Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 August 2017 - April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 August 2017 - April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016.
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Standard species / strain used for this type of study
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo, Itlay
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 175-200 g (males), 151-175 g (females)
- Fasting period before study: No
- Housing: group housed by sex , except during mating (1:1). Individual housing for females during gestation and lactation
- Diet: as libitum
- Water: ad libitum
- Acclimation period: 35 days

DETAILS OF FOOD AND WATER QUALITY: There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Records of analyses of water and diet are kept on file at RTC.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 06 September 2017 To: 13 November 2017
Route of administration:
oral: gavage
Details on route of administration:
The test item was administered orally by gavage at a dose volume of 10 mL/kg bw.
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
The test item was administered orally by gavage at a dose volume of 10 mL/kg bw. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal. During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes remained constant
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The required amount of 1,3-Propanediol, 2,2-bis(hydroxymethyl)-, allyl ether was suspended in the vehicle. The formulations were prepared weekly (concentrations of 5, 30 and 75 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied. The analytical method was validated in the present study in the range from 5 to 75 mg/mL. In this study a 28-hour stability at room temperature and an 8 day stability at +4°C were verified in the range from 5 to 75 mg/mL. The proposed formulation procedure for the test item was checked in the range from 5 to 75 mg/mL by chemical analysis (concentration and homogeneity) during the pre-treatment period to confirmthat the method was suitable. Final results for all levels were within the acceptability limits stated in RTC SOPs for concentration (80-120%) and homogeneity (CV <10%). Samples of the formulations prepared on Week 1 and Week 5 were analysed to check the homogeneity and concentration.
Duration of treatment / exposure:
Males were dosed once a day, 7 days a week, 15 days prior to pairing, during pairing with females until the day before necropsy, for a total of 30/33 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Females were treated for 15 days prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum, for a total of 48 to 61 days.

Dose volumes were adjusted once per week for each animal according to the last recorded body weight up to mating. During the gestation and lactation periods, dose volumes were calculated according to the last recorded body weight.
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
On the day of allocation (14 days prior to the start of treatment), all animals were weighed. Animals at the extremes of the weight distribution and animals showing irregular cycle were excluded to leave the required number of animals. The rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed 5 of one sex per cage. The cages were identified by a label and recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each group were distributed to minimise possible environmental effects and or contamination. In addition, 10 females showing irregular cycle during the period between allocation and the start of treatment were replaced with surplus female animals selected from the same batch.
Positive control:
Not required for this study type.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

DETAILED CLINICAL OBSERVATIONS: Yes
Once before commencement of treatment and at least once a week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). All observations were recorded for individual animals.

BODY WEIGHT: Yes
Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1, 4, 7, 13 post partum and just before necropsy.

FOOD CONSUMPTION: Yes
The weight of food consumed by each cage of males and females was recorded weekly (whenever possible) during the pre-mating period starting from allocation. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 7 and 13 post partum starting from Day 1 post partum.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
As a part of the necropsy procedure, fasted samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable litters) randomly selected from each group. The following parameters were measured: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count, Differential leucocyte count, Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells, Platelets. In addition, as part of the necropsy procedure, blood samples were taken from the abdominal vena cava of all parental male and female rats fromeach group, under isofluorane anaesthesia, to perform the determination of serum T3, T4 and TSH levels.

CLINICAL CHEMISTRY: Yes
As a part of the necropsy procedure, fasted samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable litters) randomly selected from each group. The following parameters were measured: Prothrombin time, Activated partial thromboplastin time, Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Bile acids, Inorganic phosphorus, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Grip strength and sensory reactivity to stimuli: Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength. For the females the tests were performed on Day 13 post partum (where possible). Motor activity assessment: Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group and the motor activity was measured (for approximately 5 minutes) by an automated activity recording device. For the females the tests were performed on Day 13 post partum (where possible).

IMMUNOLOGY: No

OTHER:
Oestrous cycle was monitored by vaginal smears in all stock females for 1 week before allocation in order to exclude from the study females with irregular cycle. Vaginal smears were taken in the morning from Day 1 of allocation and during treatment period, up to positive identification of mating including 15 days before the pairing. Animals that exhibited irregular cycles were not included in the study. The vaginal smear data were examined to determine anomalies of the oestrous cycle and the pre-coital interval. Vaginal smears were also taken from all females, before dispatch to necropsy.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The clinical history of the animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices)

ORGAN WEIGHTS: Yes
Weights of the adrenals, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles, spleen, testes, thymus, thyroid and uterus were recorded.

HISTOPATHOLOGY: Yes
Tissues from 5 rats/sex (control and high dose group) were examined. The livers from the remaining rats from these groups and all rats from the intermediate dose groups were also examined.
Other examinations:
A detailed qualitative examination of the testes was performed on 5 randomly selected animals from the control and high dose groups. The evaluation, taking into account the tubular stages of the spermatogenic cycle, was conducted in order to identify treatment-related effects, such as missing germcell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.PAS-stained sections were used to identify the spermatogenic stages. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
Statistics:
Standard deviationswere calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of theWilliams test. The mean values, standard deviations and statistical analysis were calculated from actual values without rounding. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if ‘n’ was more than 5.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In males, salivation and/or ataxia were observed during the pre-mating phase, mainly during the first week, in all animals at 750 mg/kg bw/d. In the females, slight to moderate ataxia and/or decreased activity and/or salivation were seen in 9/10 animals at 750 mg/kg bw/d, while salivation was observed in 3/10 females at 300 mg/kg bw/d during the pre-mating phase. Salivation was also observed in all females at 300 and 750 mg/kg bw/d during the post coitum and post partum periods. Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant changes in body weight and body weight gain were observed during the study in the treated animals, when compared to controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by treatment in both sexes during the study. A reduction of mean food consumption (statistically significant on Day 13 post partum) observed in females at 750 mg/kg bw/d during the post partum period was attributed to a single female with a amall litter (2 pups).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No relevant changes were recorded. A statistically significant increase of large unstained cells in females at 50 and 300 mg/kg bw/d was not dose related and was considered to be incidental.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Some statistically significant fluctuations of biochemical parameters were recorded, including a decrease in alkaline phosphatase in males at 50 and 750 mg/kg bw/d (18% and 22%, respectively); decreased bilirubin in males at 300 mg/kg bw/d (54%); increased glucose, chloride and sodium in females at 750 mg/kg bw/d (27%, 5% and 2%, respectively); increased chloride in females at 300 mg/kg bw/d (5%). Since no other relevant changes were recorded and findings were of minimal severity, they were considered to be of no toxicological significance. Triiodothyronine and thyroxine were increased in some males from all treated groups. Compared with controls, changes were 19% to 39%, with some relationship to dose level.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment-related alterations in motor activity, grip strength and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment. Statistically significantly lower landing footsplay was recorded in the males at 750 mg/kg bw/d, when compared to the control group. Due to the direction of the change (reduction), it was considered to be of no neurotoxicological significance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
No changes were observed in the terminal body weight of treated animals, when compared to the controls. A statistically significant increase in absolute and relative liver weight was recorded in males at 300 mg/kg bw/d and at the high dose level in both sexes, compared to controls. This finding was considered to be treatment-related on the basis of correlating histopathology.
Changes in absolute and/or relative organ weights, statistically significant in high dose males and/or females, such as brain, kidneys and thyroid were not considered to be toxicologically relevant, in the absence of correlating histopathology.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were seen in the liver of most high dose males and females sacrificed at the end of the study. The treatment-related liver changes consisted of minimal to mild centrilobular and/or midzonal hepatocytic hypertrophy, associated with cytoplasmic eosinophilia, morphologically represented by an enlarged size of the hepatocytes, possibly correlated to the hepatic enzyme induction. In the absence of degenerative changes, the hepatocytic hypertrophy was considered adaptive and not an adverse liver change.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No treatment-related anomalies were noted in the oestrous cycle.
Details on results:
There was no mortality in this study. In males, salivation and/or ataxia were observed during the pre-mating phase, mainly during the first week, in all animals at 750 mg/kg bw/d. In females, slight to moderate ataxia and/or decreased activity and/or salivation were seen in 9/10 animals at 750 mg/kg bw/d, while salivation was observed in 3/10 females at 300 mg/kg bw/d during the pre-mating phase. Salivation was also observed in all females at 300 and 750 mg/kg bw/d during the post coitum and post partum periods. Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item. Bodyweights, bodyweight gain and food consumption were unaffected by treatment. Haematology revealed a slight but statistically significant increase of large unstained cells in females at 50 and 300 mg/kg bw/d; this was not dose-related and was considered to be incidental. Clinical chemistry revealed a small number of statistically significant fluctuations, including a decrease in alkaline phosphatase in males at 50 and 750 mg/kg bw/d, decreased bilirubin in males at 300 mg/kg bw/d (54%); increased glucose, chloride and sodium in females at 750 mg/kg bw/d (27%, 5% and 2%, respectively); increased chloride in females at 300 mg/kg bw/d (5%). Since no other relevant changes were recorded and findings were of minimal severity, they were considered to be of no toxicological significance. Triiodothyronine and thyroxine were increased in some males from all treated groups. Compared with controls, changes were 19% to 39%, with some relationship to dose level. No treatment-related alterations in motor activity, grip strength and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment. Statistically significantly lower landing foot splay was recorded in the males at 750 mg/kg bw/d, when compared to the control group. Due to the direction of the change (reduction), it was considered to be of no neurotoxicological significance. A statistically significant increase in absolute and relative liver weight was recorded in males at 300 mg/kg bw/d and at the high dose level in both sexes, compared to controls. This finding was considered to be treatment-related on the basis of correlating histopathology. Changes in absolute and/or relative organ weights, statistically significant in high dose males and/or females, such as brain, kidneys and thyroid were not considered to be toxicologically relevant, in the absence of correlating histopathology. Histopathology revealed effects in the liver of most high dose males and females, consisting of minimal to mild centrilobular and/or mid-zonal hepatocyte hypertrophy, associated with cytoplasmic eosinophilia, morphologically represented by an enlarged size of the hepatocytes, possibly correlated to the hepatic enzyme induction. In the absence of degenerative changes or clinical chemistry correlates, the hepatocyte hypertrophy was considered to be adaptive and not adverse.
Key result
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
750 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver

Summary of findings

 

M

F

0

50

300

750

0

50

300

750

Bodyweight (g) pre-mate

326.9

343.5

360.4

374.2

250.5

248.3

247.5

243.5

Bodyweight (g) terminal

406.7

402.5

403.0

406.6

 

 

 

 

Bodyweight (g) GD20

 

 

 

 

385.1

386.9

395.1

381.6

Bodyweight (g) LD13

 

 

 

 

326.7

329.3

323.7

311.2

Haematology

LUC (10e3/µL)

0.008

0.018*

0.020*

0.016

0.33

0.48

0.63*

0.52

Clinical chemistry

ALP (U/L)

165.5

135.4*

142.5

128.8**

18.07

150.3

156.7

166.5

TBil (mg/dL)

0.056

0.052

0.026*

0.034

0.007

0.010

0.017

0.024

Glucose (mg/dL)

130.4

136.3

109.1

110.5

101.6

117.7

116.8

128.9*

Cl (mmol/L)

100.08

101.16

101.20

99.54

92.72

95.12

97.26**

97.78**

Na (mmol/L)

141.4

142.3

142.8

140.8

137.4

137.1

139.4

139.9*

T3 (ng/mL)

4.278

5.456*

4.606

5.966**

 

 

 

 

T4 (ng/mL)

289.1

344.0**

344.1**

372.3**

 

 

 

 

TSH (ng/mL)

2.060

1.891

2.128

2.272

 

 

 

 

 

 

 

 

 

 

 

 

 

Foot splay (cm)

7.85

7.50

7.15

6.80*

5.09

5.09

5.84

5.17

Organ weights

Liver (g)

9.71

10.22

11.18*

12.82**

8.64

9.50

8.41

10.89**

Liver (%)

2.45

2.63

2.92**

3.34**

3.08

3.35

3.15

4.03**

Kidneys (g)

2.57

2.64

2.52

2.75

1.74

1.74

1.59

1.79

Kidneys (%)

0.658

0.678

0.658

0.714**

0.619

0.612

0.601

0.664*

Thyroid (g)

0.026

0.028

0.026

0.019*

0.024

0.023

0.023

0.024

Thyroid (%)

0.007

0.007

0.007

0.005*

0.008

0.008

0.009

0.009

Histopathology

Hepatocyte hypertrophy

-

-

-

9*

-

-

-

8*

*significantly different to controls (p<0.05); **p<0.01

Conclusions:
A NOAEL of 750 mg/kg bw/d is concluded for this study in the absence of any toxicologically relevant or adverse findings.
Executive summary:

In an OECD 422 screening study, groups of Sprague-Dawley rats (10/sex) were administered 1,3-Propanediol, 2,2-bis(hydroxymethyl)-, allyl ether (in 0.5% aqueous carboxymethylcellulose) by gavage at dose levels of 0 (vehicle control), 50, 300 or 750 mg/kg bw/d. Males were dosed once a day, 7 days a week, 15 days prior to pairing, during pairing with females until the day before necropsy, for a total of 30/33 days. Females were treated for 15 days prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum, for a total of 48 to 61 days. Rats were observed daily for mortality and signs of toxicity; more detailed clinical observations were performed weekly. Neurobehavioural assessment was performed towards the end of the treatment period. Bodyweights and food consumption were measured at intervals throughout the study. Terminal blood samples were taken for the assessment of haematological and clinical chemistry parameters; T3, T4 and TSH were also measured in males. Gross necropsy was performed on all animals; weights of the adrenals, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles, spleen, testes, thymus, thyroid and uterus were recorded. Tissues from 5 rats/sex (control and high dose group) were examined histopathologically. The livers from the remaining rats from these groups and all rats from the intermediate dose groups were also examined.  There was no mortality. Clinical signs (salivation, hypoactivity and/or ataxia) were observed at 750 mg/kg bw/d; salivation was also observed in a small number of females at 300 mg/kg bw/d during the pre-mating phase. Haematology and clinical chemistry did not reveal any clear adverse effects of treatment. T3 and T4 levels were slightly increased in males from all treated groups. No treatment-related alterations in motor activity, grip strength and sensory reactivity to stimuli were observed. Gross necropsy did not reveal any effects of treatment. Increased absolute and relatibe liver weights were seen at 300 and 750 mg/kg bw/d and at 750 mg/kg bw/d corrleated with histopathological findings (minimal to mild centrilobular and/or mid-zonal hepatocyte hypertrophy, associated with cytoplasmic eosinophilia). A NOAEL of 750 mg/kg bw/d is concluded for this study in the absence of any toxicologically relevant or adverse findings.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
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)
Version / remarks:
29 July 2016.
Deviations:
no
GLP compliance:
yes
Limit test:
no
Justification for study design:
Standard species, strain and study design.

Test material

Constituent 1
Reference substance name:
1,3-Propanediol, 2,2-bis(hydroxymethyl)-, allyl ether
EC Number:
293-883-9
EC Name:
1,3-Propanediol, 2,2-bis(hydroxymethyl)-, allyl ether
Cas Number:
91648-24-7
Molecular formula:
C14H24O4
IUPAC Name:
2,2-bis(hydroxymethyl)propane-1,3-diol; 3-(prop-2-en-1-yloxy)prop-1-ene
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Standard species / strain used for this type of study
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo, Itlay
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 175-200 g (males), 151-175 g (females)
- Fasting period before study: No
- Housing: group housed by sex , except during mating (1:1). Individual housing for females during gestation and lactation
- Diet: as libitum
- Water: ad libitum
- Acclimation period: 35 days

DETAILS OF FOOD AND WATER QUALITY: There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Records of analyses of water and diet are kept on file at RTC.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 06 September 2017 To: 13 November 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
The test item was administered orally by gavage at a dose volume of 10 mL/kg bw. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal. During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes remained constant.
Details on mating procedure:
Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (spermi dentification, vaginal plug in situ or copulation plugs found in the cage tray). The female was paired with the same male until positive identification occurred or 14 days had elapsed. A parturition check was performed from Day 20 to Day 25 post coitum. Females which did not give birth after 25 days of post coitum period were sacrificed shortly after. Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of birth when the parturition was defined complete (Day 0 post partum).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations The required amount of test substance was suspended in the vehicle. The formulations were pre
pared weekly (concentrations of 5, 30 and 75 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied. The analytical method was validated in the present study in the range from 5 to 75 mg/mL. In this study a 28-hour stability at room temperature and an 8 day stability at +4°C were verified in the range from 5 to 75 mg/mL. The proposed formulation procedure for the test item was checked in the range from 5 to 75 mg/mL by chemical analysis (concentration and homogeneity) during the pre-treatment period to confirmthat the method was suitable. Final results for all levels were within the acceptability limits stated in RTC SOPs for concentration (80-120%) and homogeneity (CV <10%). Samples of the formulations prepared on Week 1 and Week 5 were analysed to check the homogeneity and concentration.
Duration of treatment / exposure:
Males were dosed once a day, 7 days a week, 15 days prior to pairing, during pairing with females until the day before necropsy, for a total of 30/33 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight. Females were treated for 15 days prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum, for a total of 48 to 61 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight up to mating. During the gestation and lactation periods, dose volumes were calculated according to the last recorded body weight.
Frequency of treatment:
Daily
Details on study schedule:
On Day 4 post partum, all pups were weighed and litters in excess of 8 offspring were culled to 8 (4 males and 4 females, where possible) by a random selection. Partial adjustment (for example, 5 males and 3 females) was performed when necessary. At least two pups (males or females, selected for culling) were sacrificed to collect blood for possible determination of serum T3, T4 and TSH levels. Pooled samples by sex were performed when necessary. Litters with less than 8 pups were not culled. Two of the pups (preference was given to remove two females in order to retain more male pups to allow nipple count on Day 14 post partum) from each litter were sacrificed to collect blood for possible determination of serum T3, T4 and TSH levels. Pooled samples by sex were performed when necessary. Retained pups in each litter were generally not less than 2 males and 2 females, with the exception of dam nos. 11 (1 male and 7 females), 39 (3 males and 1 female) and 67 (2 males only).

Males were killed after the mating of all females, after at least 29 days of treatment. Females with live pups were killed on Day 14 post partum. Females showing no evidence of copulation were killed 26 days after the last day of the mating session. Females which did not give birth were killed on Day 26 post coitum.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
On the day of allocation (14 days prior to the start of treatment), all animals were weighed. Animals at the extremes of the weight distribution and animals showing irregular cycle were excluded to leave the required number of animals. The rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed 5 of one sex per cage. The cages were identified by a label and recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each group were distributed to minimise possible environmental effects and or contamination. In addition, 10 females showing irregular cycle during the period between allocation and the start of treatment were replacedwith surplus female animals selected from the same batch.
Positive control:
Not required for this study type.

Examinations

Parental animals: Observations and examinations:
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

DETAILED CLINICAL OBSERVATIONS: Yes
Once before commencement of treatment and at least once a week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). All observations were recorded for individual animals.

BODY WEIGHT: Yes
Males were weighed weekly from allocation to termination. Females were weighed weekly from all ocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1, 4, 7, 13 post partum and just before necropsy.

FOOD CONSUMPTION: Yes
The weight of food consumed by each cage of males and females was recorded weekly (whenever possible) during the pre-mating period starting from allocation. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 7 and 13 post partum starting from Day 1 post partum.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
As a part of the necropsy procedure, fasted samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable litters) randomly selected from each group. The following parameters were measured: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count, Differential leucocyte count, Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells, Platelets.

CLINICAL CHEMISTRY: Yes
s a part of the necropsy procedure, fasted samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable litters)randomly selected from each group. The following parameters were measured: Prothrombin time, Activated partial thromboplastin time, Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Bile acids, Inorganic phosphorus, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride. In addition, as part of the necropsy procedure, blood samples were taken from the abdominal vena cava of all parental male and female rats fromeach group, under isofluorane anaesthesia, to perform the determination of serum T3, T4 and TSH levels.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Grip strength and sensory reactivity to stimuli: Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength. For the females the tests were performed on Day 13 post partum (where possible). Motor activity assessment: Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group and the motor activity was measured (for approximately 5 minutes) by an automated activity recording device. For the females the tests were performed on Day 13 post partum (where possible).

IMMUNOLOGY: No
Oestrous cyclicity (parental animals):
Oestrous cyclicity was monitored by vaginal smears in all stock females for 1 week before allocation in order to exclude from the study females with irregular cycle. Vaginal smears were taken in the morning from Day 1 of allocation and during treatment period, up to positive identification of mating including 15 days before the pairing. Animals that exhibited irregular cycles were not included in the study. The vaginal smear data were examined to determine anomalies of the oestrous cycle and the pre-coital interval. Vaginal smears were also taken from all females, before dispatch to necropsy.
Sperm parameters (parental animals):
A detailed qualitative examination of the testes was performed on 5 randomly selected animals from the control and high dose groups. The evaluation, taking into account the tubular stages of the spermatogenic cycle, was conducted in order to identify treatment-related effects, such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing ofspermatogenic cells into the lumen. PAS-stained sections were used to identify the spermatogenic stages. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
Litter observations:
As soon as possible, after parturition was considered complete (Day 0 post partum), all pups (live and dead) were counted, sexed and live pups were identified. Live pups were individually weighed on Days 1, 4, 7 and 13 post partum. Pups killed or dying during the lactation period were weighed before the despatch to necropsy. Observations were performed once daily for all litters. All pups (surviving to treatment and not selected for culling) were sacrificed with the dams on Day 14 post partum.

The AGD of each pup was measured on Day 1 post partum and normalized to the cube root of body weight collected on Day 1 post partum. The number of nipples/areolae in male pups were counted in each pup on Day 13 post partum (no nipples were observed, therefore these data were not reported).
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
The clinical history of the animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices)

ORGAN WEIGHTS: Yes
Weights of the adrenals, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles, spleen, testes, thymus, thyroid and uterus were recorded.

HISTOPATHOLOGY: Yes
Tissues from 5 rats/sex (control and high dose group) were examined. The livers from the remaining rats from these groups and all rats from the intermediate dose groups were also examined.
Postmortem examinations (offspring):
All pups found dead in the cage were examined for external and internal abnormalities. All culled pups sacrificed at Day 4 post partum were subjected to an external examination. Sex was determined by internal gonads inspection. All live pups sacrificed at Day 14 post partum were killed and examined for external abnormalities and sex confirmation by gonadal inspection. All pups with abnormalities were retained in an appropriate fixative at the discretion of the Study Director.
Statistics:
Standard deviationswere calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The mean values, standard deviations and statistical analysis were calculated from actual values without rounding. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if ‘n’ was more than 5.
Reproductive indices:
Copulatory and fertiliy index were calcuated for males and females.
Offspring viability indices:
Pre-implantation loss; post-natal loss was calculated at PND4 and PND14

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In males, salivation and/or ataxia were observed during the pre-mating phase, mainly during the first week, in all animals at 750 mg/kg bw/d. In the females, slight to moderate ataxia and/or decreased activity and/or salivation were seen in 9/10 animals at 750 mg/kg bw/d, while salivation was observed in 3/10 females at 300 mg/kg bw/d during the pre-mating phase. Salivation was also observed in all females at 300 and 750 mg/kg bw/d during the post coitum and post partum periods. Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant changes in body weight and body weight gain were observed during the study in the treated animals, when compared to controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by treatment in both sexes during the study. A reduction of mean food consumption (statistically significant on Day 13 post partum) observed in females at 750 mg/kg bw/d during the post partum period was attributed to a single female with a amall litter (2 pups).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No relevant changes were recorded. A statistically significant increase of large unstained cells in females at 50 and 300 mg/kg bw/d was not dose related and was considered to be incidental.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Some statistically significant fluctuations of biochemical parameters were recorded, including a decrease in alkaline phosphatase in males at 50 and 750 mg/kg bw/d (18% and 22%, respectively); decreased bilirubin in males at 300 mg/kg bw/d (54%); increased glucose, chloride and sodium in females at 750 mg/kg bw/d (27%, 5% and 2%, respectively); increased chloride in females at 300 mg/kg bw/d (5%). Since no other relevant changes were recorded and findings were of minimal severity, they were considered to be of no toxicological significance. Triiodothyronine and thyroxine were increased in some males from all treated groups. Compared with controls, changes were 19% to 39%, with some relationship to dose level.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment-related alterations in motor activity, grip strength and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment. Statistically significantly lower landing footsplay was recorded in the males at 750 mg/kg bw/d, when compared to the control group. Due to the direction of the change (reduction), it was considered to be of no neurotoxicological significance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were seen in the liver of most high dose males and females sacrificed at the end of the study. The treatment-related liver changes consisted of minimal to mild centrilobular and/or midzonal hepatocytic hypertrophy, associated with cytoplasmic eosinophilia, morphologicall y represented by an enlarged size of the hepatocytes, possibly correlated to the hepatic enzyme ind uction. In the absence of degenerative changes, the hepatocytic hypertrophy was considered to be adaptive and not an adverse liver change.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No treatment-related anomalies were noted in the oestrous cycle.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
No treatment-related anomalies were noted in the oestrous cycle and pre-coital interval of the treated females, when compared to controls. One control female and three females from the mid-dose group did not mate. A total of 2 females of the control group and four from the mid-dose group were found not pregnant (3 of which did not mate). The number of copulation plugs was similar between control and treated groups.

The copulatory indices were 90% for controls, 70% for animals at 300 mg/kg bw/d and 100% for animals at 50 and 750 mg/kg bw/d. The fertility indices were 90% for controls, 60% for animals at 300 mg/kg bw/d and 100% for animals at 50 and 750 mg/kg bw/d. The reduction in copulatory and fertility indices observed in animals at 300 mg/kg bw/d was considered to be incidental, since no reductions of these parameters were observed at 50 or 750 mg/kg bw/d.

Gestation periods were similar in treated groups and controls. All dams gave birth between Days 22 and 23 post coitum. Corpora lutea, implantations and pre-implantation loss, total litter size and pre-natal loss (percentage) did ot show dose-related or treatment-related differences.

Details on results (P0)

There was no mortality in this study. In males, salivation and/or ataxia were observed during the pre-mating phase, mainly during the first week, in all animals at 750 mg/kg bw/d. In females, slight to moderate ataxia and/or decreased activity and/or salivation were seen in 9/10 animals at 750 mg/kg bw/d, while salivation was observed in 3/10 females at 300 mg/kg bw/d during the pre-mating phase. Salivation was also observed in all females at 300 and 750 mg/kg bw/d during the post coitum and post partum periods. Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item. Bodyweights, bodyweight gain and food consumption were unaffected by treatment. Haematology revealed a slight but statistically significant increase of large unstained cells in females at 50 and 300 mg/kg bw/d; this was not dose-related and was considered to be incidental. Clinical chemistry revealed a small number of statistically significant fluctuations, including a decrease in alkaline phosphatase in males at 50 and 750 mg/kg bw/d, decreased bilirubin in males at 300 mg/kg bw/d (54%); increased glucose, chloride and sodium in females at 750 mg/kg bw/d (27%, 5% and 2%, respectively); increased chloride in females at 300 mg/kg bw/d (5%). Since no other relevant changes were recorded and findings were of minimal severity, they were considered to be of no toxicological significance. Triiodothyronine and thyroxine were increased in some males from all treated groups. Compared with controls, changes were 19% to 39%, with some relationship to dose level. No treatment-related alterations in motor activity, grip strength and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment. Statistically significantly lower landing foot splay was recorded in the males at 750 mg/kg bw/d, when compared to the control group. Due to the direction of the change (reduction), it was considered to be of no neurotoxicological significance. A statistically significant increase in absolute and relative liver weight was recorded in males at 300 mg/kg bw/d and at the high dose level in both sexes, compared to controls. This finding was considered to be treatment-related on the basis of correlating histopathology. Changes in absolute and/or relative organ weights, statistically significant in high dose males and/or females, such as brain, kidneys and thyroid were not considered to be toxicologically relevant, in the absence of correlating histopathology. Histopathology revealed effects in the liver of most high dose males and females, consisting of minimal to mild centrilobular and/or mid-zonal hepatocyte hypertrophy, associated with cytoplasmic eosinophilia, morphologically represented by an enlarged size of the hepatocytes, possibly correlated to the hepatic enzyme induction. In the absence of degenerative changes or clinical chemistry correlates, the hepatocyte hypertrophy was considered to be adaptive and not adverse. Fertility and reproduction parameters were unaffected by treatment.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
750 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Pallor, apparently no food intake (milk) and small appearance, black tip or damaged tail were in general the clinical signs noted in control and treated pups.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Decrease of Thyroid Stimulating Hormone was recorded in some pups at the high dose level (28% in males and 46% in females). Due to the absence of other related changes, this finding was considered to be of no toxicological relevance.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No significant differences were observed in the weight of thyroid in treated pups, when compared to controls.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Autolysed abdominal and/or thoracic organs were generally observed in pups found dead at birth and in pups which died during the lactation period. No significant findings were seen in pups killed on Day 14 post partum.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No differences in the anogenital distance (normalised value), performed on Day 1 post partum, were seen between control and treated groups both for male and female pups. No nipples were observed in male pups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

No significant differences in total litter size, live litter size, mean pup loss, sex ratio and mean pup weights were observed among the treated dams and the controls at birth and on Days 1, 4 and 13 post partum.No differences in the anogenital distance (normalised value), performed on Day 1 post partum, were seen between control and treated groups both for male and female pups. No nipples were observed in male pups. Pallor, apparently no food intake (milk) and small appearance, black tip or damaged tail were in general the clinical signs noted in control and treated pups.No significant differences were observed in the weight of thyroid in treated pups, when compared to controls. Autolysed abdominal and/or thoracic organs were generally observed in pups found dead at birth and in pups which died during the lactation period. No significant findings were seen in pups killed on Day 14 post partum. No significant differences were observed in the weight of thyroid in treated pups, when compared to controls. A decrease in Thyroid Stimulating Hormone was recorded in some pups of Group 4 (28% in males and 46% in females). Due to the absence of other related changes, this finding was considered to be of no toxicological relevance.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Summary of reproductive and developmental parameters

 

0

50

300

750

Oestrus cycles pre-mate (#)

3.0

2.7

2.9

3.0

Copulatory index (M)

90

100

70

100

Fertility index (M)

80

100

60

100

Copulatory index (F)

90

100

70

100

Fertility index (F)

80

100

60

100

Corpora lutea (#)

15.25

14.70

16.83

14.90

Implantations (#)

14.75

14.00

16.83

14.70

Pre-implantation loss (%)

3.38

5.23

0.00

1.09

Pre-natal loss (%)

11.31

9.58

3.98

12.32

Gestation length (d)

22.25

22.50

22.17

22.20

Litter size (#) PND0

13.13

12.80

16.17

13.20

Viable litter size (#) PND0

12.25

12.70

16.00

13.00

Litter size (#) PND1

12.25

12.70

15.67

13.00

Litter size (#) PND4

12.13

12.70

15.50

12.90

Loss (%) PND4

6.70

2.00

3.92

2.13

Litter size (#) PND13

7.75

7.40

8.00

7.40

Loss (%) PND13

1.56

2.50

0.00

0.00

Pup weight (g) PND1

6.95

7.02

6.48

6.61

Pup weight (g) PND4

10.48

10.41

9.03

11.90

Pup weight (g) PND13

32.03

31.37

28.33

29.43

AGD (M)

2.18

2.01

1.89

2.20

AGD (F)

1.13

1.07

1.03

1.22

T3 (ng/mL) (M)

5.84

5.49

6.08

6.36

T4 (ng/mL) (M)

177

158

140

147

TSH (M)

1.41

1.13

1.16

1.02

T3 (ng/mL) (F)

5.96

5.65

5.98

6.83

T4 (ng/mL) (F)

196

179

159

164

TSH (F)

1.53

1.35

1.11

0.84*

*significantly different to controls (p<0.05)

Applicant's summary and conclusion

Conclusions:
There was no evidence of reproductive or developmental toxicity in this screening study; a NOAEL of 750 mg/kg bw/d is determined.
Executive summary:

In an OECD 422 screening study, groups of Sprague-Dawley rats (10/sex) were administered 1,3-propanediol, 2,2- bis(hydroxymethyl) -, allyl ether (in 0.5% aqueous carboxymethylcellulose) by gavage at dose levels of 0 (vehicle control), 50, 300 or 750 mg/kg bw/d. Males were dosed once a day, 7 days a week, 15 days prior to pairing, during pairing with females until the day before necropsy, for a total of 30/33 days. Females were treated for 15 days prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum, for a total of 48 to 61 days. No treatment-related anomalies were noted in the oestrous cycle and pre-coital interval of the treated females, when compared to controls.  One control female and three females from the mid-dose group did not mate. A total of 2 females of the control group and four from the mid-dose group were found not pregnant (3 of which did not mate). The number of copulation plugs was similar between control and treated groups. The copulatory indices were 90% for controls, 70% for animals at 300 mg/kg bw/d and 100% for animals at 50 and 750 mg/kg bw/d.  The fertility indices were 90% for controls, 60% for animals at 300 mg/kg bw/d and 100% for animals at 50 and 750 mg/kg bw/d.  The reduction in copulatory and fertility indices observed in animals at 300 mg/kg bw/d was considered to be incidental, since no reductions of these parameters were observed at 50 or 750 mg/kg bw/d. Gestation periods were similar in treated groups and controls. All dams gave birth between Days 22 and 23 post coitum.  Corpora lutea, implantations and pre-implantation loss, total litter size and pre-natal loss (percentage) did not show dose-related or treatment-related differences. No significant differences in total litter size, live litter size, mean pup loss, sex ratio and mean pup weights were observed among the treated dams and the controls at birth and on Days 1, 4 and 13 post partum. No differences in the anogenital distance (normalised value), performed on Day 1 post partum, were seen between control and treated groups both for male and female pups.  No nipples were observed in male pups.  Pallor, apparently no food intake (milk) and small appearance, black tip or damaged tail were in general the clinical signs noted in control and treated pups.No significant differences were observed in the weight of thyroid in treated pups, when compared to controls.  Autolysed abdominal and/or thoracic organs were generally observed in pups found dead at birth and in pups which died during the lactation period. No significant findings were seen in pups killed on Day 14 post partum.  No significant differences were observed in the weight of thyroid in treated pups, when compared to controls.  A decrease in Thyroid Stimulating Hormone was recorded in some pups of Group 4 (28% in males and 46% in females). Due to the absence of other related changes, this finding was considered to be of no toxicological relevance. There was no evidence of reproductive or developmental toxicity in this screening study; a NOAEL of 750 mg/kg bw/d is determined.