[Name confidential or not available]

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

of 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Sprague Dawley rats, strain: Crl:CD(SD) with appropriate range of bodyweight at study start.
- Source: Charles River (UK) Ltd.
- Age at treatment start: Ca. 70 days.
- Weight at treatment start: Males: minimum 348 g, maximum 390 g,
Females: minimum 218 g, maximum 267 g.
- Housing Inside a barriered rodent facility:
all animals pre-pairing + toxicity subgroups: In groups up to 5 by sex in solid floor polycarbonate cages.
during pairing (1 male+1 female/cage): In RB3 modified polypropylene cages with stainless steel grid-floor over absorbent paper-lined trays.
males after pairing: In groups up to 5 in solid floor polycarbonate cages.
females during gestation and lactation: Females housed individually (+litter) in solid floor polycarbonate cages.
- Bedding material (in solid floor cages): Wood based bedding, sterilised by autoclaving before use.
- Cage enrichment: Aspen chew block + plastic shelter (except during pairing or post gestation day 20).
- Diet (ad libitum): Standard rodent diet (SDS VRF1 Certified) without antibiotic, chemotherapeutic or prophylactic agent.
- Fasting (diet withheld): Main phase males and Toxicity phase females overnight before blood sampling for clinical pathology.
- Water (ad libitum): Potable drinking water from the public supply.
- Acclimation period: 5 days before treatment start, under laboratory conditions.

Routine analysis of the batch of diet used and water, chew blocks and bedding material did not provide evidence of contamination that might have prejudiced the study.

IN-LIFE DATES:
- Duration of test, males & toxicity phase females: Five weeks
Duration of test, main phase females (i.e. reproductive subgroup): From 14 days prior to pairing to day 7 of lactation.
Duration of test, offspring: From birth to day 7 of lactation.

ENVIRONMENTAL CONDITIONS

Air conditioned room kept at positve pressure without re-circulation of the filtered fresh air supplied to the room.
Controlled environment, environmental conditions were set at:
- Temperature (°C): 21 ± 2°C
- Relative Humidity (%): 40 to 70%
- Photoperiod (artificial lighting): 12 hrs day / 12 hrs night
- Rate of air exchange: At least 15 changes/h
Deviations from the target ranges for temperature and relative humidity were not evident.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

- Concentration in vehicle: The concentration of the test material in vehicle varied between dose groups thus allowing constant dosage volume in terms of mL/kg bw/day.
- Amount (dose volume by gavage): 5 mL/kg bw/day..
Actual dose volumes were calculated at about weekly or shorter intervals accounting for the latest body weight. Litter animals were not dosed.

- For concentrations of test material in vehicle at different dose levels, see Table 1 in "Any other information on materials and methods incl. tables"

- Justification for choice of vehicle:
The suitability of corn oil as a vehicle was established visually and by chemical analysis during the 7-day range-finding study:
Endpoint study record "7.5.1 Repeated dose toxicity: oral - 7d_range-finding_gavage_HLS_GAH0043".
In addition, in the present main study, concentrations of dose formulations were chemically analysed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Chemical analysis of test material formulations by high performance liquid chromatography coupled with a mass spectrometer (HPLC-MS/MS).
- Concentrations (verified at first and last treatment week) of the test material formulations were confirmed at each dose level.
- Chemical analysis confirmed that the mean concentrations of WS400506 in prepared formulations were 89.5% to 99.5% of the corresponding
nominal concentration, thus confirming accuracy of formulation.

Duration of treatment / exposure:

- Treatment period, males & toxicity phase females: Daily, for five consecutive weeks, in males commencing 14 days prior to pairing
- Treatment period, main phase females (i.e. reproductive subgroup): 44 to 56 days (from 14 days prior to pairing to day 6 of lactation)
- Offspring were not dosed

Frequency of treatment:

Daily, 7 days/week (during parturition, dosing omitted as appropriate)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Toxicity phase animals: */ 5 females
Main phase animals (i.e. reproductive subgroups): 10 males / 10 females
*Explanatory note by the notifier:
Examinations assigned to the toxicity phase females to meet the requirements of a 28-day repeat dose oral toxicity study were also assigned to 5 (for some examinations to 10) main phase males per dose group. Therefore, these 5 main phase males per dose group are called also "toxicity subgroup" in the present robust study summary for clarification. After pairing with main phase females, all males were killed at the same time (Week 6).

Control animals:

yes, concurrent vehicle

Details on study design:

This study was conducted to examine both repeated dose toxicity and  reproductive/developmental toxicity as an OECD screening combined study
(OECD 422 test guideline).  Therefore, animals initially entering the study were divided into toxicity subgroup animals (toxicity phase) and reproductive subgroup animals (main phase), whereby 5 of the 10 F0 males (used for pairing) per dose group formed the toxicity male subgroups.

Dose selection was based on the results of a 7-day preliminary oral toxicity study in the rat in which dose levels of 100, 300 or 1000 mg/kg/day did not have any overt treatment-related effects on young adult animals (females nulliparous and non-pregnant).

Positive control:

Not included in the study.

Observations and examinations performed and frequency:

Clinical observations performed and frequency:
- Clinical signs : At least twice a day (before and after administration)
- Detailed physical examination
and arena observations: Before treatment start and at least once a treatment week.
- Functional Observation Battery:* During treatment week 5 (before dosing) on all toxicity subgroup animals (5 males + 5 females/group).
- Body weight, all males: Weekly throughout the study.
Body weight, Toxicity Females: Weekly throughout the study.
Body weight, Repro. Females: Weekly for pre-pairing period; on gestation days 0, 6, 13, 20; on lactation days 1 & 7.
- Food consumption, all males: Weekly for pre-pairing period and for the period after mating.
Food cons., Toxicity Females: About weekly throughout the study.
Food cons., Repro. Females: Weekly for pre-pairing period, during gestation for days 0-6, 6-13, 13-20, during lactation for days 1-4 & 4-7.

* FOB including sensory reactivity tests (approach, touch, auditory startle reflex, tail pinching), grip strength and motor activity.

Hematological examinations (only for toxicity subgroup animals) during treatment week 5 after functional observation battery:
Red blood cell count, reticulocyte count, white blood cell count, platelet count, hemoglobin concentration, hematocrit value, differential leukocyte counts,
protrombin time, activated partial thromboplastin time, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration.

Blood (plasma) chemical examinations (only for toxicity subgroup animals) during treatment week 5 after functional observation battery:
Total protein, albumin, A/G ratio, urea, creatinine, glucose, total cholesterol, total bilirubin, bile acids, sodium, potassium, chloride,
calcium, inorganic phosphorus, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transpeptidase.

This study was conducted to examine both repeated dose toxicity and  reproductive/developmental toxicity as an OECD screening combined study
(OECD 422 test guideline).  Therefore, some of the examinations were confined to toxicity subgroup animals, as indicated above.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, see below
WEIGHING OF ORGANS: Yes, see below
HISTOPATHOLOGY: Yes, see below

Terminal sacrifice
- all males and toxicity subgr. females: Killed in Week 6, after completion of the Treatment Week 5 investigations.
- reproductive subgr. females & offspring: Killed on Day 7 post partum.
2 control females which failed to mate or were without viable litter were killed on Day 25 after the last day of pairing.
Gross pathology:
- adult/parental animals: Full macroscopic examination with tissue collection.
- offspring: Full macroscopic examination including assessment of the presence of milk in the stomach, where possible.
(Missing or grossly autolysed or cannibalised offspring could not be examined).

Organs Weights:
- main phase and tox. subgr. adults: Adrenals, brain, epididymides, heart, kidneys, liver, lungs & bronchi, ovaries, pituitary, prostate, seminal vesicles
& coagulation gland, spleen, testes, thymus, thyroid with parathyroids, uterus with cervix & oviducts.

Histopathology:
- toxicity subgroups: The following organs were microscopically observed for the control and 1000 mg/kg bw/day groups:
Brain, eyes, Harderian glands, optic nerves, pituitary gland, thyroid with parathyroids, heart, thymus, liver, spleen,
adrenals, kidneys, testes, epididymides, ovaries, lung, trachea, esophagus, stomach, duodenum, jejunum, ileum,
caecum, rectum, colon, Peyer's patch, lymph node (axillary, mesenteric), urinary bladder, uterus (with cervix &
oviducts), vagina, spinal cord, sciatic nerve, skeletal muscle, skin with mammary glands, sternum with marrow,
seminal vesicle & coagulation gland, prostate. In addition, the jejunum, mesenteric lymph nodes and duodenum
were also examined for the 100 and 300 mg/kg bw/day toxicity subgroups and any gross lesions for all adult animals. - reproductive subgroups Gross lesions from all adult animals from all dose groups were examined by light microscopy.

Other examinations:

Reproductive and developmental toxicity parameters (addressed in separate endpoints).

Statistics:

Grip strength, motor activity, bodyweight, food consumption, organ weight, gestation length, litter size, survival indices & clinical pathology data were statistically analysed by adoption of the following sequence of tests:

- Parametric analysis, if Bartlett's test for variance homogeneity was not significant at the 1% level.
F1 approximate test for monotonicity of dose-response. If this F1 test was not significant at the 1% level, Williams' test for a monotonic trend was applied.
If this F1 test was significant, suggesting that the dose-response was not monotone , the Dunnett's test was performed instead.

- Non-parametric analysis, if Bartlett's test was still significant at the 1% level following logarithmic and square-root transformations.
H1 approximate test for monotonicity of dose-response. If this H1 test was not significant at the 1% level, Shirley's test for a monotonic trend was applied.
If this H1 test was significant, suggesting that the dose-response was not monotone, the Steel's test was performed instead.

-For grip strength, motor activity, survival indices and clinical pathology data, if 75% of the data (across all groups) were the same value,
pairwise comparison of each dose group against the control by Fisher’s Exact tests.

-For organ weight data, covariance analysis using terminal bodyweight as covariate (Angervall & Carlstrom, 1963).

Sex ratio
- Analysis by generalised mixed linear model with binomial errors, a logit link function and litter as a random effect (Lipsitz et al 1991).
Each treated group was compared to control using a Wald chi-square test.

Gestation length
- Exact (or asymptotic) two-tailed Linear-by-linear test (Cytel 1995), with equally spaced scores. If the test was statistically significant (p<0.05), the highest
dose group was excluded and the test re-applied until the test was no longer statistically significant (p≥0.05).

For statistical references, see next field.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

only in males at 1000 mg/kg/day. See figures on body weight attached as background material.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

See tables on food consumption attached as background material.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

See tables on haematology and clinical chemistry attached as background material.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

See tables on haematology and clinical chemistry attached as background material.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation battery including sensory reactivity tests (approach, touch, auditory startle reflex, tail pinching), grip strength, motor activity and attention to clinical signs of neurotoxicity during animal observations.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

mesenteric lymph node, duodenum, jejunum

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS, NEUROBEHAVIOUR AND MORTALITY
One control female was found dead following an intubation error. Two other control females which failed to mate or were without viable litter were killed on Day 25 after their last day of pairing. Clinical signs attributable to treatment with the test material or effects on sensory reactivity, grip strength or motor activity were not evident.

BODY WEIGHT AND WEIGHT GAIN
Overall bodyweight gain was low in males receiving 1000 mg/kg/day, but unaffected in males at lower dose levels and females at all dose levels (see Tables 2 and 3).

FOOD CONSUMPTION
Food consumption was unaffected by treatment with the test material.

CLINICAL PATHOLOGY
Haematology parameters were unaffected by treatment with the test material. Raised cholesterol levels in both sexes at 1000 mg/kg/day on their own and with no pathological correlate were considered fortuitous and toxicologically not significant.

GROSS PATHOLOGY
Macroscopic findings attributable to treatment with the test material were not evident.

ORGAN WEIGHTS
Adjusted mean adrenal weights and mean ovary weights lower than concurrent controls in all treated groups after 5 treatment weeks were considered to be fortuitous and toxicologically not significant, as there were no histopathological correlates. Following lactation, mean ovary weight was unaffected by treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
Lymphatic ectasia in the mesenteric lymph node of males at 1000 mg/kg/day and females at 300 and 1000 mg/kg/day and cystic dilation of the central lymphatic or lacteal in the villi of the duodenum and jejunum in both sexes at 300 and 1000 mg/kg/day (see Table 4) were attributed to treatment with the test material. However, only in males at 1000 mg/kg/day these findings were considered to represent an adverse effect, because in this group they may have affected digestive function thus contributing to the low body weight gain. In all groups, the intestinal changes were not associated with any inflammatory or degenerative changes.
Plasmacytosis in the mesenteric lymph node in males of all groups treated with WS400506 and in one control female and one female of the 300 mg/kg/day group was considered to be a non specific reactive change of no toxicological importance.

OTHER RESULTS
Reproductive and developmental toxicity parameters are addressed in separate endpoints.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Lymphatic ectasia in the mesenteric lymph node and cystic dilation of the central lymphatic or lacteal in the villi of the duodenum and jejunum associated with low bodyweight gain at 1000 mg/kg/day were considered to be adverse.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: NOAEL = highest dose tested. In the absence of adverse effects on bodyweight in females, histopathology findings similar to those in males were not considered to be adverse.

Critical effects observed:

not specified

 

Table 2: Bodyweight and Bodyweight Change - Group Mean Values (g) for Main and Toxicity Phase Animals – Males
Group			1		2			3		4
Compound			Vehicle Control		WS400506			WS400506		WS400506
Dose (mg/kg/day)			0		100			300		1000
Group/Sex		Week	Week	Week	Week		Week	Week		Change	Change	Change
		0	1	2	3		4	5		0-2	2-5	0-5
Statistical test:		Wi	Wi	Wi	Wi		Wi	Wi		Wi	Wi	Wi
1/M	Mean	367	401	433	449		474	489		66	56	122
	SD	11.4	13.5	17.6	16.8		23.0	33.2		8.9	18.3	26.3
	N	10	10	10	10		10	10		10	10	10
2/M	Mean	367	405	430	449		469	482		63	51	115
	SD	10.7	14.1	19.4	21.6		23.1	24.1		12.6	10.8	17.5
	N	10	10	10	10		10	10		10	10	10
3/M	Mean	367	399	424	446		471	481		57	57	114
	SD	10.5	11.7	16.9	17.3		17.1	19.4		13.1	11.0	17.5
	N	10	10	10	10		10	10		10	10	10
4/M	Mean	365	391	413*	424**		448**	453**		47**	40*	87**
	SD	13.2	15.7	16.5	16.8		17.1	16.7		8.0	14.6	16.7
	N	10	10	10	10		10	10		10	10	10

 

Statistical test Wi = Treated groups compared to Control using Williams’ test,    * p<0.05,    ** p<0.01

 

Table 3: Bodyweight and Bodyweight Change - Group Mean Values (g) for Main and Toxicity Phase Animals – Females
Group			1		2			3		4
Compound			Vehicle Control		WS400506			WS400506		WS400506
Dose (mg/kg/day)			0		100			300		1000
Group/Sex		Week	Week	Week	Week		Week	Week		Change	Change	Change
		0	1	2	3‡		4‡	5‡		0-2	2-5‡	0-5‡
Statistical test:		Wi	Wi	Wi	Wi		Wi	Wi		Wi	Wi	Wi
1/F	Mean	240	244	254	277		289	283		14	23	42
	SD	10.5	10.6	12.7	11.0		10.1	10.1		10.7	7.0	6.8
	N	15	15	15	5		5	5		15	5	5
2/F	Mean	236	241	252	264		272	274		17	23	39
	SD	11.0	12.3	13.0	9.8		13.5	13.6		7.6	9.4	7.5
	N	15	15	15	5		5	5		15	5	5
3/F	Mean	235	244	253	264		275	277		18	23	42
	SD	7.4	11.4	13.0	16.8		22.4	18.3		11.7	11.6	13.6
	N	15	15	15	5		5	5		15	5	5
4/F	Mean	237	248	256	274		287	288		20	28	50
	SD	8.2	7.9	7.4	9.0		14.2	11.5		8.6	8.1	10.8
	N	15	15	15	5		5	5		15	5	5

 

‡ Toxicity phase animals only

Statistical test Wi = Treated groups compared to Control using Williams’ test,    * p<0.05,    ** p<0.01

Table 4:Summary of Treatment Related Findings for Toxicity Phase Animals Killed after 5 Treatment Weeks
Group/Sex	1/M	2/M	3/M	4/M	1/F	2/F	3/F	4/F
Dose (mg/kg/day)	0	100	300	1000	0	100	300	1000
Mesenteric Lymph Node
Lymphatic ectasia
Minimal Slight Moderate	0 0 0	0 0 0	0 0 0	1 2 2	0 0 0	0 0 0	1 0 0	1 2 1
Total	0	0	0	5	0	0	1	4
Plasmacytosis
Minimal	0 0	3 2	3 2	2 1	0 1	0 0	0 1	0 0
Slight
Total	0	5	5	3	1	0	1	0
Number of tissues examined	5	5	5	5	5	5	5	5
Duodenum
Cystic dilation of the central lymphatic within villi
Minimal Slight Moderate	0 0 0	0 0 0	1 0 0	1 3 1	0 0 0	0 0 0	2 0 0	2 2 1
Total	0	0	1	5	0	0	2	5
Number of tissues examined	5	5	5	5	5	5	5	5
Jejunum
Cystic dilation of the central lymphatic within villi
Minimal Slight Moderate	0 0 0	0 0 0	1 0 0	1 0 3	0 0 0	0 0 0	3 0 0	1 2 2
Total	0	0	1	4	0	0	3	5
Number of tissues examined	5	5	5	5	5	5	5	5

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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)

Version / remarks:

of 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): WS400506

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Sprague Dawley rats, strain: Crl:CD(SD) with appropriate range of bodyweight at study start.
- Source: Charles River (UK) Ltd.
- Age at treatment start: Ca. 70 days.
- Weight at treatment start: Males: minimum 348 g, maximum 390 g,
Females: minimum 218 g, maximum 267 g.
- Housing Inside a barriered rodent facility:
all animals pre-pairing + toxicity subgroup: In groups up to 5 by sex in solid floor polycarbonate cages.
during pairing (1 male+1 female/cage): In RB3 modified propylene cages with stainless steel grid-floor over absorbent paper-lined trays.
males after pairing: In groups up to 5 in solid floor polycarbonate cages.
females during gestation and lactation: Females housed individually (+litter) in solid floor polycarbonate cages.
- Bedding material (in solid floor cages): Wood based bedding, sterilised by autoclaving before use.
- Cage enrichment: Aspen chew block + plastic shelter (except during pairing or post gestation day 20).
- Diet (ad libitum): Standard rodent diet (SDS VRF1 Certified) without antibiotic, chemotherapeutic or prophylactic agent.
- Fasting (diet withheld): Main phase males and Toxicity phase females overnight before blood sampling for clinical pathology.
- Water (ad libitum): Potable drinking water from the public supply.
- Acclimation period: 5 days before treatment start, under laboratory conditions.

Routine analysis of the batch of diet used and water, chew blocks and bedding material did not provide evidence of contamination that might have prejudiced the study.

IN-LIFE DATES:
- Duration of test, males & toxicity phase females: Five weeks
Duration of test, main phase females (i.e. reproductive subgroup): From 14 days prior to pairing to day 7 of lactation.
Duration of test, offspring: From birth to day 7 of lactation.

ENVIRONMENTAL CONDITIONS

Air conditioned room kept at positve pressure without re-circulation of the filtered fresh air supplied to the room.
Controlled environment, environmental conditions were set at:
- Temperature (°C): 21 ± 2°C
- Relative Humidity (%): 40 to 70%
- Photoperiod (artificial lighting): 12 hrs day / 12 hrs night
- Rate of air exchange: At least 15 changes/h
Deviations from the target ranges for temperature and relative humidity were not evident.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Treatment of parental animals by oral gavage administration. Test substance was not directly administered to F1 animals.

- Concentration in vehicle: The concentration of the test material in vehicle varied between dose groups thus allowing constant dosage volume in terms of mL/kg bw/day.
- Amount (dose volume by gavage): 5 mL/kg bw/day.
Actual dose volumes were calculated at about weekly or shorter intervals accounting for the latest body weight.

- For concentrations of test material in vehicle at different dose levels, see Table 1 in "Any other information on materials and methods incl. tables"

- Justification for choice of vehicle:
The suitability of corn oil as a vehicle was established visually and by chemical analysis during the 7-day range-finding study:
Endpoint study record "7.5.1 Repeated dose toxicity: oral - 7d_range-finding_gavage_HLS_GAH0043".
In addition, in the present main study, concentrations of dose formulations were chemically analysed.

Details on mating procedure:

- Male/female ratio per cage: 1/1
- Length of cohabitation: At the most 14 days, until proof of pregnancy was confirmed. 
- Proof of successful mating: Formation of at least one copulation plug and a sperm positive vaginal smear.
The day this was found was referred to as day 0 of gestation.
(During cohabitation, females were checked every morning for pregnancy).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Chemical analysis of test material formulations by high performance liquid chromatography coupled with a mass spectrometer (HPLC-MS/MS).
- Concentrations (verified at first and last treatment week) of the test material formulations were confirmed at each dose level.
- Chemical analysis confirmed that the mean concentrations of WS400506 in prepared formulations were 89.5% to 99.5% of the corresponding
nominal concentration, thus confirming accuracy of formulation.

Duration of treatment / exposure:

- Treatment period, males & toxicity phase females: Daily, for five consecutive weeks, in males commencing 14 days prior to mating
- Treatment period, main phase females (i.e. reproductive subgroup): 44 to 56 days (from 14 days prior to mating to day 6 of lactation)
- Offspring were not dosed

Frequency of treatment:

Daily, 7 days/week (during parturition, dosing omitted as appropriate)

Details on study schedule:

- Age at mating of the mated animals in the study: 12 to 14 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

Toxicity phase animals: */ 5 females
Main phase animals (i.e. reproductive subgroups): 10 males / 10 females
*Explanatory note by the notifier:
Examinations assigned to the toxicity phase females to meet the requirements of a 28-day repeat dose oral toxicity study were also assigned to 5 (for some examinations to 10) main phase males per dose group. Therefore, these 5 main phase males per dose group are called also "toxicity subgroup" in the present robust study summary for clarification. After pairing with main phase females, all males were killed at the same time (Week 6).

Control animals:

yes, concurrent vehicle

Details on study design:

This study was conducted to examine both repeated dose toxicity and  reproductive/developmental toxicity as an OECD screening combined study
(OECD 422 test guideline).  Therefore, animals initially entering the study were divided into toxicity subgroup animals (toxicity phase) and reproductive subgroup animals (main phase), whereby 5 of the 10 F0 males (used for pairing) per dose group formed the toxicity male subgroups.

Dose selection was based on the results of a 7-day preliminary oral toxicity study in the rat in which dose levels of 100, 300 or 1000 mg/kg/day did not have any overt treatment-related effects on young adult animals (females nulliparous and non-pregnant).

Positive control:

Not included in the study.

Examinations

Parental animals: Observations and examinations:

Clinical observations performed and frequency:
- Clinical signs : At least twice a day (before and after administration)
- Detailed physical examination
and arena observations: Before treatment start and at least once a treatment week.
- Functional Observation Battery: During treatment week 5 (before dosing) on all toxicity subgroup animals (5 parental males/group inclusively)*.
- Body weight, all males: Weekly throughout the study.
Body weight, Toxicity Females: Weekly throughout the study.
Body weight, Repro. Females: Weekly for pre-pairing period; on gestation days 0, 6, 13, 20; on lactation days 1 & 7.
- Food consumption, all males: Weekly for pre-pairing period and for the period after mating.
Food cons., Toxicity Females: Weekly throughout the study.
Food cons., Repro. Females: Weekly for pre-pairing period, during gestation for days 0-6, 6-13, 13-20, during lactation for days 1-4 & 4-7.
- Hematology: During treatment week 5 after functional observation battery*
- Blood (plasma) chemistry: During treatment week 5 after functional observation battery*

* Examinations confined to toxicity subgroup animals are marked above with an asterisk*
and are detailed in the separate endpoint study record "7.5.1 Repeated dose toxicity: oral - Repeat dose tox combined_gavage_rat_HLS_GAH0044"

Explanatory note
This study was conducted to examine both repeated dose toxicity and  reproductive/developmental toxicity as an OECD screening combined study
(OECD 422 test guideline).  Therefore, animals initially entering the study were divided into toxicity subgroup animals (toxicity phase) and reproductive subgroup animals (main phase), whereby 5 of the 10 F0 males (used for pairing) per dose group formed the toxicity male subgroups.

Oestrous cyclicity (parental animals):

Frequency of vaginal oestrus was determined by examination of vaginal smears taken daily from all main phase (i.e. reproductive subgroup) females from the beginning of the treatment period to the day of confirmed copulation.
- Regular: All observed cycles of 4 or 5 days
- Irregular: At least one cycle of 2, 3 or 6 to 10 days
- Acyclic: At least 10 days without oestrus

Sperm parameters (parental animals):

Parameters examined in male parental animals:
- testis weight,
- epididymis weight
- detailed qualitative histopathology examination of the testes taking into account the tubular stages of the spermatogenic cycle. This was to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.

Litter observations:

STANDARDISATION OF LITTERS: Not performed. The study ended on lactation day 7.

LITTER PARAMETERS EXAMINED
- From Day 20 post copulation 3 times a day checks for evidence of parturition, any difficulties and numbers of live and dead offspring.
- Total litter size on day 1 of age and mortality/live litter size on each day until 7 days after littering.
- Sex ratio expressed as percentage males and calculated for total offspring on Day 1 and for live offspring on Days 1 & 7
(No. of male pups in litter/No. of offspring in litter) x 100
- Gestation index (No. of live litters born on day 0/No. of living pregnant females) x 100
- Clinical signs, recorded daily
- Body weight of live pups (on days 1, 4 and 7 after littering) and weight change from days 1-4 and 1-7.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes, see below
WEIGHING OF ORGANS: Yes, see below
HISTOPATHOLOGY: Yes, see below

Terminal sacrifice
- all males and toxicity subgr. females: Killed in Week 6, after completion of the Treatment Week 5 investigations.
- reproductive subgr. females & offspring: Killed on Day 7 post partum.
2 control females which failed to mate or were without viable litter were killed on Day 25 after the last day of pairing.
Gross pathology:
- adult/parental animals: Full macroscopic examination with tissue collection.
In addition, recording of the number of uterine implantation sites in reproductive subgroup females.

Organs Weights:
- main phase and tox. subgr. adults: Adrenals, brain, epididymides, heart, kidneys, liver, lungs & bronchi, ovaries, pituitary, prostate, seminal vesicles
& coagulation gland, spleen, testes, thymus, thyroid with parathyroids, uterus with cervix & oviducts.

Histopathology:
- toxicity subgroups: The following organs were microscopically observed for the control and 1000 mg/kg bw/day groups:
Brain, eyes, Harderian glands, optic nerves, pituitary gland, thyroid with parathyroids, heart, thymus, liver, spleen,
adrenals, kidneys, testes, epididymides, ovaries, lung, trachea, esophagus, stomach, duodenum, jejunum, ileum,
caecum, rectum, colon, Peyer's patch, lymph node (axillary, mesenteric), urinary bladder, uterus (with cervix &
oviducts), vagina, spinal cord, sciatic nerve, skeletal muscle, skin with mammary glands, sternum with marrow,
seminal vesicle & coagulation gland, prostate. In addition, the jejunum, mesenteric lymph nodes and duodenum
were also examined for the 100 and 300 mg/kg bw/day toxicity subgroups and any gross lesions for all adult animals.
- reproductive subgroups: Gross lesions.

Postmortem examinations (offspring):

Pup survivors were killed on Day 7 post partum.
Full macroscopic examination of decedent and surviving pups including assessment of the presence of milk in the stomach, where possible.
(Missing or grossly autolysed or cannibalised pups could not be examined).

Statistics:

As detailed in Endpoint study record "7.5.1 Repeated dose toxicity: oral - Repeat dose tox combined_gavage_rat_HLS_GAH0044"

Reproductive indices:

- Pre-coital interval (pairing days until detection of mating)
- No. of animals mating (evidence of successful copulation, i.e. at least one copulation plug and a sperm positive vaginal smear)
- No. of animals achieving pregnancy
- Percentage mating (No. of animals mating/No. of animals paired) x 100
- Fertility index (No. of animals achieving pregnancy/ No. or animals paired) x 100
- Conception rate (No. of animals achieving pregnancy/No. of animals mated) x 100
- Gestation length (time elapsing between detection of mating and commencement of parturition)
- No. of living pregnant females
- For further reproductive parameters, see also the above section "Litter observations" and section "Offspring viability indices" below.

Offspring viability indices:

- Post-implantation survival index  (Total no. of pups born/Total no. of uterine implantation sites) x 100
- Live birth index (No. of live pups on day 1 after littering/Total no. of pups born) x 100
- Viability index (No. of live pups on day 4 after littering /No. of live pups on day 1 after littering) x 100
- Lactation index (No. of live pups on day 7 after littering /No. of live pups on day 1 after littering) x 100
- For further parameters indicative of the viability of the offspring, see also the above section "Litter observations"

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

only bodyweight and only in males at 1000 mg/kg/day. See figures on bodyweight F0 attached as background material.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

only bodyweight and only in males at 1000 mg/kg/day. See tables on food consumption F0 attached as background material.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

mesenteric lymph node, duodenum, jejunum

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

Slightly extended mean gestation length at 1000 mg/kg/day was considered fortuitous, as mean pup weight was unaffected. See table on mating performance attached as background material.

Details on results (P0)

MORTALITY
One control female was found dead on Day 4 of lactation following an intubation error and her offspring were humanely killed. Two other control females which failed to mate or were without viable litter were killed on Day 25 after their last day of pairing.

BODY WEIGHT AND WEIGHT GAIN
Overall bodyweight gain was low in males receiving 1000 mg/kg/day, but unaffected in males at lower dose levels and females at all dose levels.

REPRODUCTIVE ENDPOINTS
Oestrous cycles, pre-coital interval mating performance, fertility, gestation index and litter size were not affected by treatment. Mean gestation length was slightly extended in animals receiving 1000 mg/kg/day; however this was considered fortuitous as the mean pup weight at all doses of WS400506 was similar to concurrent control.

GROSS PATHOLOGY
Macroscopic findings attributable to treatment with the test material were not evident. The numbers of uterine implantation sites in main phase females recorded on lactation day 7 was unaffected by treatment with WS400506.

ORGAN WEIGHTS
Following lactation, adjusted mean liver weight marginally high and thymus weight low at 1000 mg/kg/day were considered to be fortuitous and toxicologically not significant, as there were no histopathological correlates in main phase males and toxicity subgroup females.

HISTOPATHOLOGY
Lymphatic ectasia in the mesenteric lymph node of males at 1000 mg/kg/day and of nulliparous and non-pregnant toxicity subgroup females at 300 and 1000 mg/kg/day and cystic dilation of the central lymphatic or lacteal in the villi of the duodenum and jejunum in both sexes at 300 and 1000 mg/kg/day were attributed to treatment with the test material. However, only in males at 1000 mg/kg/day these findings were considered to represent an adverse effect, because in this group they may have affected digestive function thus contributing to the low body weight gain. In all groups, the intestinal changes were not associated with any inflammatory or degenerative changes.
Plasmacytosis in the mesenteric lymph node in males of all groups treated with WS400506 and in one toxicity subgroup control female and one female of the 300 mg/kg/day group was considered to be a non specific reactive change of no toxicological importance.

Further details of histopathology findings in adult animals (main phase males and toxicity subgroup females) of the present study are given in separate endpoint study record "7.5.1 Repeated dose toxicity: oral - Repeat dose tox combined_gavage_rat_HLS_GAH0044". Main phase females (reproductive subgroup) killed on lactation day 7 were not histopathologically examined.

Effect levels (P0)

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

on bodyweight or bodyweight gain to day 7, the day of scheduled sacrifice of the pups. See figures on bodyweight F1 attached as background material.

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

No abnormality attributable to parental treatment with the test material was observed.

No abnormality in sex ratio. See table on sex ratio F1 attached as background material.

Effect levels (F1)

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NOEL = highest dose tested. Offspring development up to Day 7 of age.

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

For general toxicity, 300 mg/kg/day WS400506 represented the no-observed-adverse-effect-level (NOAEL) in males, and 1000 mg/kg/day represented the NOAEL in females. For reproductive/developmental toxicity, the no-effect-level (NOEL) was 1000 mg/kg/day in both sexes.