2-Oxetanone, 3-C14-16-alkyl 4-C15-17-alkylidene derivs.

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 July 2001 - 28 January 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to internationally accepted guidelines and under GLP. No CoA attached to study report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-Oxetanone, 3-C14-16-alkyl 4-C15-17-alkylidene derivs.

EC Number:

308-760-8

EC Name:

2-Oxetanone, 3-C14-16-alkyl 4-C15-17-alkylidene derivs.

Cas Number:

98246-87-8

Molecular formula:

C32H60O2 / C34H64O2 / C36H68O2

IUPAC Name:

4-(C15-C17) alkylidene-3-(C14-​16)​ alkyloxetan-2one

Details on test material:

Attached to the oxetane ring there is a C14 or C16 alkyl group in the 3-position and a C15 or C17 alkylidene group in the 4-position. The appropriate EC number for this substance is EC 308-760-8 (EC name: 2-Oxetanone, 3-C14-16-alkyl 4-C15-17-alkylidene derivs.)

Specific details on test material used for the study:

Name: Aquapel 3648 (technical material)
Source: (Chemie Wirtschaftsfoerdemngs-Gesellschaft) Karlstrasse 21 D-60329 Frankfurt Germany
Colour: White
Physical state: Waxy solid
Batch reference: 3LP1456
Purity (% w/w): 90.1% (This is given in:- HERCULES Technical Service Work Report: No. TSWR BLD 00-448 Dated 23 March 2001)
Storage conditions: Ambient temperature in the dark
Stability: December 2004

Test animals

Species:

rat

Strain:

other: Alpk:APfSD (Wistar-derived)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Rodent Breeding Unit, Alderley Park, Macclesfield, Cheshire, UK.
- Age at study initiation: 9- 10 weeks
- Weight at study initiation: females 200-240g, males 300-350g
- Fasting period before study: Not applicable
- Housing: The rats were housed, sexes separately, in multiple rat racks. They were housed up to 5 per cage initially, and two males or two females per cage after they had been assigned to experimental groups and during the pre-mating period: females were housed individually during pregnancy and lactation and provided with bedding material
- Diet (e.g. ad libitum): ad libitum, Diet (CTI)
- Water (e.g. ad libitum): ad libitum, mains water
- Acclimation period: minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: 24 July 2001 - 21 September 2001

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was formulated in Mazola corn oil. Dosing preparations were shaken prior to dosing, and stirred, using a magnetic stirrer, during
dosing as required.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 10, 35 or 100 mg/ml (nominal), actual concentration ± 5%
- Amount of vehicle (if gavage): 1.0ml/100g bodyweight
- Lot/batch no. (if required): Y00790/007
- Purity: no data

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: The pre-coital interval was less than 4 days for all animals i.e. all animals mated within the first oestrus cycle
- Proof of pregnancy: sperm in vaginal smear referred to as [day 0 / day 1] of pregnancy
- Further matings after two unsuccessful attempts: Not applicable, did not occur
- After successful mating each pregnant female was caged (how): females were housed individually during pregnancy and lactation and provided with bedding material
- Any other deviations from standard protocol: none

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples from all dose levels (including controls) were taken at intervals throughout the study and analysed quantitatively for Aquapel 364. The homogeneity of Aquapel 364 in the dosing formulations was determined by analysing samples from the low and high dose levels. The chemical stability of Aquapel 364 in corn oil was determined at concentrations of 1 and 100mg/ml in a preliminary study (RRO905). The method of analysis used for the quantitative determination of Aquapel 364 in Mazola Corn Oil is HPLC.

Duration of treatment / exposure:

Exposure period: At least 28 days
Premating exposure period (males): Males were dosed two weeks prior to mating continuing throughout day 29 or 30.
Premating exposure period (females): Females were dosed two weeks prior to mating, during mating, throughout pregnancy and until 4 days post partum (day 4 of lactation).

Frequency of treatment:

Once per day

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (control), 100, 350 or 1000 mg/kg bw/day
Basis:

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The dose levels selected for this study were based on the results of a dose range finding study (RR0905) in the same strain of rat carried out in this Laboratory and included an anticipated no-observed effect level and a level where Aquapel 364 was expected to produce toxicity in the parents.
- Rationale for animal assignment (if not random): random
- Other: None

Positive control:

Not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the start of the study, all rats were examined to ensure that they were normal. During the study all rats were observed daily and changes in clinical condition were recorded. Rats requiring euthanasia were killed and subjected to an examination post mortem.

BODY WEIGHT: Yes
- Time schedule for examinations: Daily

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

OTHER:
Functional observational battery
Detailed clinical assessments (during which each rat was removed from its cage and physically examined for changes in general health status) and quantitative assessments of muscle weakness (fore- and hindlimb grip strength) and sensory perception (tail-flick test) were made in week 4 of the study for five animals per sex per group. The observations were made by one observer who was 'blind' with respect to the animal's treatment, and recorded on a computer system by personnel not directly involved in the clinical observations. The presence and/or absence of all listed observations was recorded and the degree of condition noted (slight, moderate or extreme) where appropriate.
The clinical observations included, but were not restricted to, the following list of measures:
a) Assessment of signs of autonomic functions, e.g. lachrymation, salivation, piloerection, exophthalmus, urinary incontinence, diarrhoea, pupillary response to light and ptosis.
b) Description, incidence and severity of any convulsions, tremors or abnormal motor movements, both in the home cage and standard (open) arena.
c) Ranking by severity, the subject's reactivity to general stimuli such as removal from the cage or handling.
d) Ranking by severity, the subject's arousal level or state of alertness during observations of the unperturbed subject in the standard (open) arena.
e) Descriptions and incidence of posture and gait abnormalities observed in the home cage and in the standard (open) arena.
f) Assessment of audition by response to a sudden sound (e.g. clicking fingers).
g) Description and incidence of any unusual or abnormal behaviours, excessive or repetitive actions (stereotypes), emaciation, dehydration, hypotonia or hypertonia, altered fur appearance, red or crusty deposits around the eyes, nose or mouth, and any other observations that may facilitate interpretation of the data.

Motor activity
Locomotor activity was monitored by an automated activity recording apparatus. Five animals per sex per group were tested in week 4 of the study. Each observation period was divided into ten scans of five minutes duration. Treatment groups were counter balanced across test times and across devices. Motor activity was assessed in a separate room to minimise disturbances.

Urine clinical chemistry
The following parameters were measured, or determined semi-quantitatively on urine samples collected from all animals (10/sex/group) during week 4.
colour, volume, specific gravity, glucose, bilirubin, appearance, pH, protein, ketones, blood

Haematology
The following measurements were made or determined on samples collected from all animals (10/sex/group) using EDTA as an anticoagulant:
red blood cell count, haemoglobin, haematocrit, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, total white blood cell count, differential count, platelet count.
Measurements of potential clotting (prothrombin time and activated partial thromboplastin time with kaolin) were made on samples of blood collected into tubes containing trisodium citrate as an anticoagulant. Blood cell morphology, including a differential white blood cell count was assessed by automated methods for all animals. A blood film was prepared from all animals and examined where the results from the automated analysis suggested this was necessary.

Blood clinical chemistry
The following measurements were made on the plasma from blood samples collected from all animals (10/sex/group) into tubes containing lithium heparin as an anticoagulant:
urea, creatinine, glucose, albumin, total protein, albumin/globulin ratio, cholesterol, triglycerides, sodium, potassium, chloride, calcium, total bilirubin, phosphorus (as phosphate), gamma-glutamyl transferase activity, creatine kinase activity, alkaline phosphatase activity, aspartate arninotransferase activity, alanine arninotransferase activity

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

No data

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
% of pups live born
% of pups surviving to day 5
Bodyweights - Individual pup bodyweights were recorded within 24 hours of birth (day 1) and on day 5 post partum. Since pups were not individually identified, data were recorded by sex and litter.

GROSS EXAMINATION OF DEAD PUPS:
no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals on day 29/30 of the study.
- Maternal animals: All surviving animals day 4 of lactation.

GROSS NECROPSY
- All animals were subjected to a full exarnination post mortem. A full exarnination post mortem is defined in this Laboratory as one which involves an external observation and a detailed examination of all cranial, thoracic and abdominal organs and structures. For all females (10/sex/group) which had been mated the number of implantation sites was recorded and the number of corpora lutea counted.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

From all animals (10/sex/group) surviving to scheduled termination, the following organs were removed, trimmed free of extraneous tissue and weighed (Paired organs were weighed together):
adrenal glands, brain, epididymides, heart, kidneys, liver, spleen, thymus, testes, ovaries

At necropsy the full range of tissues, see below was taken on all animals (i.e. 10/sex/group) but the tissues from odd numbered animals were stored for possible future examination. Removal and storage of tissues.
abnormal tissue, adrenal gland, bone marrow (femoral bone), brain (cerebrum, cerebellum, pons), caecum, cervix, colon, duodenum, epididymis, heart
ileum, jejunum, kidney, liver, lung, lymph nodes (cervical and mesenteric), mammary gland (females only), ovary, Peyer's patches, pituitary gland, prostate gland, rectum, sciatic nerve, seminal vesicle including coagulating gland, spinal cord, spleen, stomach, testis, thymus, thyroid gland, trachea, urinary bladder, uterus, vagina

Tissue processing and examination
All submitted tissues from all animals found dead or killed intercurrently and for 5 males and 5 females (even numbered animals) per group at scheduled termination were trimmed, embedded in paraffin wax and 5pm sections cut, stained with haematoxylin and eosin and examined by light microscopy. Compound-related changes were observed in some tissues, listed below, in the high dose group and therefore relevant tissues from the mid and low dose groups (even numbered animals) were trimmed, embedded in paraffin wax and 5 p sections cut, stained with haematoxylin and eosin and examined by light microscopy. Tissues processed from the mid and low dose groups:
Males: Kidney, liver, mesenteric lymph node, spleen and heart.
Females: Adrenals, cervix, heart, jejunum, kidney, liver, cervical and mesenteric lymph nodes, Peyer's patch, spleen, thymus and uterus.

Postmortem examinations (offspring):

None

Statistics:

All analyses were carried out in SAS (1999). For Fisher's Exact Tests the proportion in each treated group was compared to the control group proportion. Analyses of variance and covariance allowed for the replicate structure of the study design.

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

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One control female was killed for humane reasons following paturition, no further mortalities. There were no compound related clinical chenges.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- Males days 11-29
Bodyweights in males given 1000mg/kg/day were slightly lower than controls during the last week of the study. There were no other compound-related effects on bodyweights in males.
- Females pre-mating
There were no compound-related effects on bodyweights in females.
- Females during gestation
Group mean female bodyweights during gestation were similar to control values.
- Females during lactation
During the lactation period there were no effects that were attributable to treatment with Aquapel 364. Daily bodyweights were recorded prior to littering for animals number 59 71 and 79 and since littering was not completed during the working day, bodyweightspost partum were not recorded on day 1 for these animals. The pre-littering bodyweights were excluded from the data analysis.

- Males pre-mating
Food consumption in all treated male groups was similar to controls during the pre-mating period.
- Females pre-mating
There were no adverse effects on food consumption during the pre-mating period.
- Females during gestation
Group mean food consumption during gestation was similar to control values.
- Females during lactation
Food consumption in females during lactation showed some variation between the groups but there was no clear indication of a compound-related effect.
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Mating
The pre-coital interval was less than 4 days for all animals i.e. all animals mated within the first oestrus cycle.
- Fertility
There were no compound-related effects.
- Gestation
There was no effect on the length of gestation.
- Reproductive performance
In females, however, there was an inflammation of the ovaries, uterus and cervix. Along with the inflammations, the ratio of corpora lutea to implantation sites was reduced. This pre-implantation loss, was observed in all treated groups, but the effect was not dose-related. . The number of corpora lutea was slightly higher in females given 1000 mg/kg b.w./day than in the control, 100 and 350 mg/kg b.w./day dose groups. However, the ratio of implantation sites to corpora lutea was similarly reduced in all treated groups when compared to controls. There were no effects on post-implantation loss. The litter size was smaller in all treated groups with the effect being most marked at the low- and mid-dose where the number of corpora lutea and hence the number of implantation sites, was lower than at the high-dose (see table 3) Individual animal data (see Table 4) seems to confirm a correlation between inflammation of the uterus and cervix and the observed pre-implantation losses. Five animals per group were subjected to micropathology. To analyse the data, a threshold for significant pre-implantation losses was set to five pre-implantation losses, since it is the mean + S.D. for pre-implantation losses in the control group.
- Whole litter losses
There was one whole litter loss, in the 100mg/kg/day group. There were no whole litter losses in the higher dose groups and therefore this is not a compound-related effect.
- Pups live born
There were no compound-related effects.
- Litter size
There were no compound-related effects.

ORGAN WEIGHTS (PARENTAL ANIMALS) (table 1)
There was a dose-related elevation in spleen weights at all dose levels in males and females. In females there was an elevation in kidney and liver weights at all dose levels and an elevation in ovary weights in animals given 1000 or 350 mg Aquapel 364kg/day

GROSS PATHOLOGY (PARENTAL ANIMALS)
All animals survived to the scheduled termination except for one control female which was killed on humane grounds. Implantation sites were absent from one(350 mg Aquapel 364kg) or two (1000 mg Aquapel 364kg) females in the two higher dose groups whereas all controls and females receiving 100 mg Aquapel 364kg had implantation sites visible at necropsy. Enlargement of the spleen and a number of lymph nodes were observed in females in all treatment groups. The affected lymph nodes were as follows: hepatic (100, 350 and 1000mg Aquapel 364/kg), mesenteric (350, 1000mgkg), pancreatic (1000 mgkg), renal (100, 350 and 1000 mg/kg), thymic (1000 mg/kg). In most cases the incidence of the observation was related to dose level. No change which could be related to treatment was observed in males.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Treatment related inflammatory changes including histiocytosis were observed in a variety of tissues of animals at all dose levels. Overall the incidence, severity and distribution of the changes were greater in females. In females the liver and mesenteric lymph node were most consistently affected i.e. in 100% of females at all dose levels. The other tissues affected (listed in order of decreasing frequency with which they were involved) were cervix, kidney, Peyer's patch, spleen, uterus, hepatic lymph node, cervical lymph node and jejunum. Isolated incidences of changes similar to those seen in other tissues were also seen in the adrenal gland, ileum, pancreatic, renal and thymic lymph nodes and thymus. In males the range of tissues affected was smaller. Changes similar to those observed in females were present in the mesenteric lymph node and liver at all dose levels. Other tissues affected were kidney in animals receiving 350 and 1000 mg Aquapel 364/kg and spleen in animals receiving 1000 mg Aquapel 364kg only. No treatment related changes were observed in the male reproductive organs. Increased extramedullary haemopoiesis in the spleen was also observed in all treated females at all dose levels and in the majority of males receiving 350 and 1000 mg Aquapel 364/kg. In addition there was a small increase in incidence of minor changes in the heart (degenerative cardiomyopathy and mononuclear cell infiltration) in treated animals of both sex compared with controls although this was not closely related to dose level. In all tissues the inflammatory changes consisted predominantly of mononuclear cell infiltration often with focal accumulation of macrophages. The latter feature was particularly prominent in the liver of females where large multinucleate giant cells were also frequently observed. Similar accumulation of macrophages was observed as histiocytosis in the spleen, lymph nodes and Peyer's patches of a number of animals at the affected dose levels. In affected lymph nodes histiocyte accumulation was most prominent in the paracortex and medullary cords. In the heart inflammatory cell infiltration was sometimes associated with degenerative changes; in these cases the term degenerative cardiomyopathy was used. In male livers the change consisted of a few very small foci of mononuclear cells without macrophage accumulation. The term "mononuclear cell infiltration" was used to describe this to differentiate it from the considerably more diffuse change (diffuse mononuclear cell hepatitis) seen in females. There was no consistent dose response; especially in females where the incidence and severity of most changes was not dose related. In males a dose response was a little more evident with changes at the lowest dose level (100mg Aquapel 364/kg) restricted to the liver and mesenteric lymph node.

OTHER FINDINGS (PARENTAL ANIMALS)
- Urine clinical chemistry
There was a slight reduction in urine pH in males given 1000mg Aquapel 364 kg/day. This change is slight and is considered to be unrelated to administration of Aquapel 364. All other urine parameters in males and females were similar to controls.
- Haematology (table 2)
There were no effects on red blood cell parameters in either sex. The decreased platelet count seen in females receiving 350 mg/kg/day was not sustained at 1000 mgkglday so was considered not to be treatment-related. A dose-responsive increase was seen for white blood cell, lymphocyte, large unstained cells and monocyte counts in both sexes at all doses, although in males, the lymphocyte and monocyte counts were only statistically significantly raised at 1000mg/kg/day. In females there was a dose related increase in basophil numbers, but whilst all dose groups of males had increased basophil numbers compared with controls this increase was only statistically significant at 100 mg/kg/day. The automated haematology analyser indicated alerts for atypical white blood cells in individual animals, which triggered the manual examination of blood smears. The manual verification of blood films demonstrated the presence of immature myeloid cells and atypical monocytes. Only a single male at each of 100 and 350 mg/kg/day required evaluation, but all dosed females except for one female at each of 100 and 350 mg/kg/day required evaluation. In females there was a dose-related increase in immature myeloid cells, but the incidence of atypical monocytes did not vary across the groups. Prothrombin times were slightly lower than control values in both sexes given 1000 mg Aquapel 364kg/day and in males given 350 mg/kg/day. These changes were considered not to be adverse and are of no toxicological significance.
- Blood clinical chemistry
In females, increases were seen for albumin and total protein in animals receiving 350 and 1000 mg/kg/day, leading to a decreased albumin globulin ratio in these groups. Increases were also seen for plasma calcium values at these doses in this sex.
Following exclusion of high values for females 60 and 70 in the intermediate dose groups, total bilirubin was increased in both sexes receiving 1000 mg/kg/day. Increases were also seen for cholesterol in both sexes receiving 1000 mgkglday and in females receiving 350 mg/kg/day. Enzyme activity was increased for alkaline phosphatase in females receiving 350 and 1000 mg/kg/day and males receiving 1000 mg/kg/day, for alanine aminotransferase in males receiving 350 and 1000 mg/kg/day and females receiving 1000 mg/kg/day and for aspartate aminotransferase in both sexes receiving 350 and 1000 mg/kg/day. Generally these increases showed a dose response. The increased gamma glutamyl transferase activity in females receiving 1000 mgkglday was small in magnitude and considered not to be of any biological or toxicological significance.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

< 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

histopathology: non-neoplastic

Remarks on result:

not determinable

Remarks:

no NOAEL identified

2

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

100 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: LOAEL based on inflammatory changes in a variety of tissues in both genders.

3

Dose descriptor:

NOAEL

Remarks:

reproductive performance

Effect level:

< 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

reproductive performance

Remarks on result:

not determinable

Remarks:

no NOAEL identified

4

Dose descriptor:

LOAEL

Remarks:

reproductive performance

Effect level:

100 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: LOAEL based on reduced proportion of implantation sites compared to the numbers of corpora lutea.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects seen

Remarks on result:

other: No effects at highest dose 1000 mg/kg bw/day

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: Body weights and Organ weights (only organs with significant changes are listed) 

	Dose [mg/kg b.w./day]
	0	100	350	1000
TERMINAL B.W.
MALES
Mean	467.9	463.5	459.2	446.3
S.D.	30.7	31.4	33.1	33.1
FEMALES
Mean	331.2	338.9	330.0	319.8
S.D.	11.3	13.1	25.1	26.2
LIVER
MALES
MEAN	18.4	18.2	18.5	18.5
S.D.	1.5	1.6	1.8	1.5
Organ Weight Adjusted	18.1	18.0	18.5	19.0*
For Bodyweight
FEMALES
MEAN	13.4 15.7*	17.0**	16.8**
S.D.	1.0	1.1	2.8	2.4
Organ Weight Adjusted	13.3	14.9**	17.0**	17.6**
For Bodyweight
KIDNEYS
MALES
Mean	3.21	3.08	3.20	3.20
S.D.	0.23	0.27	0.23	0.38
Organ Weight Adjusted	3.16	3.05	3.20	3.28
For Bodyweight
FEMALES
Mean	1.94	2.18**	2.10*	2.09*
S.D.	0.11	0.17	0.20	0.13
Organ Weight Adjusted	1.93	2.13**	2.10**	2.15**
for Bodyweight
SPLEEN
MALES
Mean	0.966	1.096*	1.184**	1.200**
S.D.	0.100	0.132	0.156	0.116
Organ Weight Adjusted	0.943	1.084**	1.184**	1.235**
for Bodyweight
FEMALES
Mean	0.708	1.110**	1.403**	1.405**
S.D.	0.085	0.203	0.227	0.253
Organ Weight Adjusted	1.93	2.13**	2.10**	2.15**
for Bodyweight
OVARIES
Mean	0.107	0.118	0.130**	0.127**
S.D.	0.015	0.014	0.015	0.012
Organ Weight Adjusted	0.107	0.116	0.130**	0.129**
for Bodyweight

** Statistically significant difference from the control group mean at the 1% level (Student's t-test, two sided)

 

Table 2: Haematology(in cell counts x10^-9/L) (only parameter with significant changes are listed)

			Dose in mg/kg b.m./day
Gender	Parameter	control	100	350	1000
♂ day 30	White blood cells	7.56	9.02*	9.50**	10.29**
	Neutrophil	1.46	2.34**	2.36**	2.10**
	Lymphocyte	5.71	6.02	6.49	7.41**
	Monocyt	0.123	0.174	0.192	0.290**
	Large unstained cells	0.092	0.238**	0.246**	0.275**
♀ day 5 post partum	White blood cells	5.41	8.32**	9.96**	11.40**
	Lymphocyte	53.23	5.29**	6.73**	7.55**
	Monocyt	0.066	0.332**	0.395**	0.536**
	Basophil	0.012	0.133**	0.152**	0.160**
	Large unstained cells	0.063	0.609**	0.697**	0.922**

** Statistically significant difference from the control group mean at the 1% level (Student's t-test, two sided)

Table 3: Corpora lutea and implantation sites and incidences of pre-impantation losses

	Dose level of Aquapel 364 [mg/kg b.w./day]
	0 (control)	100	350	1000
No. of corpora lutea
MEAN	13.9	14.1	12.2	16.0
S.D.	2.4	2.9	4.5	3.1
N	9	10	9	8
No. of implantations
MEAN	11.2	9.3	9.1	11.3
S.D.	3.6	4.6	3.1	3.4
N	9	9	8	8
Pre-implantation loss
Prop. of implants affected	24/125	54/141**	36/110*	47/128**
Percentage
MEAN	19.8	39.4	28.1	35.6
S.D.	19.8	27.8	27.1	29.9
N	9	10	9	8
No. of live + dead pups
MEAN	11.2	7.0*	7.1	9.1
S.D.	3.7	4.5	4.7	4.9
N	10	10	9	8

Table 4: Pre-implantation losses and inflammations of uterus and/or cervix in animals with micropathological examination (five animals per group were examined)

Dose	Animal no	Cervix	Uterus	Corpora lutea		Implantations		Pre-implantation. losses^a
				Left	Right	Left	Right
0	42	0	0	4	10	4	8	2
	44	0	0	9	7	3	6	7
	46	0	0	3	8	3	5	3
	48	other		6	10	6	9	1
	50	0	0	6	6	5	0	7
100	52	0	0	6	5	0	5	6
	54	1	1	6	7	2	3	8
	56	2	1	10	8	2	2	14
	58	0	0	3	9	3	9	0
	60	other	other	5	4	0	3	6
350	62	1	1	4	9	2	4	7
	64	2	1	6	8	2	4	8
	66	2	2	7	5	3	4	5
	68	1	1	small	small	1	0	0
	70	0	0	small	small	0	0	0
1000	72	1	1	8	8	8	5	3
	74	1	1	5	8	1	1	11
	76	1	1	7	7	4	7	3
	78	1	1	small	small	0	0	0
	80	1	0	5	10	5	10	0

 ^a bold letters indicate losses above threshold of 5 (mean+S.D. of control), Italic, red fonts indicate significant losses with inflammation of uterus and/or cervix

Cervix/Uterus

 “severity of the lesions”

1 minimal

2 slight

3 moderate

4 marked

Applicant's summary and conclusion

Conclusions:

Oral administration of 1000 mg/kg/day Aquapel 364 for at least 28 days in males and throughout pregnancy to day 5 lactation in females produced inflammatory changes in a number of tissues together with increases in specified organ weights, increases in white blood cell counts and splenic extramedullary haemopoiesis. In addition there was a mild perturbation of some clinical chemistry parameters and a decreased proportion of implantation sites compared to the observed number of corpora lutea. There were no adverse effects on pup parameters.
The majority of changes were seen at decreased severity and/or incidence in animals given 350 or 100 mg Aquapel 364/kg/day. However some of the histopathological changes and the reduced ratio of implantation sites to corpora lutea showed no evidence of a dose-response relationship.
A NOAEL was not achieved in this study.

Executive summary:

A 28 day repeated dose combined with repoductive/developmental screening study was performed according to OECD 422 and under GLP.

Study design

Groups of 10 male and 10 female (parents) Alpk:APfSD (Wistar-derived) rats were dosed with 0 (control), 100, 350 or 1000mg/kg/day Aquapel 364 in Mazola Corn Oil. After 2 weeks, the animals were mated and allowed to rear the ensuing litters to 5 dayspost parturn. Males were dosed during mating and to termination on day 29/30 of the study. Females were dosed during mating, gestation and to day 4 of lactation. Litters were weighed and examined on days 1 and 5 postpartum.

In addition the following were carried out on all animals. Clinical observations, bodyweights and food consumption were measured throughout the study. Urine samples were collected during week 4 for the assessment of urine clinical pathology. Detailed clinical observations, including quantitative assessments of sensory perception and muscle weakness, and assessment of motor activity were performed on day 28 on 5 animals /sex/group. At the end of the scheduled period, the animals were killed and subjected to an examination post mortern. Cardiac blood samples were taken for clinical pathology, selected organs were weighed, corpora lutea were counted in pregnant females and specified tissues were taken for

subsequent histopathology examination.

Results

The analyses of stability, achieved concentration and homogeneity of the test substance in the dosing preparations were satisfactory. At a dose level of 1000 mg Aquapel 364/kg/day in adults there were no mortalities, no clinical changes and no adverse effects on bodyweight or food consumption and no neurotoxic effects. There were no effects on breeding parameters with the exception of a reduced proportion of implantation sites compared to the numbers of corpora lutea. In both sexes there was an increase in total white blood cell count due to increases in large unstained cells, lymphocytes and monocytes and in females there was an increase in basophils. There were increases in total bilirubin and cholesterol in both sexes and in albumin, total protein and plasma calcium in females. In addition there were increases in some enzymes in both sexes. In both sexes there was an increase in spleen weight and in liver, kidney and ovary weights in females. Macroscopic enlargement of the spleen and lymph nodes was seen in females. Microscopically, inflammatory changes were seen in a variety of tissues in both sexes but the

overall incidence, severity and distribution was greater in females. There were no effects on pups. At a dose level of 350mg Aquapel 364/kg/day in adults there was a reduced proportion of implantation sites compared to the numbers of corpora lutea. In both sexes there was an increase in total white blood cell count due to increases in large unstained cells, lymphocytes and monocytes and in females there was an increase in basophils. There was an increase in cholesterol, albumin, total protein and plasma calcium in females. In both sexes there was an increase in spleen weight and in liver, kidney and ovary weights in females.

Macroscopic enlargement of the spleen and lymph nodes was seen in females. Microscopically, inflammatory changes were seen in a variety of tissues in both sexes but the overall incidence, severity and distribution was greater in females. At a dose level of 100mg Aquapel 364kg/day in adults there was a reduced proportion of implantation sites compared to the numbers of corpora lutea. In both sexes there was an increase in total white blood cell count due to increases in large unstained cells, lymphocytes and monocytes and in females there was an increase in basophils. There was an increase in spleen weight and in liver and kidney weights in females. Macroscopic enlargement of the spleen and lymph nodes was seen in females. Microscopically, inflammatory changes were seen in a variety of tissues in both sexes but the overall incidence, severity and distribution was greater in females.

Conclusion

Oral administration of 1000 mg/kg/day Aquapel 364 for at least 28 days in males and throughout pregnancy to day 5 lactation in females produced inflammatory changes in a number of tissues together with increases in specified organ weights, increases in white blood cell counts and splenic extramedullary haemopoiesis. In addition there was a mild perturbation of some clinical chemistry parameters and a decreased proportion of implantation sites compared to the observed number of corpora lutea. There were no adverse effects on pup parameters. The majority of changes were seen at decreased severity and/or incidence in animals given 350 or 100mg Aquapel 364/kg/day. However some of the histopathological changes and the reduced ratio of implantation sites to corpora lutea showed no evidence of a dose-response relationship.

A NOAEL was not achieved in this study. A LOAEL for reproductive performance was defined at 100 mg/kg bw based on reduced proportion of implantation sites compared to the numbers of corpora lutea.