dose / conc.:

300 other: active ingredient (mg/kg bw/d)

Dose / conc.:

900 other: active ingredient (mg/kg bw/d)

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor. No data are available after repeat exposure with the test substance The sponsor does not have test data, but information on the test item at lower purity is available:
rat acute oral toxicity study: LD50 (rat): > 3500 mg/kg,
skin irritation (rabbit): severe irritant,
eye irritation (rabbit): severe irritant,
mouse micronucleus study (oral gavage): dose-level 900 mg/kg.
Based on the dose-level chosen for the main micronucleus test, 900 mg/kg was considered an appropriate high dose-level for testing the test item with a the present purity.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each animal was checked for mortality and morbidity once a day during the pre-treatment period and at least twice a day during the treatment period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was observed once a day, at approximately the same time, for the recording of clinical signs

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded once before group allocation and then twice weekly until the end of the study.

FOOD CONSUMPTION:
- The quantity of food consumed by the animals of each cage was recorded twice a week during the study.

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: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

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, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

HISTOPATHOLOGY: No

Other examinations:

None

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
At 100, 300 and 900 mg/kg/day, all animals (male and female rats) exhibited ptyalism. Ptyalism was recorded from day 6, 2 and 2 in males and from day 11, 2 and 2 in females at 100, 300 and 900 mg/kg/day, respectively. This treatment-related finding was considered to be of minor toxicological importance and non adverse.
At 100 mg/kg/day, soft faeces was recorded in 1/3 males.
At 300 mg/kg/day, soft faeces and soil coat was recorded in 1/3 males and soil coat in 1/3 females.
At 900 mg/kg/day, piloerection was observed in all animals, thin appearance and loud breathing were noted in 2/3 males, soft faeces in 2/3 males and 1/3 females, and liquid faeces in 1/3 males. Soiled coat was noted in 1/3 females.
All these findings were considered to be treatment-related and of marked toxicological importance at 900 mg/kg/day.
A few clinical signs commonly observed in this strain of rats (alopecia or thinning of hair) were recorded in one female treated at 100 or 300 mg/kg/day.

BODY WEIGHT AND WEIGHT GAIN
In males only, there was a mean body weight loss on day 5 resulted in a statistically significant lower mean body weight change at 900 mg/kg/day during the period of days 1-5 (-18 g vs. +29 g in controls, p<0.05). This resulted in an overall (not statistically significant) moderate decrease in mean body weight on day 14 (-15.8 % vs. controls). This finding was considered to be of toxicological importance.

FOOD CONSUMPTION
There were statistically significant reduction mean food consumptions on days 1-4 in males and females (-54.8 % vs. controls for males and -30 % vs. controls for females) at 900 mg/kg/day, on days 5-7 in males at 900 mg/kg/day (29.2 % vs. controls) and on days 8-10 in females at 300 mg/kg/day (-20.4 % vs. controls). Taking into account their amplitudes, these findings were considered to be of toxicological importance at 900 mg/kg/day.

ORGAN WEIGHTS
There were decreases in the mean absolute and relative heart weights (up to -27%) in high-dose females. In the absence of similar variations in males, and since the individual relative heart weights were within the range of CIT reference control values, these variations were not considered to be related to the test item administration.
Other organ weight changes were not considered to be related to the test item as they were small in amplitude, had no gross correlates, were not dose-related in magnitude, and/or were not consistent for the sexes.

GROSS PATHOLOGY
The few macroscopic findings noted at the end of the treatment period were of those commonly recorded in the Sprague-Dawley rat and none were considered to be related to the test item administration.

Remarks on result:

other: the dose-level of 900 mg/kg/day was considered to be an excessively high dose-level for a further OECD 422 study.

Critical effects observed:

not specified

Conclusions:

Based on the results obtained in this study, the dose-level of 900 mg/kg/day was considered to be an excessively high dose-level for a further OECD 422 study.

Executive summary:

In a range-finding assay, the test substance diluted in corn oil was administrated daily by oral gavage to Sprague-Dawley rats (3 animals/sex/dose) for a period of 14 days at the dose of 0; 100; 300 and 900 mg/kg bw/d.

No mortality ocurred during the study. At 100, 300 and 900 mg/kg/day, all animals (male and female rats) exhibited ptyalism. However this treatment-related finding was considered to be of minor toxicological importance and non adverse. Soft faeces were observed in males and females at all tested doses.

At 900 mg/kg/day, piloerection was observed in all animals, thin appearance and loud breathing were noted in 2/3 males, and liquid faeces in 1/3 males. Soiled coat was noted in 1/3 females.

All these findings were considered to be treatment-related and of marked toxicological importance at 900 mg/kg/day.

In males only, there was a mean body weight loss on day 5 resulted in a statistically significant lower mean body weight change at 900 mg/kg/day during the period of days 1-5. This resulted in an overall (not statistically significant) moderate decrease in mean body weight on day 14 (-15.8 % vs. controls). This finding was considered to be of toxicological importance.

There were statistically significant reduction mean food consumptions on days in both sexes at 900 mg/kg/day, and in females at 300 mg/kg/day. Taking into account their amplitudes, these findings were considered to be of toxicological importance at 900 mg/kg/day.

There was no toxicologically relevant effect on organ weight and no macroscopic findings were recorded.

Based on these results, the dose-level of 900 mg/kg/day was considered to be an excessively high dose-level for a further OECD 422 study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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

GLP compliance:

yes (incl. QA statement)

Remarks:

2011-07-19

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2,4,4-trimethylpentyl)phosphinic acid

EC Number:

280-445-7

EC Name:

Bis(2,4,4-trimethylpentyl)phosphinic acid

Cas Number:

83411-71-6

Molecular formula:

C16H35O2P

IUPAC Name:

bis(2,4,4-trimethylpentyl)phosphinic acid

Test material form:

other: colorless to pale yellow liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: breeder: Charles River Laboratories France, Saint-Germain sur L’Arbresle, France.
- Age/weight at study initiation: the males were approximately 10 weeks old and had a mean body weight of 405 g (range 385 g to 424 g) and the females were approximately 9 weeks old and had a mean body weight of 222 g (range 206 g to 241 g).
- Fasting period before study: no
- Housing: individually housed, except during pairing, or with their litter during lactation, in polycarbonate cages (Tecniplast, 2154, 940 cm²) with stainless steel lids and containing autoclaved sawdust.
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access) except for one male on day 22 due to a mal positioned bottle.
- Acclimation period: at least 6 days before the beginning of the study.

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

IN-LIFE DATES: 18 April 2012 to 14 June 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test was administered as a solution in the vehicle. The test item was mixed with the required quantity of vehicle. No correction factor was applied.
The dose formulations were prepared on a daily basis, stored and delivered at room temperature in brown flasks.

VEHICLE
- Concentration in vehicle: 13.3, 40.0 and 120.0/80.0 mg/ml
- Amount of vehicle: 5 ml/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance concentrations in the administered dose formulations analyzed in weeks 1, 3, 4 and 5 remained within an acceptable range of -12.9 % to +3.8 % when compared with the nominal values (± 15 %), except for group 3 which was found at -19.7 % on week 3. Taking into account the amplitude of this variation when compared with the nominal values, this was considered of non significant impact on the dose administered. The test substance was not detected in control samples.

Duration of treatment / exposure:

in the males (36 days in total):
− 2 weeks before pairing,
− during the pairing period (up to 3 weeks),
− until sacrifice (at least 5 weeks in total).

in the females (42 to 57 days in total):
− 2 weeks before pairing,
− during the mating period (up to 3 weeks),
− during gestation,
- during lactation until day 5 post-partum (p.p.) inclusive,
- until sacrifice for non-pregnant females.

Day 1 corresponds to the first day of the treatment period.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

600 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 dose-levels were selected in agreement with the Sponsor based on a 2-week dose range finding study by oral route (gavage) in rat.
In this preliminary study, the following findings were recorded:
- clinical signs: all test item-treated animals had ptyalism. Ptyalism was considered to be treatment-related but of non toxicological significance. In addition, soft feces were observed from 100 mg/kg/day; soiled fur was observed from 300 mg/kg/day and piloerection (all animals) and loud breathing (2/3 males) were observed at 900 mg/kg/day
- body weight: there were no statistically significant differences between test item-treated groups and control group. However, when compared with the control group there were -13.7 % (day 8) to -15.8 % (day 14) reductions in mean body weight in the 900 mg/kg/day group in males only. There were no treatment-related effects in females
- food consumption: there was a dose related reduction in mean food consumption from 300 mg/kg/day in both sexes, males being more affected than females. This reduction resulted in severe effect at the top dose (-54.8 % in males and -30.7 % in females vs. controls). Females recovered soon thereafter (no effect on day 5) while male stilled affected up to termination
- pathology: there were no treatment-related macroscopic findings but statistically significant changes in mean organ weights at 900 mg/kg/day: increased liver (+58.8 % relative increase in males and +57.4 % relative increase in females, both p < 0.01 vs. controls, respectively) and decreased heart weight (-25.14% relative decrease in females, p < 0.05 vs. controls).

Overall, 900 mg/kg/day was considered be an excessive high dose-level (especially in males) for the further OECD 422 study while 600 mg/kg/day could be an appropriate high dose-level.

Therefore, 600 mg/kg/day was selected as the high dose-level. The low-dose and mid-dose were selected using a ratio representing a 3-fold interval (i.e. 66.7 and 200 mg/kg/day).

After treatment initiation, a range of severe clinical signs (hypoactivity, dyspnea, abdominal breathing and/or reflux) were observed in two males which were found dead on study day 3 or 10, respectively. Similar signs but with less severity were also recorded in high-dose females. Therefore, it has been decided to decrease the dose-level in the high-dose group from 600 to 400 mg/kg bw/d.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day during the treatment period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day during the treatment period.

BODY WEIGHT: Yes
- Time schedule: Males: on the first day of treatment, then once a week until sacrifice. Females: on the first day of treatment, then once a week until mating (or until sacrifice), on Days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum.

FOOD CONSUMPTION:
- Time schedule: once a week until sacrifice, with the exception of the mating period (not recorded).

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during FOB test
- Dose groups that were examined: The first five males and the first five females to deliver from each group. once at the end of the treatment period. For females, this was performed on day 5 p.p. after sacrifice of the pups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of sacrifice
- Anaesthetic used for blood collection: Yes light isoflurane anesthesia
- Animals fasted: Yes
- How many animals: from the first five males and the first five females to deliver from each group
- Parameters checked: hematology parameters

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of sacrifice.
- Animals fasted: Yes
- How many animals:from the first five males and the first five females to deliver from each group
- Parameters checked: blood biochemistry

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once 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 deliver from each group during FOB test
- Battery of functions tested:
The following parameters were assessed and graded:
- "touch escape" or ease of removal from the cage,
-I n the hand: fur appearance, salivation, lachrymation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis),
- In the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia.


-Reactivity to manipulation or to different stimuli
The following parameter measurements, reflexes and responses were recorded:
- touch response,
- forelimb grip strength,
- pupillary reflex,
- visual stimulus response,
- auditory startle reflex,
- tail pinch response,
- righting reflex,
- landing foot splay,
- at the end of observation: rectal temperature.

Motor activity: motor activity of all animals was measured once by automated infra-red sensor equipment over a 60-minute period.

Sacrifice and pathology:

Post-mortem examination
SACRIFICE
- Male animals: all surviving animals after the end of the mating period
- Female animals: all surviving animals = day 5 post-partum or, for females which had not delivered yet, day 25 post-coitum (mothers with litter dying entirely were sacrificed as appropriate)

ORGAN WEIGHTS: necropsy observation
The body weight of each animal sacrificed as scheduled was recorded before sacrifice, and the organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection.
The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.
The organ weights were recorded by excess for one female.

GROSS PATHOLOGY:
A complete macroscopic post-mortem examination was performed on all parent animals including aminals that died during the study or were sacrificed prematurely. 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 and the neck with its associated organs and tissues.

HISTOPATHOLOGY:
- on all tissues listed in the table 7.5.1/3 for the first five control and high dose animals (groups 1 and 4) sacrificed as scheduled
- on all macroscopic lesions
- all females sacrificed because of non-delivery to investigate possible causes

Other examinations:

none

Statistics:

See attached document: statistical method

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Mortality In males at 600.0 mg/kg/day, two males were found dead after treatment on study day 3 or 10. Hypoactivity, abdominal breathing, dyspnea, ptyalism, reflux at dosing, soiled hindlimbs and mouth area were noted for the first one and hypoactivity and ptyalism for the second one just before death. At macroscopic post-mortem examination only spleen reduced in size was noted in one male. At 200.0 mg/kg/day, one male was prematurely sacrificed on study day 31. Piloerection, hypoactivity, abdominal breathing, dyspnea, loud breathing, ptyalism, chromorhinorrhea and eyes half-closed were noted before sacrifice. At macroscopic post-mortem examination gelatinous mandibular lymph node, red discoloration of the thymus and yellow deposit in the subcutaneous tissue were noted.
In females at 600.0 mg/kg/day, one female was sacrificed on day 25 post-coitum (p.c.) due to difficulties to deliver. Piloerection, emaciated appearance, round back, pallor of extremities, hypoactivity, dyspnea, loud breathing, ptyalism, soiled urogenital area, eyes half-closed or pallor were noted before death. At macroscopic post-mortem examination thymus reduced in size was noted. Clinical signs In males, piloerection, abdominal or loud breathing, eyes half-closed were observed in some animals treated at the dose-levels of 200.0 or 600.0/400.0 mg/kg/day. Ptyalism was recorded in almost test item-treated males while hypoactivity was recorded in almost mid- and high-dose groups. In females, abdominal breathing, eyes half-closed, piloerection, sedation and/or dyspnea were observed in some animals treated at the dose levels of 200.0 or 600.0/400.0 mg/kg/day. Hypoactivity was recorded in some treated females in mid- and high-dose groups and ptyalism was recorded in almost test item-treated females. These clinical signs recorded from 200.0 mg/kg/day were considered to be related to the treatment with the test item.

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality In males at 600.0 mg/kg/day, two males were found dead after treatment on study day 3 or 10. Hypoactivity, abdominal breathing, dyspnea, ptyalism, reflux at dosing, soiled hindlimbs and mouth area were noted for the first one and hypoactivity and ptyalism for the second one just before death. At macroscopic post-mortem examination only spleen reduced in size was noted in one male. At 200.0 mg/kg/day, one male was prematurely sacrificed on study day 31. Piloerection, hypoactivity, abdominal breathing, dyspnea, loud breathing, ptyalism, chromorhinorrhea and eyes half-closed were noted before sacrifice. At macroscopic post-mortem examination gelatinous mandibular lymph node, red discoloration of the thymus and yellow deposit in the subcutaneous tissue were noted. In females At 600.0 mg/kg/day, one female was sacrificed on day 25 post-coitum (p.c.) due to difficulties to deliver. Piloerection, emaciated appearance, round back, pallor of extremities, hypoactivity, dyspnea, loud breathing, ptyalism, soiled urogenital area, eyes half-closed or pallor were noted before death. At macroscopic post-mortem examination thymus reduced in size was noted. Clinical signs In males, piloerection, abdominal or loud breathing, eyes half-closed were observed in some animals treated at the dose-levels of 200.0 or 600.0/400.0 mg/kg/day. Ptyalism was recorded in almost test item-treated males while hypoactivity was recorded in almost mid- and high-dose groups. In females, abdominal breathing, eyes half-closed, piloerection, sedation and/or dyspnea were observed in some animals treated at the dose levels of 200.0 or 600.0/400.0 mg/kg/day. Hypoactivity was recorded in some treated females in mid- and high-dose groups and ptyalism was recorded in almost test item-treated females. These clinical signs recorded from 200.0 mg/kg/day were considered to be related to the treatment with the test item.

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There were decreased mean food consumptions from 200.0 mg/kg/day in males only

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

moderate decrease in forelimb grip strength in males and delayed landing foot splay in females at 600/400 mg/kg/day. No effect on motor activity measurements (horizontal movements and rearing).

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

thymus (female), liver, adrenal (male)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

the thymus was reduced in size in a single male and a single female from the high-dose group. This correlated with low thymus weights in both animals.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

tubular hyaline droplets in males at all dose-levels, increase severity of lymphoid atrophy in the thymus.

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
At 600.0 mg/kg/day, two males were found dead after treatment (day 3 and day 10). Hypoactivity, abdominal breathing, dyspnea, ptyalism, reflux at dosing, soiled hindlimbs or mouth area were noted for the first one and hypoactivity and ptyalism for the second one just before death. The cause of death of these males was considered to be related to a dosing error. At 200.0 mg/kg/day, one male was prematurely sacrificed on study day 31. Piloerection, hypoactivity, abdominal breathing, dyspnea, loud breathing, ptyalism, chromorhinorrhea and eyes half-closed were noted before sacrifice. At macroscopic post-mortem examination gelatinous mandibular lymph node, red discoloration of the thymus and yellow deposit in the subcutaneous tissue were noted. A dosing error (esophageal perforation) was the cause of death.
In the 600.0/400.0 mg/kg/day group, one female was sacrificed on day 25 post-coitum due to difficulties to deliver. Piloerection, emaciated appearance, round back, pallor of extremities, hypoactivity, dyspnea, loud breathing, ptyalism, soiled urogenital area, eyes half-closed or pallor were noted before sacrifice. At macroscopic post-mortem examination thymus reduced in size was noted. In the control group, one female was sacrificed on day 25 post-coitum for absence of delivery (at necropsy, this female was found to be non pregnant).
In males, piloerection, abdominal or loud breathing, eyes half-closed were observed in some animals treated at the dose-levels of 200.0 and/or 600.0/400.0 mg/kg/day, cutaneous lesion was observed in two control animals. Ptyalism was recorded in almost test item-treated males while hypoactivity was recorded in almost mid- and high-dose groups.
In females, abdominal or loud breathing, eyes half-closed, area of hair loss on forelimb, piloerection, sedation, dyspnea, cutaneous lesion on neck, were observed in some animals treated at the dose-levels of 200.0 and/or 600.0/400.0 mg/kg/day. Hypoactivity was recorded in some treated females in mid- and high-dose groups and ptyalism was recorded in almost test item treated females.
With the exception of cutaneous lesion on neck and area of hair loss on forelimb, all clinical signs recorded from 200.0 mg/kg/day were considered to be related to the treatment with the test item and of toxicological significance. Ptyalism recorded from 66.7 mg/kg/day was also treatment related but considered to be of non toxicological importance.

BODY WEIGHT AND WEIGHT GAIN:
Statistically significant decreases in mean body weight and mean body weight change, in males only, were noted in mid- (-54.3%) and high- (-77.1%, statistically significant p < 0.001) dose groups between days 1 and 8, when compared to controls. The body weight loss was noted until the end of the treatment period.
There were no effects in mean body weight and body weight changes in female.

FOOD CONSUMPTION:
There were decreased mean food consumptions from 200 mg/kg/day in males only, which correlated with their decreased mean body weights and mean body weight gains.
There were no effects on mean food consumption in females.

OPHTHALMOSCOPIC EXAMINATION:
There were no relevant differences in treated group animals when compared with control animals

HAEMATOLOGY:
Overall, there were a few statistically significant differences observed in males only, poorly dose related and of small amplitudes. Therefore a treatment-related effect with the test item was considered unlikely.

CLINICAL CHEMISTRY:
In the high-dose group, there was a statistically significant increase in bile acid in females and a decrease in cholesterol in males. In the absence of similar finding in the opposite sex and of microscopic correlates, a relationship to the treatment with the test item was considered unlikely.

Overall, there was a tendency towards hepatic enzyme induction (ALP and ALAT) which were considered to be non adverse in the absence of correlates atmicroscopic examination of the liver.

NEUROBEHAVIOUR:
There were no relevant differences in motor activity (horizontal movements and rearing) in treated animals when compared with controls.

ORGAN WEIGHTS:
When compared with controls, the mean relative weights of kidneys were statistically significantly increased in all male groups given the test item.

There were decreased mean absolute and relative thymus weights in females given the test item at 66.7 or 600.0/400.0 mg/kg/day. These variations at the lowest and the highest dose-levels were not statistically significant, and were not seen at the mid-dose of 200.0 mg/kg/day.
In males, the individual absolute and relative thymus weights of male Y21954 were low.

There were statistically significantly higher mean relative weights of liver and adrenals in males given the test item at 200.0 or 600.0/400.0 mg/kg/day. Although this was partly due to the decreased body weight (up to -11% at the high-dose), a relationship to the test item could not be completely excluded.

The statistically significant increases in relative (to body weight) organ weights in males for brain (+19% at the high-dose), epididymis (+13% at the mid- and high-doses) and testis (+21% at the high-dose) were considered to be a reflection of reductions in body weight (up to -11% at the high dose) and not due to test item-related organ toxicity (Levin et al., 1993). There were no significant changes in the absolute weights of these organs.

GROSS PATHOLOGY:
The thymus was reduced in size in a single male and a single female from the high-dose group. This correlated with low thymus weights in both animals.
The other macroscopic findings had no histologic correlates or correlated with common histologic findings in control Sprague-Dawley rats, and were considered to be incidental.

HISTOPATHOLOGY: NON-NEOPLASTIC:
Tubular hyaline droplets were seen with dose-related incidence and severity in some males given the test item at all dose-levels, as shown in the table 7.5.1/9, and correlated with increases in relative kidney weights. This was characterized by the presence of dense eosinophilic droplets in proximal tubular epithelium. At the lowest dose-level, a single male was affected.
In the thymus, increased severity of lymphoid atrophy (moderate versus minimal), characterized by decreased cellularity and decreased thickness of the cortex and medulla, was seen in 2/5 females given the test item at 600.0/400.0 mg/kg/day, and correlated with lower thymus weights at this dose-level (table 7.5.1/10). There were no significant differences in the incidence and severity of lymphoid atrophy in the thymus at the low- and mid-dose when compared with controls.
In one high-dose male, there was moderate chronic broncho-alveolar inflammation of the lungs similar to that seen in male decedents, and which was also considered to be secondary to misdosing. In addition, there was moderate subacute inflammation and atrophy (correlated with reduced in size) of the seminal vesicles associated with slight prostate atrophy. Minimal centrilobular inflammation and pigment were found in the liver. All these isolated changes were considered to be incidental and unrelated to the test item administration. Stress-associated, marked lymphoid atrophy was found in the thymus, and correlated with low weights and reduced size at necropsy.

Other microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the Sprague-Dawley rat.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

66.7 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

2

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

gross pathology

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Clinical signs

Clinical signs
Sex	Male				Female
Dose-level (mg/kg/day)	0	66.7	200.0	600.0/400.0	0	66.7	200.0	600.0/400.0
Piloerection	0	0	1	0	0	0	0	1 (P/L)
Emaciated appearance	0	1	0	0	0	0	0	0
Hypoactivity	0	0	5	8	0	0	2 (P/G) 1 (L)	6 (P) 3 (G/L)
Sedation	0	0	0	0	0	0	1 (G)	1 (G)
Dyspnea	0	0	0	0	0	0	0	1 (L)
Abdominal breathing	0	0	2	3	0	0	1 (P)	1 (G)
Loud breathing	0	0	0	1	0	0	0	1 (G)
Ptyalism	0	8	9	8	0	1 (P) 6 (G) 2 (L)	7(P) 10 (G) 8 (L)	10 (P) 9 (G/L)
Reflux at dosing	0	0	0	1	1 (G)	0	2 (G)	0
Eyes half-closed	0	0	4	6	0	1 (G)	1 (P) 3 (G) 1 (L)	2 (P) 4 (G/L)
Cutaneous lesion on neck	2	0	0	0	0	0	0	1 (G)
Area of hair loss on forelimb	0	0	0	0	2 (G/L)	1 (G) 1(L)	1 (P/G/L)	0

 

Body weight

Mean body weights and mean body weight changes (g)

Sex

Male

Female

Dose-level (mg/kg/day)

0

66.7

200.0

600.0/400.0

0

66.7

200.0

600.0/400.0

- Mean body weight

Day 1

406

402

406

407

220

223

222

222

Day 8

441

435 (-1.4)

422 (-4.3)

397# (-10.0)

229

236

233

235

Day 15

465

460 (-1.1)

431* (-7.3)

422** (-9.2)

240

245

241

249

Day 36

526

524 (-0.4)

485 (-7.8)

471* (-10.5)

/

/

/

/

- Mean body weight change

Days 1 - 8

+35

+34 (-2.9)

+16 (-54.3)

-8# (-77.1)

+8

+13

+10

+13

Days 1 - 15

+59

+59 (0.0)

+25** (-57.6)

+18** (-69.5)

+19

+22

+19

+27

Days 15 - 36

+61

+64 (0.5)

+61 (0.0)

+49 (-19.7)

/

/

/

/

Days 1 - 36

+120

+123 (+2.5)

+80 (-33.3)

+66* (-45.0)

/

/

/

/

Dose-level (mg/kg/day)

0

66.7

200.0

600.0/400.0

- Mean body weight

Day 0p.c.

246

246

249

265*

Day 20p.c.

400

391

381

392

Day 1p.p.

298

298

296

306

Day 5p.p.

312

309

311

319

- Mean body weight change

Days 0 - 20p.c.

+154

+146

+132

+127

Days 1 - 5p.p.

+14

+11

+15

+13

( ): percent of changesvs.controls, %.

/: not applicable.

Statistically significant: *: p<0.05, **: p<0.01, #: p<0.001.

p.c.: post-coitum.

p.p.: post-partum.

Food consumption

Mean food consumption (g/animal/day)
Sex	Male						Female
Dose-level (mg/kg/day)	0	66.7		200.0	600.0/400.0		0		66.7	200.0		600.0/400.0
Days 1 - 8	31	29 (-6.5)		25** (-19.4)	19# (-38.7)		16		18	16		15
Days 8 -15	28	27 (-3.6)		23# (-17.9)	26 (-7.1)		16		17	17		17
Dose-level (mg/kg/day)			0			66.7		200.0			600.0/400.0
- Mean food consumption (g/animal/day)
Days 0 - 7p.c.			21			19		21			21
Days 7 - 14p.c.			22			22		22			21
Days 14 - 20p.c.			23			23		22			22
Days 1 - 5p.p.			37			35		35			33

 

Laboratory investigations

Sex	Male				Female
Dose-level (mg/kg/day)	0	66.7	200.0	600.0/ 400.0	0	66.7	200.0	600.0/ 400.0
Mean cell hemoglobin (pg)	17.8	17.9 (+0.6)	17.1* (-3.9)	17.4 (-2.2)	19.0	18.7	18.8	18.8
Mean cell hemoglobin concentration (g/dL)	34.2	33.9 (-0.9)	33.1* (-3.2)	33.0** (-3.5)	34.1	34.0	34.1	34.0
Fibrinogen (g/L)	3.50	3.17 (-9.4)	2.91 (-16.9)	2.55* (-27.1)	3.64	3.43	3.28	3.14
Bile acid (µmol/L)	80.2	72.9	50.4	35.3	31.9	50.9 (+59.6)	49.7 (+55.8)	107.3** (3.4-fold)^a
Cholesterol (mmol/L)	1.8	1.4* (-22.2)	1.5 (-16.7)	1.3** (-27.8)	1.6	1.5	1.6	1.9
ALP (IU/L)	398	459 (+15.3)	495 (+24.4)	683** (+71.6)	301	276	294	384
ALAT (IU/L)	45	50 (+11.1)	48 (-6.7)	79* (+75.6)	69	82 (+18.8)	71 (+2.9)	112** (x1.6)^a

 

Pathology

Sex	Male			Female
Dose-level (mg/kg/day)	66.7	200.0	600.0/400.0	66.7	200.0	600.0/400.0
Body weight	0	-8	-11*	+1	0	+1
- Kidneys
. absolute	+12	+5	0	+87	+3	-2
. relative	+13**	+17**	+22**	+86	+2	-1
- Thymus
. absolute	-8	-11	-13	-22	+8	-30
. relative	-6	-1	+4	-23	+5	-29
- Adrenals
. absolute	+13	+13	+17	-8	+5	-11
. relative	+15	+24*	+46**	-8	+4	-10
- Liver
. absolute	+1	+9	+9	-20	-1	+9
. relative	+2	+21**	+33**	-21	-2	+10

Statistically significant from controls: *: p < 0.05, **: p < 0.01

Statistical significance determined for organ weights values and not percent changes.

 

Incidence and severity of tubular hyaline droplets in the renal epithelium in males (n = 5)

Group	1	2	3	4
Dose-level (mg/kg/day)	0	66.7	200.0	600.0/400.0
Hyaline droplets; tubular epithelium
Minimal (grade 1)	-	-	1	-
Slight (grade 2)	-	1	2	2
Moderate (grade 3)	-	-	-	2

-: no animal affected.

 

Incidence and severity of lymphoid atrophy in the thymus in females (n = 5)

Group	1	2	3	4
Dose-level (mg/kg/day)	0	66.7	200.0	600.0/400.0
Lymphoid atrophy
Minimal (grade 1)	4	2	3	3
Slight (grade 2)	-	2	1	-
Moderate (grade 3)	-	-	-	2

-: no animal affected.

Applicant's summary and conclusion

Conclusions:

Based on the experimental conditions of this study:
- NOAEL for male parental toxicity was considered to be 66.7 mg/kg/day based on clinical signs and effects on body weight and food consumption in males from 200.0 mg/kg/day,
- NOAEL for female parental toxicity was considered to be 200 mg/kg/day based on the decrease of the thymus associated with a lymphoid atrophy in females at 600/400 mg/kg/day.

Executive summary:

In a combined repeated dose toxicity study with the reproductive/developmental toxicity screening test by oral route performed in compliance with the GLP and in accordance with the OECD 422 test guideline, the test material,diluted in corn oil was administered daily by gavage to Sprague Dawley rats (10 animals/sex/dose) at doses of 0; 66.7; 200 and 600 mg/kg bw/d.The high dose-level group received 600 mg/kg bw/d for 10 days from the beginning of the treatment period only. Because of excessive toxicity at this dose-level, the high‑dose group was treated at 400 mg/kg bw/d for all the remaining treatment period. Males were exposed 2 weeks before pairing, during pairing and until sacrifice (at least 5 weeks in total) whereas females were exposed 2 weeks before pairing, during pairing, during gestation and during lactation until day 5 post-partum (7-8 weeks in total).

Animals were checked daily for clinical signs, mortality, and detailed clinical observations were conducted weekly. Body weights and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. A Functional Observation Battery was performed on five males and females per group at the end of the study. Prior to sacrifice, blood samples were also taken from these animals for analysis of hematology and blood biochemistry parameters. The males were sacrificed after completion of the mating period. Body weights and selected organs weights were recorded and a complete macroscopic post-mortem examination performed. A microscopic examination was also conducted on selected organs from the first five males in the control group and the high-dose group. Microscopic examination was conducted on all macroscopic lesions from all groups. Dams were sacrificed on day 6 p.p.. Body weights and selected organs weights were recorded and a complete macroscopic examination was performed. A microscopic examination was then conducted on selected organs from the first five females to deliver in the control group and the high-dose group and on any macroscopic lesions from all groups.

At 600 mg/kg bw/d, two males were found dead after treatment on study day 3 or 10 and one male treated at 200 mg/kg bw/d was prematurely sacrificed on study day 31. Therefore, it has been decided to decrease the dose-level in the high-dose group from 600 to 400 mg/kg bw/d. After macroscopic examination the cause of death of these males was in fact considered to be related to a dosing error. However, clinical signs such as hypoactivity, dyspnea, abdominal breathing and/or reflux in animals of both sexes of the high dose group of 600 mg/kg bw/d justify also this decrease of dosing. In males, piloerection, abdominal or loud breathing, eyes half-closed were observed in some animals treated at the dose-levels of 200 and/or 600/400 mg/kgbw/d. Ptyalism was recorded in almost test item-treated males while hypoactivity was recorded in almost mid- and high-dose groups. In females, abdominal or loud breathing, eyes half-closed, piloerection, sedation, dyspnea, were observed in some animals treated at the dose-levels of 200 and/or 600/400 mg/kg bw/d. Hypoactivity was recorded in some treated females in mid- and high-dose groups and ptyalism was recorded in almost test item treated females. All of these clinical signs recorded from 200 mg/kg/day were considered to be related to the treatment with the test item and of toxicological significance. Ptyalism recorded from 66.7 mg/kg/day was also treatment related but considered to be of non-toxicological importance.

There were decreased mean food consumptions from 200 mg/kg bw/d in males only, which correlated with their decreased mean body weights and mean body weight gains. There was a moderate decrease in forelimb grip strength in males and delayed landing foot splay in females at 600/400 mg/kg bw/d which was considered to be related to the treatment with the test item. There was no effect of the test item on hematologic parameters and blood chemistry.

 

Tubular hyaline droplets were seen with dose-related incidence and severity in some males given the test item at all dose-levels and correlated with increases in relative kidney weights. This was characterized by the presence of dense eosinophilic droplets in proximal tubular epithelium. At the lowest dose-level, a single male was affected. These hyaline droplets, occasionally seen in untreated male rats (but not in the current study), are consistent with α2u globulin and are known to increase after treatment with a wide range of drugs or chemicals (Greaves, 2007). These findings were not considered to be adverse in the absence of associated degenerative changes. Although human excrete proteins of a similar nature, they are found in only trace amounts and therefore this finding is considered to be non-relevant for human.

In females given the test item at 600/400 mg/kg bw/d, decreased thymus weights correlated with increased severity of lymphoid atrophy (decreased cellularity and decrease thickness of the cortex and medulla). An effect of the test item could not be excluded for the decreased thymus weights at 600/400 mg/kg bw/d, while the same variations at 200 mg/kg bw/d were considered to be most likely incidental and unrelated to the test item. Low thymus weights for a single male at 600/400 mg/kg bw/d correlated with marked lymphoid atrophy which was considered to be stress-associated in view of the other inflammatory lesions in this animal.

There were statistically significantly higher mean relative weights of liver and adrenals in males given the test item at 200 or 600/400 mg/kg bw/d. Although this was partly due to the decreased body weight (up to -11% at the high-dose), a relationship to the test item could not be completely excluded.

Based on the results of this study, 66.7 mg/kg bw/d of test item was established as the no-observed-adverse effect-level (NOAEL) considering the clinical signs and effects on body weight and food consumption observed in males from 200 mg/kg bw/d. The NOAEL for female parental toxicity was considered to be 200 mg/kg bw/day based on the decrease of the thymus associated with a lymphoid atrophy in females at 600/400 mg/kg bw/d.