Amidinourea phosphate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 November 2017 to 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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 (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Amidinourea phosphate

EC Number:

241-659-6

EC Name:

Amidinourea phosphate

Cas Number:

17675-60-4

Molecular formula:

C2H6N4O.xH3O4P

IUPAC Name:

diaminomethylideneurea;phosphoric acid

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Name: Guanylurea phosphate (GUP)
Product: Amidinourea phosphate
CAS No.: 17675-60-4
Batch No.: 16VL8189
Physical State: solid, powder
Molecular Weight: 200.09 g/mol
Colour: white
pH value: 5
Purity: 98 % (was not taken into account)
Expiry Date: 02 April 2018

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar Crl: WI(Han)

Details on species / strain selection:

A commonly used species and strain of rat employed in this type of study globally.

Sex:

male/female

Details on test animals or test system and environmental conditions:

1. Full barrier in an air-conditioned room
2. Temperature: 22 ± 3 °C
3. Relative humidity: 55 ± 10 %
4. Artificial light, sequence being 12 hours light, 12 hours dark
5. Air change: 10 x / hour
6. Free access to Altromin 1324 maintenance diet for rats and mice
7. Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
8. Animals were housed in groups of 5 animals / sex / cage in type IV polysulphone cages or in double decker IVC cages during the premating period for both males and females and during post-mating period for males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages and males were returned to their original cage. In each cage Altromin saw fibre was used as bedding.
9. Nesting material were provided latest on GD 18 for all mated females
10. Adequate acclimatisation period (at least 5 days) under laboratory conditions

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage, this is a potential route of incidential exposure in man.

Vehicle:

water

Details on oral exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days in females, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration analysis of formulation samples was determined at three concentrations, 20 mg/mL, 60 mg/mL and 200 mg/mL in study weeks 1, 3, 5 and in the last week of the study. The mean recoveries observed for the LD dose group was between 89.54 % and 94.31 % of the nominal value, between
92.24% and 97.51% for the MD dose group and between 87.16 % and 97.28 % of the nominal value for HD dose group. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 91.0 %, 94.1 %, and 94.0 % of the nominal concentration, respectively.

Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 15 %.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days in females, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Control: application volume for all groups was 5 mL/kg body weight

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Low Dose: application volume for all groups was 5 mL/kg body weight

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Medium Dose: application volume for all groups was 5 mL/kg body weight

Doses / concentrations 4

Dose / conc.:

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

Remarks:

High Dose: application volume for all groups was 5 mL/kg body weight

No. of animals per sex per dose:

10 males and 10 females.

Control animals:

yes, concurrent vehicle

Details on study design:

The study followed the OECD 422 Guideline.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

Clinical Observations
General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

Once before the first exposure, and at least once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded. 

Body Weight and Food Consumption
The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post¬partum), on PND 4, PND 9 and PND 13 along with pups. All animals were weighed directly before termination.

Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating p eriod in males and females and the post-mating period in males.

Functional Observations
Multiple detailed behavioural observations were made in the week before the first treatment and dur ing the last week of the treatment in 5 randomly selected males and during the last week of the lac tation period in 5 randomly selected females (only lactating females were evaluated) of each group outside the home cage using a functional observational battery of tests.

Haematology
Haematological parameters were examined in 5 randomly selected males and females (only lactat ing females were evaluated) from each group at the end of the treatment prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in EDTA-coa ted tubes.
Blood Coagulation

Coagulation parameters from 5 randomly selected males and females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in citrate tubes.

Clinical Biochemistry
Parameters of clinical biochemistry from 5 randomly selected males and females (only lactating femal es were evaluated) of each group were examined at the end of the treatment prior to or as part of t he sacrifice of the animals (11.20). Blood from the abdominal aorta of the animals was collected i n serum separator tubes. From 2 preferably female pups/litter on day 4 after birth from all dams a nd 2 pups/litter at termination on day 13 and from all adult males at termination, blood samples we re collected from the defined site in serum separator tubes. All blood samples were stored under ap propriate conditions. Blood samples from the day 13 pups and the adult males were assessed for seru m levels for thyroid hormones (T4). Pup blood was pooled by litter for thyroid hormone analysis. Fu rther assessment of T4 in blood samples from the dams and day 4 pups was not deemed necessary based on the fact that no major histopathological findings were observed in thyroid/parathyroid gland of selected male and female adult animals and T4 hormone levels of males and day 13 pups Two pups per litter were sacrificed on day 4 after birth and blood samples were taken for possible serum hormone assessments. No pups were eliminated as litter size droped below 8 pups. As there was only one pup available above a litter size of 8, only one pup was sacrificed.

Urinalysis
A urinalysis was performed with samples from 5 randomly selected males and females (only lactating females were evaluated) prior to or as part of the sacrifice of the animals. Additionally, urine colour/ appearance were recorded.

Estrous cyclicity (parental animals)
Estrous cycles were monitored before treatment initiation to select for the study females with regular estrus cyclicity. Vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating.

Sperm parameters (parental animals)
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.

Litter observations
The duration of gestation was recorded and is calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after the delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of littering (PND 0) and on PND 4 and PND 13. Live pups were identified by tattooing. In addition to the observations of the parent animals, any abnormal behaviour of the offspring were recorded.

The anogenital distance (AGD) of each pup was measured on PND 0. Pup body weight measured on PND 0 was converted to cube root and used for the calculation of relative AGD (Relative AGD = A GD/Cube root of pup weight). The number of nipples/areolae in male pups was counted on PND 12.

Sacrifice and pathology:

Pathology

All males were sacrificed any time after the completion of the mating period (after a minimum dosing period of 28 days) and females were sacrificed on the respective PND 13 by using anesthesia (ket amine/xylazine). All surviving pups were killed by cervical dislocation on PND 13.
Vaginal smears were examined on the day of necropsy to determine the stage of estrous cycle.
Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.
All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coag ulating glands as a whole), the thyroid/parathyroid glands and all organs showing macroscopic lesio ns of all adult animals were preserved in 4 % neutral-buffered formaldehyde, except for testes and epididymides which were preserved in modified Davidson's Solution for 24 hours and then transferred to 70 % ethanol.

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Organ Weights

The wet weight of the organs (Table 8) of 5 randomly selected male and female animals (only lac tating females were evaluated) from each group was recorded as soon as possible. Paired organs were weighed together. Organ weights of the animal euthanised for animal welfare reasons were not recorded.
Reproductive organs (testes, epididymides, prostate with seminal vesicles and coagulating glands, uterus with cervix and ovaries) were weighed from all animals.
Thyroid/parathyroid glands from 1 pup/sex/litter/group (if possible) (sacrificed on PND 13) and from al l adult males and females were preserved. Weight of thyroid/parathyroid glands was measured after f ixation.

Histopathology

A full histopathology was carried out on the preserved organs and tissues of all animals of the control and high dose groups which were sacrificed at the end of the treatment period. Thyroid/parathyroid glands from pups and from the adult animals were examined. Thyroid/parathyroid glands from pups and from the remaining non-selected adult animals were not examined as no major histopathological findings were observed in thyroid/parathyroid gland of selected male and female adult animals and T4 hormone levels of males and day 13 pups.
A full histopathology was carried out on the preserved organs and tissues of all animals which died during the study.

Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed, embedded into paraffin, cut at an approximated thi ckness of 2-4 um and stained with hematoxylin and examined in control and HD animals and in non-pregnant female animals of the LD and MD animals. Testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland) were also examined in the mating partners of the non-pregnant female LD and MD animals.
These examinations were extended to animals of all other dosage groups for treatment-related changes in the stomach that are observed in the high dose group.
Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for histopathology). The study phases from test site 1 and 2 were performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1] (Status as of 01 December 2012) [10]. Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP's of the test sites

Postmortem examinations (offspring)

Pathology

All surviving pups were killed by cervical dislocation on PND 13.
Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.
All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

Organ Weights

Thyroid/parathyroid glands from 1 pup/sex/litter/group (if possible) (sacrificed on PND 13) and from all adult males and females were preserved. Weight of thyroid/parathyroid glands was measured after fixation.

Histopathology

Thyroid/parathyroid glands from pups and from the adult animals were examined. Thyroid/parathyroid glands from pups and from the remaining non-selected adult animals were not examined as no maj or histopathological findings were observed in thyroid/parathyroid gland of selected male and female adult animals and T4 hormone levels of males and day 13 pups.

Other examinations:

Reproductive indices: Copulation and fertility indices were calculated (see results for full details).

Offspring viability indices: Assessed throughout the post-partum period.

Statistics:

The findings of this study were evaluated in terms of the observed effects, the necropsy and the microscopic findings.

The gestation length, pre-coital interval, the number of live births and post-implantation loss, the number of pups with grossly visible abnormalities, the number of implantations, corpora lutea, AGD, litter size and litter weights were summarized in tabular form.

Parameters like body weight gain and food consumption were calculated as the difference in weight measured from one week to the next. The relative organ weights were calculated in relation to the body weight (measured at necropsy) and were presented as percentage.

All results were reported in a tabular form (summarised in mean or summary tables and/or listed in individual data tables).

A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non -parametric Kruskal-Wallis Test and a post-hoc Dunn's Test, based on the results of homogeneity and normality tests. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.1.3 software (p<0.05 was considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

On several study days, all animals of the LD, MD and HD group were observed moving the bedding with the nose or/and salivating immediately after administration. The clinical signs salivation and moving the bedding in males and females were observed immediately after the dose administration and therefore were a sign of a local reaction to the test item rather than a systemic adverse effect and has no toxicological relevance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female animal from the LD group (no. 51) was sacrificed moribund on PMD 10 and showed moderately reduced spontaneous activity, moderate piloerection and hunched posture. No other mortalities were recorded at the other dose levels.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The test item had no effect on body weight development of the male animals in this study. Body weights of male and female animals were in the normal range of variation for this strain throughout the treatment of this study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The test item had no effect on food consumption in this study. Mean daily food intake of male and female animals was in the normal range of variation throughout the treatment period of this study.

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

The test item had no toxicologically relevant effect on haematology parameters analysed at the end of the treatment period of this study. However, the mean MCH value from the LD female group is slightly but significantly higher than the control. This value is in the range (16.6 - 21.9 pg / Ery) of the biological variation of this strain, sex and age, does not follow any dose dependency and is therefore not considered to be test item related.

The test item had no toxicologically relevant effect on blood coagulation parameters analysed at the end of the treatment period of this study.

All haematological parameters were within the normal range of variation and differences are not assumed to be biologically relevant.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The test item had no statistical significant or toxicologically relevant effect on parameters of clinical bio chemistry analysed at the end of the treatment period of this study.

The slightly higher mean ASAT value in the female LD group (114.64 U/L) compared to the control (87.82 U/L) was due to one single dam no. 57 with a high individual value (154.1 U/L). The slightly higher mean ASAT value in the male HD group (105.32 U/L) compared to the control (92.04 U/L) wa s due to one male no. 32, which has a high individual value (135.4 U/L). However, parameter was wit hin the normal range of variation for this strain and is not assumed to be biologically relevant.

The mean potassium values of the female animals follow a dose-dependent decrease from the control group to the HD group. However, parameter was within the normal range of variation for this strain and is not assumed to be biologically relevant.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The test item had no toxicologically relevant effect on urinary parameters analysed at the end of th e treatment period of this study. However female no. 49 of the control group had a high number of erythrocytes (approximately 250 cells/µL) and protein (500 mg/dl) in the urine. The erythrocytes might be from a blood contamination during obtaining the urine with a syringe through the bladder wall. Despite, findings were only observed in the control group and therefore not considered to be test item related

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

See clinical signs described above.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The test item had no toxicologically relevant effect on organ weights determined at the end of the treatment period of this study.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A small number of male rats had some findings, but these findings were not correlated to any histological changes and therefore were not considered to be test item related.

A similar situation was recorded for female rats.

There were some findings in the kidneys and these findings correlated with pelvic dilatation in the histopathology evaluation and were therefore considered incidental.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the stomach minimal to moderate multifocal mixed inflammatory cell infiltrates in the submucosa of the glandular stomach were seen and in the limiting ridge. Whereas in control animals slight mixed inflammatory cell infiltrates were observed in the limiting ridge of one animal only. The accumulation of inflammatory cells in the submucosa of the glandular stomach was observed in animals from the high dose group only and was most likely induced by the local irritating effect of the test item.

In kidneys from male rats hyaline droplets were seen and represent an accumulation of secondary lysosomes within the cytoplasm and contain alpha-2u-globulin that reversibly binds to the inducing xenobiotics and/or metabolites. Hyaline droplets deposition is a male rat specific event, and it is of no toxicological relevance in humans.

The test item did not produce any histological evidence of toxicity in the reproductive organs and tis sues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, oviducts, uterus and cervix, and vagina. Furthermore, there were no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse systemic toxicity

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Reproductive function / performance (P0)

Reproductive function: estrous cycle:no effects observed

Test item had no biologically significant effect on the oestrous cycle analysed for 2 weeks premating period after the first administration in treatment groups when compared to the controls. There were no considerable differences in the length or sequence of cycle stages between the treatment groups and the control group.

Reproductive function: sperm measures:no effects observed

There were no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure.

The test item did not produce any histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, oviducts, uterus and cervix, and vagina.

Reproductive performance:no effects observed

There were no test item related effects on the reproductive indices (copulation, fertility, viability and delivery indices) in the dose groups when compared to the control group. All indices were 100 %.

Functional Observations (parental rats)

In males and females, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period when compared with the controls. Th ere were no biologically relevant differences observed in body temperature between the groups.

General toxicity (F1)

Clinical signs:no effects observed

Mortality / viability:no mortality observed

Body weight and weight changes:effects observed, non-treatment-related

A slight lower mean pup weight on PND 0 for male and female pups was observed in the HD group resulting in a slightly lower mean cube root of pup weight. The mean pup weight and cube root of pup weight of the female pups on PND 0 was also significantly lower in the LD group than control. However, values were in the normal range of biological variance and therefore not considered to be test item related.

Clinical biochemistry findings:no effects observed

Thyroid Hormone (T4) Analysis:

No test item related effect of toxicological relevance or statistical significance was observed on pup thyroid weight, male and PND 13 pup thyroxine hormone (T4) in the treatment groups when compared to the controls.

Sexual maturation:no effects observed

No statistically significant effect of toxicological relevance was observed on nipple retention in the pups of any of the groups when compared with the controls.

TABULATED DATA

Clinical Observations - Females – Summary

Clinical Finding	C	LD	MD	HD
Total number of animals examined	10	10	10	10
salivation increased	-	5	7	10
moving the bedding	-	10	10	10
piloerection	-	2	-	-
spontaneous activity reduced	-	1	-	1
hunced posture	-	1	-	-
regurgitation of test item	-	1	-	1
abnormal breathing	-	1	-	-
crust (neck)	-	1	-	-
alopecia	-	-	-	6

 

Mean Body Weight (g) – Males

Group		Premating			Mating and Postmating		Terminal Sacrifice
		Day 1	Day 7	Day 14	Day 7	Day 14
C	Mean	391.30	403.10	416.60	419.60	426.70	430.10
	SD	16.59	19.63	21.32	23.44	23.77	24.28
	N	10	10	10	10	10	10
LD	Mean	388.00	398.50	410.20	414.80	424.90	426.70
	SD	15.16	15.32	13.39	13.60	15.39	14.37
	N	10	10	10	10	10	10
	%	99	99	98	99	100	99
MD	Mean	386.00	395.10	407.50	409.20	416.10	417.70
	SD	12.67	13.61	14.58	16.59	17.36	17.73
	N	10	10	10	10	10	10
	%	99	98	98	98	98	97
HD	Mean	389.30	397.90	409.30	408.40	420.00	422.00
	SD	15.99	16.55	18.98	22.48	23.76	24.06
	N	10	10	10	10	10	10
	%	99	99	98	97	98	98

Mean Body Weight (g) – Females

Group		Premating			Gestation
		Day 1	Day 7	Day 14	Day 0	Day 7	Day 14	Day 20
C	Mean	236.30	238.40	244.30	244.10	261.20	282.90	345.30
	SD	6.02	4.84	5.03	5.26	6.88	8.03	12.16
	N	10	10	10	10	10	10	10
LD	Mean	235.90	238.30	243.78	241.22	262.78	285.89	353.44
	SD	5.28	7.45	8.70	7.43	6.96	11.75	15.73
	N	10	10	9	9	9	9	9
	%	99.8	100.0	99.8	98.8	100.6	101.1	102.4
MD	Mean	234.90	241.60	247.90	245.70	267.40	293.00	352.30
	SD	8.28	6.83	8.49	9.33	9.24	10.22	16.61
	N	10	10	10	10	10	10	10
	%	99.4	101.3	101.5	100.7	102.4	103.6	102.0
HD	Mean	237.80	246.10	250.90	252.50	271.90	296.40*	358.00
	SD	7.13	11.61	11.50	13.99	11.54	14.08	21.40
	N	10	10	10	10	10	10	10
	%	100.6	103.2	102.7	103.4	104.1	104.8	103.7

 

Group		Lactation
		Day 0	Day 4	Day 9	Day 13
C	Mean	263.22	285.50	298.50	303.30
	SD	13.50	9.55	8.18	7.97
	N	9	10	10	10
LD	Mean	265.78	289.78	302.11	307.89
	SD	11.17	15.54	17.87	14.87
	N	9	9	9	9
	%	101.0	101.5	101.2	101.5
MD	Mean	279.10*	294.30	304.10	310.10
	SD	9.67	10.67	9.86	12.80
	N	10	10	10	10
	%	106.0	103.1	101.9	102.2
HD	Mean	281.30*	300.30	308.10	314.40
	SD	16.85	14.40	15.52	9.63
	N	10	10	10	10
	%	106.9	105.2	103.2	103.7

Asterisks indicate statistically significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

Mean Food Consumption (g/week) – Males

Group		Premating
		Day 1-7	Day 7-14
C	Mean	23.00	23.50
	SD	0.00	0.71
	N	2	2
LD	Mean	22.50	23.00
	SD	0.71	0.00
	N	2	2
MD	Mean	22.50	23.00
	SD	0.71	0.00
	N	2.00	2.00
HD	Mean	22.00	24.00
	SD	0.00	0.00
	N	2	2

N = no. of cages with measurement of food consumption (5 rats per cage)
Premating period excluded from statistics due to low sample number (N = 2)

Mean Food Consumption (g/week) – Females

Group		Premating (5 animals per cage)		Gestation
		Day 1-7 (g/Animal)	Day 7-14 (g/Animal)	Day 0-7 (g/Animal)	Day 7-14 (g/Animal)	Day 14-20 (g/Animal)
C	Mean	15.75	15.84	18.49	19.77	22.08
	SD	0.16	0.67	1.63	1.63	1.93
	N	2	2	10	10	10
LD	Mean	16.05	16.17	18.92	19.86	23.30
	SD	1.15	0.20	1.72	2.15	3.21
	N	2	2	9	9	9
MD	Mean	16.50	16.69	19.51	21.11	22.55
	SD	0.57	0.53	1.45	1.54	1.61
	N	2.00	2.00	10.00	10.00	10.00
HD	Mean	16.42	17.00	21.20**	22.21*	23.27
	SD	0.16	1.41	1.75	1.71	2.76
	N	2	2	10	10	10

 

Group		Lactation
		Day 0-4 (g/Animal)	Day 4-9 (g/Animal)	Day 9-13 (g/Animal)
C	Mean	31.72	47.72	55.55
	SD	3.83	5.49	4.87
	N	9	10	10
LD	Mean	33.31	49.47	57.64
	SD	4.09	4.60	6.06
	N	9	9	9
MD	Mean	33.00	45.58	54.13
	SD	6.65	4.89	5.41
	N	10.00	10.00	10.00
HD	Mean	31.83	47.54	57.43
	SD	3.80	4.18	4.43
	N	10	10	10

Asterisks indicate statistically significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.
Premating period excluded from statistics due to low sample number (N = 2)

Estrous Cyclicity Premating - Summary

Group				4-Day Cycle		5-Day Cycle		Less than 4-Days		More than 5-Days		Mean Cycle Length (Days)		No. of Cycle		No. of Abnormal Cycle		No. of Normal Cycle
C		Mean		2.40		0.00		0.00		0.00		4.00		2.40		0.00		2.40
		SD		0.52		0.00		0.00		0.00		0.00		0.52		0.00		0.52
		N		10		10		10		10		10		10		10		10
LD		Mean		2.44		0.00		0.00		0.00		4.00		2.44		0.00		2.44
		SD		0.73		0.00		0.00		0.00		0.00		0.73		0.00		0.73
		N		9		9		9		9		9		9		9		9
MD		Mean		2.20		0.10		0.20		0.00		3.95		2.50		0.20		2.30
		SD		0.79		0.32		0.42		0.00		0.16		0.53		0.42		0.67
		N		10		10		10		10		10		10		10		10
HD		Mean		2.30		0.10		0.00		0.00		4.03		2.40		0.00		2.40
		SD		0.48		0.32		0.00		0.00		0.11		0.52		0.00		0.52
		N		10		10		10		10		10		10		10		10

Statistical analysis was performed of the mean cycle length, abnormal and normal cycle

Litter Data - Summary

Group		PND 0							PND 4 (before interim sacrifice)				PND 4
		Total No. of Pups (live & Dead)	No. of Male (live & Dead)	No. of Female (live & Dead)	Sex Ratio (m/f) ((live & Dead))	Live Pups	Still Birth	Runt	No. of Male	No. of Female	Live Pups	Sex Ratio (m/f)	Live Pups
													Post interim sacrifice
C	Mean	11.20	5.50	5.70	1.24	11.20	0.00	0.00	5.50	5.70	11.20	1.24	9.30
	SD	1.55	1.35	2.00	0.96	1.55	0.00	0.00	1.35	2.00	1.55	0.96	1.42
	N	10	10	10	10	10	10	10	10	10	10	10	10
LD	Mean	11.44	5.33	6.11	1.04	11.44	0.00	0.00	5.33	6.11	11.44	1.04	9.78
	SD	1.94	1.50	2.32	0.55	1.94	0.00	0.00	1.50	2.32	1.94	0.55	1.39
	N	9	9	9	9	9	9	9	9	9	9	9	9
MD	Mean	11.00	4.90	6.10	1.12	10.80	0.20	0.00	4.80	6.00	10.80	1.11	9.20
	SD	2.54	2.51	2.88	0.97	2.44	0.63	0.00	2.57	2.79	2.44	0.98	1.81
	N	10	10	10	10	10	10	10	10	10	10	10	10
HD	Mean	11.20	5.90	5.30	1.23	11.10	0.10	0.00	5.90	5.20	11.10	1.25	9.50
	SD	2.04	1.52	1.64	0.53	2.08	0.32	0.00	1.52	1.62	2.08	0.52	1.51
	N	10	10	10	10	10	10	10	10	10	10	10	10

Litter Data – Summary (Continued)

Group		PND 13
		No. of Male	No. of Female	Live Pups	Sex Ratio (m/f)
C	Mean	5.50	3.80	9.30	2.37
	SD	1.35	1.81	1.42	2.47
	N	10	10	10	10
LD	Mean	5.33	4.44	9.78	1.54
	SD	1.50	1.94	1.39	1.06
	N	9	9	9	9
MD	Mean	4.80	4.40	9.20	2.12
	SD	2.57	2.41	1.81	2.98
	N	10	10	10	10
HD	Mean	5.90	3.60	9.50	2.14
	SD	1.52	1.35	1.51	1.80
	N	10	10	10	10

Mean Litter Weight

					PND 0		PND 4		PND 13			PND 0
Group		Pup Mean weight on Day 0 (g)	Pup Mean weight on Day 4 (g)	Pup Mean weight on Day 13 (g)	Number of alive Males	Number of alive Females	Number of alive Males	Number of alive Females	Number of alive Males	Number of alive Females	Total No. of alive Pups	Total No. of Pups
C	Mean	6.63	11.62	30.82	5.50	5.70	5.50	5.70	5.50	3.80	9.30	11.20
	SD	0.43	0.98	2.97	1.35	2.00	1.35	2.00	1.35	1.81	1.42	1.55
	N	10	10	10	10	10	10	10	10	10	10	10
LD	Mean	6.39	11.41	30.40	5.33	6.11	5.33	6.11	5.33	4.44	9.78	11.44
	SD	0.47	1.43	2.87	1.50	2.32	1.50	2.32	1.50	1.94	1.39	1.94
	N	9	9	9	9	9	9	9	9	9	9	9
MD	Mean	6.52	11.74	30.68	4.80	6.00	4.80	6.00	4.80	4.40	9.20	11.00
	SD	0.64	1.44	3.24	2.57	2.79	2.57	2.79	2.57	2.41	1.81	2.54
	N	10	10	10	10	10	10	10	10	10	10	10
HD	Mean	6.22	11.09	29.61	5.90	5.20	5.90	5.20	5.90	3.60	9.50	11.20
	SD	0.74	1.69	3.36	1.52	1.62	1.52	1.62	1.52	1.35	1.51	2.04
	N	10	10	10	10	10	10	10	10	10	10	10

Mean Litter Weight (Continued)

		PND 0			PND 4			PND 13
Group		Total Litter Weight (g)	Male Litter Weight (g)	Female Litter Weight (g)	Total Litter Weight (g)	Male Litter Weight (g)	Female Litter Weight (g)	Total Litter Weight (g)	Male Litter Weight (g)	Female Litter Weight (g)
C	Mean	73.75	37.33	36.42	129.12	64.75	64.37	284.02	170.81	113.21
	SD	7.00	8.90	11.76	11.36	15.77	20.66	25.93	44.05	51.68
	N	10	10	10	10	10	10	10	10	10
LD	Mean	72.47	35.17	37.30	128.54	62.70	65.84	294.53	166.16	128.38
	SD	9.36	9.97	12.35	13.34	19.68	20.47	28.12	49.95	48.15
	N	9	9	9	9	9	9	9	9	9
MD	Mean	69.79	32.54	37.25	124.54	57.74	66.80	278.34	147.34	131.00
	SD	14.62	18.35	14.85	22.35	31.54	26.35	38.30	77.28	67.45
	N	10	10	10	10	10	10	10	10	10
HD	Mean	67.87	37.36	30.51	120.49	65.29	55.20	277.51	175.06	102.45
	SD	7.99	8.76	6.86	12.51	15.04	12.87	23.35	42.48	34.90
	N	10	10	10	10	10	10	10	10	10

Mean Precoital Interval and Duration of Gestation

Group		Precoital Interval (Days)	Duration of Gestation (Days)
C	Mean	2.30	22.50
	SD	1.16	0.53
	N	10	10
LD	Mean	2.78	22.33
	SD	1.30	0.50
	N	9	9
MD	Mean	2.70	22.30
	SD	1.06	0.48
	N	10	10
HD	Mean	2.30	22.10
	SD	1.16	0.57
	N	10	10

Pre- and Post-Natal Data - Summary

Group		Corpora Lutea (CL)	Implantation Sites (IS)	Live Pups on PND 0	Live Pups on PND 4 (before)	Live Pups on PND 4 (after)	Live Pups on PND 13	Pre Implantation Loss (%)	Post Implantation Loss (%)
C	Mean	13.60	12.10	11.20	11.20	9.30	9.30	9.81	6.72
	SD	2.37	1.91	1.55	1.55	1.42	1.42	13.27	9.94
	N	10	10	10	10	10	10	10	10
LD	Mean	13.63	13.50	11.44	11.44	9.78	9.78	0.83	15.33
	SD	1.19	1.07	1.94	1.94	1.39	1.39	2.36	15.53
	N	8	8	9	9	9	9	8	8
MD	Mean	13.50	13.10	10.80	10.80	9.20	9.20	2.93	18.25
	SD	1.27	1.52	2.44	2.44	1.81	1.81	7.35	12.97
	N	10	10	10	10	10	10	10	10
HD	Mean	13.22	13.00	11.10	11.10	9.50	9.50	1.65	16.19
	SD	1.64	1.66	2.08	2.08	1.51	1.51	3.27	16.43
	N	9	9	10	10	10	10	9	9

Mean Viability Indices

Group		Viability Index (%) 0-4	Viability Index (%) 4-13
C	Mean	100.00	100.00
	SD	0.00	0.00
	N	10	10
LD	Mean	100.00	100.00
	SD	0.00	0.00
	N	9	9
MD	Mean	100.00	100.00
	SD	0.00	0.00
	N	10	10
HD	Mean	100.00	100.00
	SD	0.00	0.00
	N	10	10

Viability Index (%) = (No. of live offspring at day 4 / No. of live offspring at birth) X 100

Mean Pup Survival Data

Group		Post Natal Day
		0			1					2
		Number of alive Males	Number of alive Females	Total	Number of alive Males	Number of alive Females	Total	Mortality	% Mortality	Number of alive Males	Number of alive Females	Total	Mortality	% Mortality
C	Mean	5.50	5.70	11.20	5.50	5.70	11.20	0.00	0.00	5.50	5.70	11.20	0.00	0.00
	SD	1.35	2.00	1.55	1.35	2.00	1.55	0.00	0.00	1.35	2.00	1.55	0.00	0.00
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
LD	Mean	5.33	6.11	11.44	5.33	6.11	11.44	0.00	0.00	5.33	6.11	11.44	0.00	0.00
	SD	1.50	2.32	1.94	1.50	2.32	1.94	0.00	0.00	1.50	2.32	1.94	0.00	0.00
	N	9	9	9	9	9	9	9	9	9	9	9	9	9
MD	Mean	4.80	6.00	10.80	4.80	6.00	10.80	0.00	0.00	4.80	6.00	10.80	0.00	0.00
	SD	2.57	2.79	2.44	2.57	2.79	2.44	0.00	0.00	2.57	2.79	2.44	0.00	0.00
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
HD	Mean	5.90	5.20	11.10	5.90	5.20	11.10	0.00	0.00	5.90	5.20	11.10	0.00	0.00
	SD	1.52	1.62	2.08	1.52	1.62	2.08	0.00	0.00	1.52	1.62	2.08	0.00	0.00
	N	10	10	10	10	10	10	10	10	10	10	10	10	10

Mean Pup Survival Data (Continued)

Group		Post Natal Day										Total Mortality (PND 0-4) %
		3					4
		Number of alive Males	Number of alive Females	Total	Mortality	% Mortality	Number of alive Males	Number of alive Females	Total	Mortality	% Mortality
C	Mean	5.50	5.70	11.20	0.00	0.00	5.50	5.70	11.20	0.00	0.00	0.00
	SD	1.35	2.00	1.55	0.00	0.00	1.35	2.00	1.55	0.00	0.00	0.00
	N	10	10	10	10	10	10	10	10	10	10	10
LD	Mean	5.33	6.11	11.44	0.00	0.00	5.33	6.11	11.44	0.00	0.00	0.00
	SD	1.50	2.32	1.94	0.00	0.00	1.50	2.32	1.94	0.00	0.00	0.00
	N	9	9	9	9	9	9	9	9	9	9	9
MD	Mean	4.80	6.00	10.80	0.00	0.00	4.80	6.00	10.80	0.00	0.00	0.00
	SD	2.57	2.79	2.44	0.00	0.00	2.57	2.79	2.44	0.00	0.00	0.00
	N	10	10	10	10	10	10	10	10	10	10	10
HD	Mean	5.90	5.20	11.10	0.00	0.00	5.90	5.20	11.10	0.00	0.00	0.00
	SD	1.52	1.62	2.08	0.00	0.00	1.52	1.62	2.08	0.00	0.00	0.00
	N	10	10	10	10	10	10	10	10	10	10	10

Mean Pup Survival Data (Continued)

Group		Post Natal Day								Total Mortality (PND 4-13) %
		4			13
		Number of alive Males after interim sacrifice	Number of alive Females after interim sacrifice	Total	Number of aliveMales	Number of alive Females	Total	Mortality	% Mortality
C	Mean	5.50	3.80	9.30	5.50	3.80	9.30	0.00	0.00	0.00
	SD	1.35	1.81	1.42	1.35	1.81	1.42	0.00	0.00	0.00
	N	10	10	10	10	10	10	10	10	10
LD	Mean	5.33	4.44	9.78	5.33	4.44	9.78	0.00	0.00	0.00
	SD	1.50	1.94	1.39	1.50	1.94	1.39	0.00	0.00	0.00
	N	9	9	9	9	9	9	9	9	9
MD	Mean	4.80	4.40	9.20	4.80	4.40	9.20	0.00	0.00	0.00
	SD	2.57	2.41	1.81	2.57	2.41	1.81	0.00	0.00	0.00
	N	10	10	10	10	10	10	10	10	10
HD	Mean	5.90	3.60	9.50	5.90	3.60	9.50	0.00	0.00	0.00
	SD	1.52	1.35	1.51	1.52	1.35	1.51	0.00	0.00	0.00
	N	10	10	10	10	10	10	10	10	10

Mean Anogenital Distance and Nipple Retention

		Male Pups					Female Pups
Group		Mean Pup Weight on PND 0 (g)	Cube Root of Pup Weight	Anogenital Distance (mm) of Pups	Relative Anogenital Distance Pups	Pup nipple retention (N) on PND 12	Mean Pup Weight on PND 0 (g)	Cube Root of Pup Weight	Anogenital Distance (mm) of Pups	Relative Anogenital Distance Pups
C	Mean	6.79	1.89	2.86	1.51	0.67	6.39	1.85	0.73	0.40
	SD	0.56	0.05	0.39	0.18	1.02	0.51	0.05	0.14	0.08
	N	55	55	55	55	55	57	57	57	57
LD	Mean	6.59	1.87	2.77	1.48	0.56	6.10*	1.83*	0.72	0.40
	SD	0.61	0.06	0.29	0.14	0.97	0.51	0.05	0.11	0.06
	N	48	48	48	48	48	55	55	55	55
MD	Mean	6.78	1.89	2.79	1.47	0.42	6.21	1.84	0.71	0.39
	SD	0.65	0.06	0.26	0.12	0.74	0.63	0.06	0.09	0.05
	N	48	48	48	48	48	60	60	60	60
HD	Mean	6.33***	1.85***	2.80	1.51	0.42	5.87***	1.80***	0.72	0.40
	SD	0.66	0.06	0.38	0.16	0.79	0.74	0.07	0.12	0.06
	N	59	59	59	59	59	52	52	52	52

Asterisks indicate statistically significant differences to control group C, with * p<0.05, ** p<0.01 and *** p<0.001

Mean Thyroxine (T4) Analysis – Males and Pups (Day 13)

Group		Male- Thyroxine (T4)	Pup-Thyroxine (T4)
	Units	nmoL/L	nmoL/L
C	Mean	88.49	104.57
	SD	23.36	29.74
	N	10	10
LD	Mean	79.48	108.88
	SD	23.22	9.72
	N	10	9
MD	Mean	82.56	123.70
	SD	18.63	22.00
	N	10	10
HD	Mean	82.94	115.19
	SD	11.05	18.19
	N	10	10

Mean Haematology – Males

Group		RBC	HB	HCT	MCV	MCH	MCHC	PLT	WBC	Neu	Lym	Mono	Eos	Baso	Luc	RE
	Units	10^12/L	g/dL	%	fL	pg/Ery	g/dL	10^9/L	10^9/L	%	%	%	%	%	%	%
C	Mean	9.42	16.32	50.06	53.12	17.32	32.60	681.60	4.58	17.50	78.92	2.26	0.30	0.12	0.92	1.69
	SD	0.07	0.22	1.35	1.15	0.15	0.63	34.69	1.68	2.99	3.25	0.50	0.12	0.11	0.15	0.35
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
LD	Mean	9.14	16.00	49.44	54.26	17.56	32.38	643.40	4.97	17.06	78.52	1.86	0.22	0.26	2.12	1.55
	SD	0.67	0.48	1.92	2.74	1.11	0.50	49.89	1.06	5.22	4.66	0.74	0.08	0.13	1.39	0.21
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
MD	Mean	9.56	16.44	50.10	52.46	17.24	32.86	727.60	4.53	16.64	79.58	2.36	0.38	0.20	0.86	1.58
	SD	0.28	0.38	1.07	1.74	0.63	0.57	78.04	1.04	2.24	2.21	0.26	0.18	0.10	0.33	0.20
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
HD	Mean	8.97	15.80	48.26	53.78	17.62	32.74	663.00	5.51	24.88	70.82	2.50	0.48	0.18	1.16	1.60
	SD	0.44	0.72	2.34	1.28	0.26	0.40	52.79	2.13	13.04	14.52	1.15	0.69	0.08	0.27	0.22
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5

Mean Coagulation – Males

Group		PT	aPTT
	Units	sec	sec
C	Mean	20.46	9.22
	SD	0.33	2.02
	N	5	5
LD	Mean	20.90	11.26
	SD	0.91	3.53
	N	5	5
MD	Mean	20.74	9.20
	SD	1.12	1.18
	N	5	5
HD	Mean	20.96	8.78
	SD	1.05	1.01
	N	5	5

Mean Haematology – Females

Group		RBC	HB	HCT	MCV	MCH	MCHC	PLT	WBC	Neu	Lym	Mono	Eos	Baso	Luc	RE
	Units	10^12/L	g/dL	%	fL	pg/Ery	g/dL	10^9/L	10^9/L	%	%	%	%	%	%	%
C	Mean	7.38	13.86	45.40	61.56	18.80	30.56	710.20	3.56	30.42	66.10	2.36	0.32	0.12	0.70	2.77
	SD	0.46	0.73	3.25	2.95	0.52	0.88	172.06	0.86	8.14	7.85	0.92	0.29	0.08	0.31	0.48
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
LD	Mean	7.02	14.00	45.44	64.74	19.92*	30.82	724.40	3.01	35.74	61.24	1.94	0.36	0.28	0.48	2.34
	SD	0.10	0.50	2.30	3.58	0.69	1.31	90.17	0.84	12.74	13.36	1.08	0.17	0.15	0.25	0.58
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
MD	Mean	7.04	13.86	44.48	63.16	19.72	31.26	711.80	3.58	26.86	69.98	2.22	0.12	0.22	0.60	2.80
	SD	0.36	0.68	2.93	2.82	0.73	1.06	49.52	0.87	3.37	3.71	0.48	0.08	0.08	0.14	0.40
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
HD	Mean	7.23	13.66	42.64	58.96	18.94	32.10	745.40	4.33	31.84	64.54	2.18	0.20	0.26	1.00	2.39
	SD	0.21	0.17	1.85	1.88	0.64	1.44	66.29	1.68	9.60	9.45	0.31	0.17	0.21	0.41	0.65
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5

Mean Coagulation – Females

Group		PT	aPTT
	Units	sec	sec
C	Mean	20.15	15.10
	SD	0.83	1.68
	N	4	4
LD	Mean	19.80	14.06
	SD	1.50	1.55
	N	5	5
MD	Mean	20.18	14.63
	SD	0.49	0.95
	N	4	4
HD	Mean	19.80	15.30
	SD	0.42	2.83
	N	2	2

HD group excluded from statistics due to low sample number (N = 2

 

Mean Clinical Biochemistry – Males

Group		ASAT	ALAT	AP	Crea	TP	Alb	Urea	TBA	Chol	Gluc	Na	K
	Units	U/L	U/L	U/L	µmol/L	g/L	g/L	mmol/L	µmol/L	mmol/L	mmol/L	mmol/L	mmol/L
C	Mean	92.04	35.16	168.86	19.80	56.48	29.23	8.16	25.23	1.80	16.45	133.80	4.02
	SD	12.13	6.58	41.12	2.59	1.90	0.87	0.83	13.91	0.44	1.74	1.10	0.24
	N	5	5	5	5	5	5	5	5	5	5	5	5
LD	Mean	95.94	32.58	176.12	20.60	58.76	31.07	7.18	23.10	1.49	15.76	135.00	4.13
	SD	23.59	8.61	32.17	1.82	1.73	1.72	0.84	8.61	0.14	3.57	1.87	0.26
	N	5	5	5	5	5	5	5	5	5	5	5	5
MD	Mean	82.30	29.58	149.48	18.40	57.52	29.40	7.81	21.36	1.90	17.75	134.20	3.96
	SD	19.21	4.16	39.19	1.95	2.48	1.24	0.73	7.98	0.21	1.85	1.30	0.13
	N	5	5	5	5	5	5	5	5	5	5	5	5
HD	Mean	105.32	33.32	226.16	19.40	56.04	29.80	8.05	28.77	2.02	17.07	135.20	4.03
	SD	24.25	4.43	37.72	1.95	0.43	0.52	1.39	22.57	0.28	2.79	1.10	0.18
	N	5	5	5	5	5	5	5	5	5	5	5	5

Mean Clinical Biochemistry – Females

Group		ASAT	ALAT	AP	Crea	TP	Alb	Urea	TBA	Chol	Gluc	Na	K
	Units	U/L	U/L	U/L	µmol/L	g/L	g/L	mmol/L	µmol/L	mmol/L	mmol/L	mmol/L	mmol/L
C	Mean	87.82	70.92	386.34	18.80	47.60	25.70	11.69	32.06	1.91	6.67	128.80	6.51
	SD	27.15	26.10	204.23	3.42	5.82	3.55	1.68	9.54	0.30	1.17	12.87	0.77
	N	5	5	5	5	5	5	5	5	5	5	5	5
LD	Mean	114.64	62.96	264.42	16.40	45.44	24.15	10.59	36.84	1.76	6.02	127.20	6.30
	SD	32.78	18.54	157.45	5.73	7.61	4.24	0.98	18.94	0.29	1.56	17.68	1.59
	N	5	5	5	5	5	5	5	5	5	5	5	5
MD	Mean	94.12	58.70	273.44	16.80	49.48	26.13	11.02	27.21	1.98	7.39	130.40	5.90
	SD	18.84	11.44	35.13	2.77	3.35	2.14	1.44	13.55	0.28	1.65	6.47	1.19
	N	5	5	5	5	5	5	5	5	5	5	5	5
HD	Mean	94.78	65.58	285.83	18.40	51.70	27.44	11.63	21.55	1.85	8.23	139.40	5.45
	SD	22.90	13.56	119.62	2.61	3.22	1.47	1.54	7.16	0.25	1.70	3.21	0.77
	N	5	5	5	5	5	5	5	5	5	5	5	5

Applicant's summary and conclusion

Conclusions:

Based on this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with Guanylurea phosphate (GUP) in male and female Wistar rats with dose levels of 0, 100, 300, and 1000 mg/kg body weight day the following conclusions can be made. No adverse effects of test item were found on male and female clinical observations, functional observations , body weight development, food consumption, oestrous cyclicity, litter data, litter weight data, pre¬-coital interval and duration of gestation, pre and post-natal data, reproductive indices, pup survival da ta, anogenital distance and nipple retention, pup thyroid weight and thyroid hormone analysis in pa rental males and pups sacrificed on PND 13, pup external findings, haematology and coagulation, clinical biochemistry, urinalysis, gross macroscopic findings at necropsy, organ weights and histopath ology in all treatment groups.

The Systemic NOAEL of Guanylurea phosphate (GUP) in this study for general toxicity and reproductive toxicity screening is 1000 mg/kg body weight.

Executive summary:

The aim of this study was to assess the possible effects of Guanylurea phosphate (GUP)on male and female fertility and embryo-fetal development after repeated dose administration in Wistar rats in an OECD 422 study design. The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. Animals of an additional control group were handled identically as the dose groups but received sterile water, the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats.

Methods

The following doses were evaluated: 0 (control), 100, 300 and 1000 mg/kg bw/day (low dose, medium dose and high dose).

Before dosing all females were screened for two weeks for regular oestrous cyclicity and animals (10 females/ group) with regular oestrous cycle (4-5-day cycle) were used in the study.

During the period of administration, the animals were observed each day for signs of toxicity.

Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals.

Haematological and clinical biochemistry evaluations were performed on blood samples collected at terminal sacrifice from five randomly selected males and females from each group. Urinalysis was performed on samples collected at terminal sacrifice from five randomly selected males from each group.

Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behaviour observations were performed in the week before the treatment from all animals and in the last week of treatment in five randomly selected males and females of each group.

After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually.

Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition, on day 4 and day 13 post-partum. The anogenital distance (AGD) of each pup was measured on PND 0. The number of nipples/areolae in male pups was counted on PND 12.

From 2 pups/litter on day 4 after birth; from all dams and 2 pups /litter at termination on day 13 and from all adult males at termination, blood samples were collected from the defined site. All blood samples were stored under appropriate conditions. Blood samples from the day 13 pups and from the adult males were assessed for serum levels for thyroid hormones (T4). Further assessment of T4 in blood samples from adult females and day 4 pups were not deemed necessary. Pup blood was pooled by litter for thyroid hormone analysis.

The males were sacrificed after completion of the mating period on treatment day 29 and the females along with their pups were sacrificed on post-natal day 13. Non-pregnant females were sacrificed on day 26.

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Pups sacrificed on post-natal day 4 or 13 and those found dead, were carefully examined for gross external abnormalities.

A full histopathological evaluation of the preserved tissues was performed on high dose and control animals and dead animal. These examinations were extended to animals of all other dosage groups as treatment-related changes were observed in stomach of the high dose group. For the testes, a detailed qualitative examination was made; considering the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides. All gross lesions macroscopically identified were examined microscopically in all animals.

 

Results

Mortality:

One female (no. 51 from LD group) was sacrificed because of animal welfare reasons during premating because of a technical error during gavage.

Clinical Observations:

HD female no. 73 was seen with moderately reduced spontaneous activity on PMD 4. HD female no. 75 regurgitated test item on GD 7 but recovered afterwards. Both females survived till the end of the scheduled study end.

In terminally sacrificed females and males of the LD, MD and HD group major clinical signs observed during the treatment period were slight to moderate increased salivation and moving the bedding which were observed immediately after the dose administration and therefore were a sign of a local reaction to the test item rather than a systemic adverse effect and has no toxicological relevance.

None of the females showed signs of abortion or premature delivery.

During the weekly detailed clinical observation, no relevant differences between the groups were found.

Functional Observations:

In males and females, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period when compared with the controls. There were no biologically relevant differences observed in body temperature between the groups.

Body Weight Development:

In both males and females, there was no test item treatment related effect observed on body weight and body weight gain in the dose groups during the study period. There were no statistically significant differences observed for body weight and body weight gain between the dose groups and the control group.

Food Consumption:

No test item related or statistically significant effect on food consumption was observed in males and females during the whole study period.

Oestrus Cyclicity:

Test item had no biologically significant effect on the estrous cycles for 2 weeks premating period after the first administration in treatment groups when compared to the controls. There were no considerable differences in the length or sequence of cycle stages between the treatment groups and the control group.

Litter Data:

There was no test item treatment related or statistically significant effects observed in treatment groups on litter data parameters like group mean total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, still birth, runt on PND 0 as well as number of live pups, male pups, number of female pups and sex ratio on PND 4 and PND 13 when compared with the controls.

Litter Weight Data:

There was no test item related effect on pup mean weight, total litter weight, female litter weight on PND 0, PND 4 and PND 13 observed in treatment groups when compared with the controls. There was no statistically significant change in dose groups compared to corresponding controls.

Pre-coital Interval and Duration of Gestation:

There were no test item related or statistically significant effects observed on the duration of pre-coital interval and the duration of gestation in the dose groups when compared to the control group.

Pre and Post-Natal Data:

There were no test item treatment related effects observed on the number of corpora lutea, implantation sites, live pups on PND 0, 4 and 13, percent preimplantation loss and post implantation loss in treatment groups when compared with the control group.

Reproductive Indices:

There were no test item related effects on the reproductive indices (copulation, fertility, viability and delivery indices) in the dose groups when compared to the control group.

Pup Survival Data:

No test item related effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13 in treatment groups when compared to the control group was noted.

Anogenital Distance and Nipple Retention:

A slight but statistical significant lower mean pup weight on PND 0 for male and female pups were observed in the HD group resulting in a slight but significant lower mean cube root of pup weight. The mean pup weight and cube root of pup weight of the female pups on PND 0 was also significantly lower in the LD group than control. However, values are in the normal range of biological variance and therefore not considered to be test item related. In male and females, parameters like litter size and sex ratio were not affected in treatment groups and in the light of absence of effect on both absolute and relative anogenital distance in male and female pups, this slight effect on pup weight and cube root in HD group was not considered to be adverse.

No statistically significant effect of toxicological relevance was observed on nipple retention in the pups of any of the groups when compared with the controls.

Thyroid Hormone (T4) Analysis:

No test item related effect of toxicological relevance or statistical significance was observed on pup thyroid weight, male and PND 13 pup thyroxine hormone (T4) in the treatment groups when compared to the controls.

Pup External Findings:

No test item related gross external abnormalities of toxicological relevance on PND 0-12 were observed in the pups of any of the groups.

Haematology and Coagulation:

In females and males sacrificed at the end of treatment period, no test item related effect or statistically significant effect observed on any of the haematology parameters.

No test item related effect was observed on coagulation parameters in males and females when compared with the respective controls.

Clinical Biochemistry:

In males and females sacrificed at the end of treatment period, no test item related or statistically significant effect on any clinical biochemistry parameter in treatment groups was observed when compared with the control.

Urinalysis:

The urinalysis performed in selected male and female animals sacrificed at the end of treatment period revealed no test item treatment related effect and all urinary parameters were in the normal range of variation.

Pathology:

A small number of male rats had some findings, but these findings were not correlated to any histological changes and therefore were not considered to be test item related.

A similar situation was recorded for female rats. 

There were some findings in the kidneys and these findings correlated with pelvic dilatation in the histopathology evaluation and were therefore considered incidental.

Few more specific macroscopic changes were recorded for the male and female animals which based on microscopic examination were not considered to be of test item treatment relevance.

Organ Weight:

There was no test item related effect on individual or mean organ weights of all animals assigned to terminal sacrifice. All single values are in the range of biological variation of this strain and age.

Histopathology:

In the stomach minimal to moderate multifocal mixed inflammatory cell infiltrates in the submucosa of the glandular stomach were seen and in the limiting ridge. Whereas in control animals slight mixed inflammatory cell infiltrates were observed in the limiting ridge of one animal only. The accumulation of inflammatory cells in the submucosa of the glandular stomach was observed in animals from the high dose group only and was most likely induced by the local irritating effect of the test item.

 

The test item did not produce any histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, oviducts, uterus and cervix, and vagina. Furthermore, there were no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure.

Dose Formulation Analysis:

Dose formulation analysis for nominal concentration revealed that nominal concentrations for all formulations were confirmed throughout the study period as measured concentrations were within acceptance criterion of 15%.

Conclusion

Based on a combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with Guanylurea phosphate (GUP) in male and female Wistar rats with dose levels of 0, 100, 300, and 1000 mg/kg body weight day the following conclusions can be made. No adverse effects of test item were found on male and female clinical observations, functional observations, body weight development, food consumption, estrous cyclicity, litter data, litter weight data, pre-coital interval and duration of gestation, pre and post-natal data, reproductive indices, pup survival data, anogenital distance and nipple retention, pup thyroid weight and thyroid hormone analysis in parental males and pups sacrificed on PND 13, pup external findings, haematology and coagulation, clinical biochemistry, urinalysis, gross macroscopic findings at necropsy, organ weights and histopathology in all treatment groups.

The Systemic NOAEL of Guanylurea phosphate (GUP) in this study for general toxicity and reproductive toxicity screening is 1000 mg/kg body weight.