Praseodymium(III,IV) oxide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 August 2012 - 1 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:CD (SD)
- Age at study initiation: On dispatch from the supplier, the males were approximately 5 - 6 weeks old and the females were approximately 4 - 6 weeks old. At initiation of dosing, the animals were approximately 8 - 10 weeks old.
- Weight at study initiation: On dispatch from the supplier, the males weighed 158 - 200 g and the females weighed 112 - 156 g. At initiation of dosing, the animals weighed 330 - 460 g for males and 192 - 269 g for females.
- Housing: Animals were housed in cages, suspended on a series of racks. Male and female cages were racked separately.
Animals were housed in polycarbonate cages with stainless steel grid tops and solid bottoms, with approximate dimensions of 61 x 43.5 x 24 cm. Cages were suspended on moveable racks and fitted with water bottles and integrated stainless steel food hoppers.
The animals were initially housed 2 or 3 per cage. A few days prior to pairing for mating, males were transferred to individual cages with a stainless steel grid insert. After mating, the males were re-housed with their original cage mates.
Mated females were transferred to individual solid bottom cages. White paper tissue was supplied as nesting material from Day 20 of gestation.
- Diet (e.g. ad libitum): Rat and Mouse Breeder Diet No. 3 (Expanded), supplied by Special Diets Services Limited, Witham, Essex, UK.was provided ad libitum.
- Water (e.g. ad libitum): Water taken from the public supply (Scottish Water, Edinburgh, UK) was available ad libitum.
- Acclimation period: 27 days.
- Source: Charles River Limited, Margate, Kent, UK.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23 °C
- Humidity (%): 40 - 70 %
- Air changes (per hr): A minimum of 10 air changes per hour.
- Photoperiod (hrs dark / hrs light): A 12 hour light/dark cycle was in operation.

IN-LIFE DATES: From: 27 August 2012 To: 12 October 2012

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % w/v Methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The dosing formulations were prepared weekly, stored at ambient temperature and dispensed daily. All formulations were used within the 8 day stability period that was established previously at the testing laboratory.
The dosing formulations were stirred for at least 30 minutes prior to and throughout dosing.

VEHICLE
- Concentration in vehicle: 10, 30 or 100 mg/mL for the 100, 300 and 1000 mg/kg/day dose levels, respectively.
- Dose volume: 10 mL/kg. The volume administered to each animal was determined on each day by the weight of that animal recorded immediately prior to dosing, except during late gestation; from Day 16 of gestation until when parturition was complete, the dose volume was determined by the weight of the animal on Day 16 of gestation.

Details on mating procedure:

- M/F ratio per cage: Pairings were on a one male to one female basis.
- Length of cohabitation: A few days prior to the initiation of mating, the males were separated into individual grid bottom cages. Animals were paired in ascending numerical order within each group. Each female was transferred to the cage of its appropriate co group male near the end of the work day, where it remained until mating had occurred or 14 nights had elapsed.
- Proof of pregnancy: Vaginal lavages were taken daily early each morning from the day of pairing until mating occurred and the stage of oestrus observed in each lavage was recorded. The day of presence of sperm in such a lavage was designated Day 0 of gestation. The time taken for each female to show a positive mating sign was evaluated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYTICAL METHOD
Analyses were performed by ICP-Optical Emission Spectroscopy using a validated analytical procedure. Samples to be analysed were transferred at ambient temperature to the analytical laboratory.

CONCENTRATION AND HOMOGENEITY ANALYSIS
Duplicate 0.5 mL samples were collected from the top, middle and bottom (duplicate middle only for control) from each formulation at each sampling time point (control samples not collected at Weeks 5 and 6) and were sent to the analytical laboratory.
Additional 0.5 mL triplicate samples were collected from the top, middle and bottom (triplicate middle only from control) from each formulation at each sampling time point (control samples not collected at Weeks 5 and 6) and were retained as back-up samples.
The results of the sample concentration were considered acceptable if they were within ± 10 % of theoretical concentration. For homogeneity, the criterion for acceptability was a relative standard deviation (RSD) of concentration of ≤10 % for each group.

Duration of treatment / exposure:

The males were dosed for 4 weeks, starting 2 weeks prior to mating. The females were dosed 2 weeks prior to mating, throughout mating, gestation and through to at least day 4 of lactation.
Several females were not dosed on their respective days of parturition due to the animals starting to give birth prior to the commencement of dosing on that day.

Frequency of treatment:

Once daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 animals per sex per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were agreed after a review of existing relevant toxicological data, including a 14 day dose range finding study conducted at the testing facility in which dose levels up to 1000 mg/kg/day produced no adverse reaction to treatment.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality/Moribundity checks on all animals were carried out early morning and as late as possible each day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week starting in pre-trial, all animals received a detailed clinical examination. All animals were examined for reaction to treatment at approximately hourly intervals up to 4 hours post dose on each day of dosing.0

BODY WEIGHT: Yes
- Time schedule for examinations: Bodyweights were recorded one week prior to the start of treatment. From the start of treatment, the individual bodyweights were recorded daily.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was measured for both sexes weekly, starting 1 week prior to dosing until pairing for mating. After pairing, the female food consumption was measured over Days 0 - 7, 7 - 14 and 14 - 20 of gestation and Days 0 - 4 of lactation. Male food consumption did not recommence after pairing for mating.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OTHER: Ophthalmoscopic examinations were carried out, haematology, coagulation and clinical chemistry parameters were evaluated and full functional tests to investigate any neurotoxic effects of the test material were also conducted.

Oestrous cyclicity (parental animals):

As part of the mating procedure, vaginal lavages were taken daily early each morning from the day of pairing until mating occurred (for up to 14 days) and the stage of oestrus observed in each lavage was recorded.

Sperm parameters (parental animals):

Parameters examined in [P] male parental generations: testis and epididymis weight.

Litter observations:

F1 GENERATION
- Litter size and sex: The numbers of live and dead pups born in each litter was recorded as soon as possible after completion of parturition on Day 0 of lactation.
- Clinical observations: The live pups were counted and examined for the presence of milk in the stomach and for any externally visible abnormalities daily. Where practicable, any pups that were found dead or killed during lactation were sexed and appropriately examined. All pups were externally normal and were discarded following examination.
- Bodyweights: Each litter was weighed en masse (by sex) on Days 1 and 4 of lactation.

OBSERVATIONS ON FEMALES WITH LITTERS DURING LACTATION
The females were allowed to litter normally. Any observed difficulty or prolongation of parturition was recorded. The day of birth of the litter (day on which the first pups were born) was designated as Day 0 of lactation. The duration of gestation was calculated.
Deficiencies in maternal care were recorded: inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups, or apparently inadequate lactation or feeding. White paper tissue was supplied to each mother for incorporation in the nest which was replaced when it became soiled.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: The males were killed when mating was completed and the animals had been dosed for at least 4 weeks.
- Maternal animals: The females were killed between Day 5 and 7 of lactation. Animals were killed by exposure to carbon dioxide followed by exsanguination.

GROSS NECROPSY
- All adult animals were subjected to a complete necropsy examination which included evaluation of the carcass and musculoskeletal system, all external surfaces and orifices, cranial cavity and external surfaces of the brain and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. The reproductive tracts of all females were examined.
The following organs were weighed at necropsy for all adult animals before sampling and preservation: brain, epididymis, adrenal gland, pituitary gland, prostate gland, thyroid gland, heart, kidney, liver, lung, ovary, spleen, testis, thymus and uterus.

HISTOPATHOLOGY
Representative samples of the following tissues were collected from all adult animals and preserved as appropriate: animal identification (microchip), aortic artery, bone marrow smear, bone marrow (femur and sternum), femur (bone), rib (bone), sternum (bone), brain, cervix, epididymis, eye, adrenal gland, harderian gland, lacrimal gland, mammary gland, parathyroid gland, pituitary gland, prostate gland, salivary gland, seminal vesicle gland, thyroid gland, gross lesions/masses, gut-associated lymphoid tissue, heart, kidney, large intestine (caecum, colon and rectum), larynx, liver, lung, lymph node (mandibular and mesenteric), skeletal muscle, nasal cavity, optic nerve, sciatic nerve, oesophagus, ovary, oviduct, pancreas, pharynx, skin, small intestine (duodenum, ileum and jejunum), spinal cord, spleen, stomach, testis, thymus, tongue, trachea, ureter, urinary bladder, uterus and vagina.
Histopathological evaluation of all tissues were undertaken for the 5 selected males and females in the control and high dose groups (the same animals that were used for the laboratory investigations).

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were killed by intra-peritoneal injection of sodium pentobarbitone between Days 5 and 7 of lactation.

GROSS NECROPSY
- Where practicable, animals found dead or killed prematurely were sexed and then checked for the presence of milk in the stomach and for the presence of any externally visible abnormalities. Surviving pups were also examined for externally visible abnormalities. All pups were then discarded.

Statistics:

Where required to assist with interpretation, tests were applied to determine the statistical significance of observed differences between control and groups receiving test material. Unless otherwise stated, all statistical tests were two-sided and performed at the 5 % significance level using in-house software. Pairwise comparisons were only performed against the control group.

Bodyweight and food consumption data, haematology, coagulation, clinical chemistry and selected FOB and motor activity data was analysed for homogeneity of variance using the ‘F Max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test; i.e., pairwise comparisons were made only if the overall F test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).

Organ weight data was analysed as above, and by analysis of covariance (ANCOVA) using terminal bodyweight as the covariate.

Reproductive indices:

For each group:
Male fertility index = Number siring a litter / Number paired
Female fertility index = Number pregnant / Number paired
Gestation index = Number bearing live pups / Number pregnant

Offspring viability indices:

For each litter and group:
Birth index = Total number of pups born (live and dead) / Number of implantation scars
Live birth index = Number of pups live on Day 0 of lactation / Total number born (live and dead)
Viability index = Number of pups live on Day 4 of lactation / Number live on Day 0

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see "Details on Results (parental animals)"

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see "Details on Results (parental animals)"

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see "Details on Results (parental animals)"

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths during the course of this study.
There were no clinical signs during the course of the study that were considered to be related to administration of the test material.
Animal 60 (female, 100 mg/kg/day) was not dosed on Days 2 - 4 of lactation, prior to scheduled euthanasia on Day 5 of lactation, due to displaying signs of pale skin and eyes, walking on tiptoes, dark/red discharge from the vagina, weight loss, staining on fur (ventral abdomen), body hunched and piloerection.
Animal 68 (female: 300 mg/kg/day) was not dosed on Day 1 of lactation due to displaying signs of pale skin and eyes, walking on tiptoes, staining on fur (ventral abdomen), abnormal vocalisation (in hand of technician) and body hunched. At Day 2 of lactation, the animal suffered a total litter loss, however these signs were no longer present and the animal was dosed Days 2 - 4 of lactation, prior to scheduled euthanasia on Day 5 of lactation.
These signs were considered to be related to difficulties experienced by these animals during parturition and/or the early part of lactation, and were considered to be incidental given their low absolute incidence and the absence of similar signs in animals receiving 1000 mg/kg/day.

BODY WEIGHT AND FOOD CONSUMPTION
Bodyweight gains were similar between control animals and animals receiving the test material.
Mean bodyweight gain was slightly higher than controls in females receiving 1000 mg/kg/day over gestation Day 0 - 20 (approximately 13 % more than the control group mean); however this was considered to be incidental given the considerable inter-animal variation within both the control and 1000 mg/kg/day dose groups.
Food consumption was similar between control animals and animals receiving the test material.
Mean food consumption was noted to be higher than the controls in all dose groups between Days 0 - 4 of lactation; however this was considered incidental and a consequence of a slightly low mean Control group value.

REPRODUCTIVE FUNCTION
Mating performance, fertility indices, corpora lutea and implantation counts, duration of gestation, and the mean number of live pups born per litter, were similar between control females and females receiving the test material. The data are summarised in Tables 1 and 2.

ORGAN WEIGHTS
No test-material related organ weight changes were noted. There were isolate organ weight values that were different from their respective controls. There were, however, no patterns, trends or correlating data to suggest these values were toxicologically relevant. Thus, the organ weight differences observed were considered incidental and unrelated to administration of the test material.

GROSS PATHOLOGY
No test-material related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rat on this type of study, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of the test material.

HISTOPATHOLOGY
Minimal granulomas containing pigmented material were observed in the lungs of two animals given 1000 mg/kg/day (32 Male and 71 Female). This was considered to be some type of foreign inhaled material, presumably due to reflux of test material during the dosing procedure.
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat on this type of study, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test material.
Thymic atrophy and gastritis in females were considered to be secondary to stress associated with parturition.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects were seen on reproductive performance up to the maximum test concentrations.

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

see "Details on Results (parental animals)"

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

LITTER SIZE AND SURVIVAL, LITTER AND PUP WEIGHTS
The mean number of live pups born per litter was similar between control females and those receiving the test material.
Litter survival, litter weights and mean pup weights were similar between litters derived from control females and litters derived from females receiving the test material.
Total litter losses were suffered by 1/10 females at 100 mg/kg/day, and 1/10 females at 300 mg/kg/day versus none in the control females. Given the low absolute incidence and the absence of any litter losses at 1000 mg/kg/day, these litter losses were considered incidental.
The data is summarised in Tables 3 and 4.

OBSERVATIONS AMONG DAMS/PUPS
The nature and incidence of the observations recorded for dams and their pups were similar between control females and females receiving the test material.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects observed.

Reproductive effects observed:

not specified

Table 1: Mating Performance and Fertility Indices

Number of Nights to Positive Mating Sign	Dose Level (mg/kg/day)
	0	100	300	1000
	Number of Animals (number not becoming pregnant)
1 2 3 4	0 6 1 3	1 5 2 2	1 2 4 3	1 5 2 2
No clear indication of mating Median no. nights to positive mating sign Number passing one oestrus	0 2 0	0 2 0	0 3 0	0 2 0
Number of males paired Number of siring males Male Fertility Index (%) Number of females paired Number pregnant Female Fertility Index (%)	10 10 100 10 10 100	10 10 100 10 10 100	10 10 100 10 10 100	10 10 100 10 10 100

 

Table 2: Group Mean Duration of Gestation and Overall Litter Performance

	Dose Level (mg/kg/day)
	0	100	300	1000
Number Pregnant	10	10	10	10
Duration of Gestation (Days) 20 21 22 23 Mean Duration	1 5 4 0 21.3	0 5 5 0 21.5	0 3 6 1 21.8	0 3 6 1 21.8
Number of females producing a live litter Gestation index as %	10 100	10 100	10 100	10 100
Mean number of corpora lutea sites* per pregnancy ± SD	17.6 ± 1.4	18.3 ± 2.1	17.7 ± 3.5	19.2 ± 3.9
Mean number of implant sites* per pregnancy ± SD	15.6 ± 1.3	16.3 ± 1.7	15.6 ± 2.3	15.1 ± 2.3
Mean total number of pups born* per litter ± SD	13.8 ± 1.7	14.2 ± 1.9	13.7 ± 2.7	13.5 ± 3.1
Mean number of live pups* per litter ± SD Day 0 of lactation Day 1 of lactation Day 4 of lactation	13.8 ± 1.7 13.8 ± 1.7 13.5 ± 1.7	13.8 ± 1.5 13.6 ± 1.5 13.6 ± 1.5	13.7 ± 2.7 13.4 ± 2.7 13.3 ± 2.6	13.4 ± 3.2 13.3 ± 3.1 13.1 ± 3.1
Total no. males** on Day 1 of lactation (%) Total no. females** on Day 1 of lactation (%)	57 (46)  66 (54)	45 (42) 62 (58)	54 (50) 53 (50)	58 (44) 75 (56)

* Excludes litters where all pups died

** Excludes litters where all pups died, excludes litters with mis-counted/sexed pups

 

Table 3: Group Mean F1 Survival Indices

		Dose Level (mg/kg/day)
		0	100	300	1000
Birth Index	Mean Litter Index Number losing >2 pups Number of litters	89 1 10	87 3 10	86 5 10	89 2 10
Live Birth Index	Mean Litter Index (%) Number losing >1 pup Number of Litters	100 0 10	97 1 10	100 0 10	99 0 10
Viability Index Days 1 - 4	Mean Litter Index (%) Number losing >3 pups Number of Litters	98 0 10	89 1 10	88 1 10	98 0 10

 

Table 4: Group Mean Litter and Pup Weight (g) ± Standard Deviation

Day of Lactation	Dose Level (mg/kg/day)
	0	100	300	1000
Litter Day 1 Day 4	86 ± 7 123 ± 19	83 ± 7 125 ± 13	88 ± 15 128 ± 23	88 ± 16 125 ± 24
Mean of Litter Mean Pup Weight
Males Day 1 Day 4	6.6 ± 1.0 9.5 ± 1.8	6.5 ± 0.5 9.6 ± 1.1	6.9 ± 0.8 10.0 ± 1.5	7.0 ± 1.0 10.1 ± 1.6
Females Day 1 Day 4	6.3 ± 0.9 9.0 ± 1.8	6.0 ± 0.2 9.0 ± 0.7	6.5 ± 0.7 9.6 ± 1.5	6.6 ± 0.9 9.5 ± 1.6

Conclusions:

Under the conditions of this study in Sprague-Dawley strain rats the No Observed Adverse Effect Level (NOAEL) for reproductive effects was considered to be 1000 mg/kg/day for both males and females.

Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the test material in accordance with the standardised guideline OECD 422.

Three groups of 10 male and 10 female Sprague-Dawley rats of the Crl:CD(SD) strain were dosed once daily, by oral gavage, with the test material at dose levels of 100, 300, or 1000 mg/kg/day. Another group of 10 male and 10 female rats were dosed with the vehicle (0.5 % w/v methylcellulose) following the same dosing regimen as the treated animals and were used as controls.

The males were treated for 2 weeks prior to mating, then through mating, until the day prior to necropsy (ca. 4 weeks of treatment). Females were treated for 2 weeks prior to mating, then through mating, gestation and until at least Day 4 of lactation (ca. 6 weeks of treatment). 

The following parameters and end points were evaluated: clinical signs, bodyweights, bodyweight changes, food consumption, ophthalmology, detailed functional tests and observations, clinical pathology parameters (haematology, coagulation and clinical chemistry), gross necropsy findings, organ weights, and histopathological examinations, mating and pregnancy performance, fertility, maternal care and pup performance (litter survival and pup weights).

Mating performance, fertility indices, corpora lutea and implantation counts, duration of gestation, and the mean number of live pups born per litter were similar between control and treated females. Litter survival, litter weights and mean pup weights were similar between litters derived from control and treated females.

In conclusion, under the conditions of this study the No Observed Adverse Effect Level (NOAEL) for systemic toxicity (parents) was considered to be 1000 mg/kg/day and the reproductive/developmental No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day, for both males and females.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Due to the absence of adverse effects on fertility in an OECD 422 screening reproductive / developmental toxicity study, the Two Generation Reproductive Toxicity study is considered scientifically unjustified and hence no further testing is proposed for this endpoint.

Short description of key information:

No adverse effects on fertility were observed in an OECD 422 screening reproductive / developmental toxicity study.

Effects on developmental toxicity

Description of key information

No effects on developmental toxicity were observed in an OECD 422 screening reproductive / developmental toxicity study.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 August 2012 - 1 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:CD (SD)
- Age at study initiation: On dispatch from the supplier, the males were approximately 5 - 6 weeks old and the females were approximately 4 - 6 weeks old. At initiation of dosing, the animals were approximately 8 - 10 weeks old.
- Weight at study initiation: On dispatch from the supplier, the males weighed 158 - 200 g and the females weighed 112 - 156 g. At initiation of dosing, the animals weighed 330 - 460 g for males and 192 - 269 g for females.
- Housing: Animals were housed in cages, suspended on a series of racks. Male and female cages were racked separately.
Animals were housed in polycarbonate cages with stainless steel grid tops and solid bottoms, with approximate dimensions of 61 x 43.5 x 24 cm. Cages were suspended on moveable racks and fitted with water bottles and integrated stainless steel food hoppers.
The animals were initially housed 2 or 3 per cage. A few days prior to pairing for mating, males were transferred to individual cages with a stainless steel grid insert. After mating, the males were re-housed with their original cage mates.
Mated females were transferred to individual solid bottom cages. White paper tissue was supplied as nesting material from Day 20 of gestation.
- Diet (e.g. ad libitum): Rat and Mouse Breeder Diet No. 3 (Expanded), supplied by Special Diets Services Limited, Witham, Essex, UK was provided ad libitum.
- Water (e.g. ad libitum): Water taken from the public supply (Scottish Water, Edinburgh, UK) was available ad libitum.
- Acclimation period: 27 days.
- Source: Charles River Limited, Margate, Kent, UK.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23 °C
- Humidity (%): 40 - 70 %
- Air changes (per hr): A minimum of 10 air changes per hour.
- Photoperiod (hrs dark / hrs light): A 12 hour light/dark cycle was in operation.

IN-LIFE DATES: From: 27 August 2012 To: 12 October 2012

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % w/v Methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The dosing formulations were prepared weekly, stored at ambient temperature and dispensed daily. All formulations were used within the 8 day stability period that was established previously at the testing laboratory.
The dosing formulations were stirred for at least 30 minutes prior to and throughout dosing.

VEHICLE
- Concentration in vehicle: 10, 30 or 100 mg/mL for the 100, 300 and 1000 mg/kg/day dose levels, respectively.
- Dose volume: 10 mL/kg. The volume administered to each animal was determined on each day by the weight of that animal recorded immediately prior to dosing, except during late gestation; from Day 16 of gestation until when parturition was complete, the dose volume was determined by the weight of the animal on Day 16 of gestation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYTICAL METHOD
Analyses were performed by ICP-Optical Emission Spectroscopy using a validated analytical procedure. Samples to be analysed were transferred at ambient temperature to the analytical laboratory.

CONCENTRATION AND HOMOGENEITY ANALYSIS
Duplicate 0.5 mL samples were collected from the top, middle and bottom (duplicate middle only for control) from each formulation at each sampling time point (control samples not collected at Weeks 5 and 6) and were sent to the analytical laboratory.
Additional 0.5 mL triplicate samples were collected from the top, middle and bottom (triplicate middle only from control) from each formulation at each sampling time point (control samples not collected at Weeks 5 and 6) and were retained as back-up samples.
The results of the sample concentration were considered acceptable if they were within ± 10 % of theoretical concentration. For homogeneity, the criterion for acceptability was a relative standard deviation (RSD) of concentration of ≤10 % for each group.

Details on mating procedure:

- M/F ratio per cage: Pairings were on a one male to one female basis.
- Length of cohabitation: A few days prior to the initiation of mating, the males were separated into individual grid bottom cages. Animals were paired in ascending numerical order within each group. Each female was transferred to the cage of its appropriate co group male near the end of the work day, where it remained until mating had occurred or 14 nights had elapsed.
- Proof of pregnancy: Vaginal lavages were taken daily early each morning from the day of pairing until mating occurred and the stage of oestrus observed in each lavage was recorded. The day of presence of sperm in such a lavage was designated Day 0 of gestation. The time taken for each female to show a positive mating sign was evaluated.

Duration of treatment / exposure:

The males were dosed for 4 weeks, starting 2 weeks prior to mating. The females were dosed 2 weeks prior to mating, throughout mating, gestation and through to at least day 4 of lactation.
Several females were not dosed on their respective days of parturition due to the animals starting to give birth prior to the commencement of dosing on that day.

Frequency of treatment:

Once daily

Duration of test:

Approximately 4 weeks for the males and approximately 6 weeks for the females.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 animals per sex per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were agreed after a review of existing relevant toxicological data, including a 14 day dose range finding study conducted at the testing facility in which dose levels up to 1000 mg/kg/day produced no adverse reaction to treatment.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality/Moribundity checks on all animals were carried out early morning and as late as possible each day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week starting in pre-trial, all animals received a detailed clinical examination. All animals were examined for reaction to treatment at approximately hourly intervals up to 4 hours post dose on each day of dosing. Furthermore, once during the pre-treatment period (week -1) and weekly thereafter, a more detailed examination was made on all animals.

BODYWEIGHT: Yes
- Time schedule for examinations: Bodyweights were recorded one week prior to the start of treatment. From the start of treatment, the individual bodyweights were recorded daily.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was measured for both sexes weekly, starting 1 week prior to dosing until pairing for mating. After pairing, the female food consumption was measured over Days 0 - 7, 7 - 14 and 14 - 20 of gestation and Days 0 - 4 of lactation. Male food consumption did not recommence after pairing for mating.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- The females were killed between Day 5 and 7 of lactation. Animals were killed by exposure to carbon dioxide followed by exsanguination.
- All adult animals were subjected to a complete necropsy examination which included evaluation of the carcass and musculoskeletal system, all external surfaces and orifices, cranial cavity and external surfaces of the brain and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. The reproductive tracts of all females were examined.
The following organs were weighed at necropsy for all adult animals before sampling and preservation: brain, adrenal gland, pituitary gland, thyroid gland, heart, kidney, liver, lung, ovary, spleen, thymus and uterus.

OTHER: Ophthalmoscopic examinations were carried out, haematology, coagulation and clinical chemistry parameters were evaluated and full functional tests to investigate any neurotoxic effects of the test material were also conducted.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes. The reproductive tracts of all females were examined for signs of implantation, with the number of implantation sites being recorded. In addition , the total number of corpora lutea graviditatis on the ovaries of each female was recorded.

Fetal examinations:

F1 GENERATION
- Litter size and sex: The numbers of live and dead pups born in each litter was recorded as soon as possible after completion of parturition on Day 0 of lactation.
- Clinical observations: The live pups were counted and examined for the presence of milk in the stomach and for any externally visible abnormalities daily. Where practicable, any pups that were found dead or killed during lactation were sexed and appropriately examined. All pups were externally normal and were discarded following examination.
- Bodyweights: Each litter was weighed en masse (by sex) on Days 1 and 4 of lactation.

Statistics:

Where required to assist with interpretation, tests were applied to determine the statistical significance of observed differences between control and groups receiving test material. Unless otherwise stated, all statistical tests were two-sided and performed at the 5 % significance level using in-house software. Pairwise comparisons were only performed against the control group.

Bodyweight and food consumption data, haematology, coagulation, clinical chemistry and selected FOB and motor activity data was analysed for homogeneity of variance using the ‘F Max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test; i.e., pairwise comparisons were made only if the overall F test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).

Organ weight data was analysed as above, and by analysis of covariance (ANCOVA) using terminal bodyweight as the covariate.

Indices:

For each group:
Male fertility index = Number siring a litter / Number paired
Female fertility index = Number pregnant / Number paired
Gestation index = Number bearing live pups / Number pregnant

For each litter and group:
Birth index = Total number of pups born (live and dead) / Number of implantation scars
Live birth index = Number of pups live on Day 0 of lactation / Total number born (live and dead)
Viability index = Number of pups live on Day 4 of lactation / Number live on Day 0

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths during the course of this study.
There were no clinical signs during the course of the study that were considered to be related to administration of the test material.
Animal 60 (female, 100 mg/kg/day) was not dosed on Days 2 - 4 of lactation, prior to scheduled euthanasia on Day 5 of lactation, due to displaying signs of pale skin and eyes, walking on tiptoes, dark/red discharge from the vagina, weight loss, staining on fur (ventral abdomen), body hunched and piloerection.
Animal 68 (female: 300 mg/kg/day) was not dosed on Day 1 of lactation due to displaying signs of pale skin and eyes, walking on tiptoes, staining on fur (ventral abdomen), abnormal vocalisation (in hand of technician) and body hunched. At Day 2 of lactation, the animal suffered a total litter loss, however these signs were no longer present and the animal was dosed Days 2 - 4 of lactation, prior to scheduled euthanasia on Day 5 of lactation.
These signs were considered to be related to difficulties experienced by these animals during parturition and/or the early part of lactation, and were considered to be incidental given their low absolute incidence and the absence of similar signs in animals receiving 1000 mg/kg/day.

BODY WEIGHT AND FOOD CONSUMPTION
Bodyweight gains were similar between control animals and animals receiving the test material.
Mean bodyweight gain was slightly higher than controls in females receiving 1000 mg/kg/day over gestation Day 0 - 20 (approximately 13 % more than the control group mean); however this was considered to be incidental given the considerable inter-animal variation within both the control and 1000 mg/kg/day dose groups.
Food consumption was similar between control animals and animals receiving the test material.
Mean food consumption was noted to be higher than the controls in all dose groups between Days 0 - 4 of lactation; however this was considered incidental and a consequence of a slightly low mean Control group value.

REPRODUCTIVE FUNCTION
Mating performance, fertility indices, corpora lutea and implantation counts, duration of gestation, and the mean number of live pups born per litter, were similar between control females and females receiving the test material. The data are summarised in Tables 1 and 2.

ORGAN WEIGHTS
No test-material related organ weight changes were noted. There were isolate organ weight values that were different from their respective controls. There were, however, no patterns, trends or correlating data to suggest these values were toxicologically relevant. Thus, the organ weight differences observed were considered incidental and unrelated to administration of the test material.

GROSS PATHOLOGY
No test-material related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rat on this type of study, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of the test material.

HISTOPATHOLOGY
Minimal granulomas containing pigmented material were observed in the lungs of two animals given 1000 mg/kg/day (32 Male and 71 Female). This was considered to be some type of foreign inhaled material, presumably due to reflux of test material during the dosing procedure.
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat on this type of study, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test material.
Thymic atrophy and gastritis in females were considered to be secondary to stress associated with parturition.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
REPRODUCTIVE FUNCTION
Mating performance, fertility indices, corpora lutea and implantation counts, duration of gestation, and the mean number of live pups born per litter, were similar between control females and females receiving the test material. The data are summarised in Tables 1 and 2.

LITTER SIZE AND SURVIVAL, LITTER AND PUP WEIGHTS
The mean number of live pups born per litter was similar between control females and those receiving the test material.
Litter survival, litter weights and mean pup weights were similar between litters derived from control females and litters derived from females receiving the test material.
Total litter losses were suffered by 1/10 females at 100 mg/kg/day, and 1/10 females at 300 mg/kg/day versus none in the control females. Given the low absolute incidence and the absence of any litter losses at 1000 mg/kg/day, these litter losses were considered incidental.
The data is summarised in Tables 3 and 4.

OBSERVATIONS AMONG DAMS/PUPS
The nature and incidence of the observations recorded for dams and their pups were similar between control females and females receiving the test material.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related efffects observed.

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Mating Performance and Fertility Indices

Number of Nights to Positive Mating Sign	Dose Level (mg/kg/day)
	0	100	300	1000
	Number of Animals (number not becoming pregnant)
1 2 3 4	0 6 1 3	1 5 2 2	1 2 4 3	1 5 2 2
No clear indication of mating Median no. nights to positive mating sign Number passing one oestrus	0 2 0	0 2 0	0 3 0	0 2 0
Number of males paired Number of siring males Male Fertility Index (%) Number of females paired Number pregnant Female Fertility Index (%)	10 10 100 10 10 100	10 10 100 10 10 100	10 10 100 10 10 100	10 10 100 10 10 100

 

Table 2: Group Mean Duration of Gestation and Overall Litter Performance

	Dose Level (mg/kg/day)
	0	100	300	1000
Number Pregnant	10	10	10	10
Duration of Gestation (Days) 20 21 22 23 Mean Duration	1 5 4 0 21.3	0 5 5 0 21.5	0 3 6 1 21.8	0 3 6 1 21.8
Number of females producing a live litter Gestation index as %	10 100	10 100	10 100	10 100
Mean number of corpora lutea sites* per pregnancy ± SD	17.6 ± 1.4	18.3 ± 2.1	17.7 ± 3.5	19.2 ± 3.9
Mean number of implant sites* per pregnancy ± SD	15.6 ± 1.3	16.3 ± 1.7	15.6 ± 2.3	15.1 ± 2.3
Mean total number of pups born* per litter ± SD	13.8 ± 1.7	14.2 ± 1.9	13.7 ± 2.7	13.5 ± 3.1
Mean number of live pups* per litter ± SD Day 0 of lactation Day 1 of lactation Day 4 of lactation	13.8 ± 1.7 13.8 ± 1.7 13.5 ± 1.7	13.8 ± 1.5 13.6 ± 1.5 13.6 ± 1.5	13.7 ± 2.7 13.4 ± 2.7 13.3 ± 2.6	13.4 ± 3.2 13.3 ± 3.1 13.1 ± 3.1
Total no. males** on Day 1 of lactation (%) Total no. females** on Day 1 of lactation (%)	57 (46)  66 (54)	45 (42) 62 (58)	54 (50) 53 (50)	58 (44) 75 (56)

* Excludes litters where all pups died

** Excludes litters where all pups died, excludes litters with mis-counted/sexed pups

 

Table 3: Group Mean F1 Survival Indices

		Dose Level (mg/kg/day)
		0	100	300	1000
Birth Index	Mean Litter Index Number losing >2 pups Number of litters	89 1 10	87 3 10	86 5 10	89 2 10
Live Birth Index	Mean Litter Index (%) Number losing >1 pup Number of Litters	100 0 10	97 1 10	100 0 10	99 0 10
Viability Index Days 1 - 4	Mean Litter Index (%) Number losing >3 pups Number of Litters	98 0 10	89 1 10	88 1 10	98 0 10

 

Table 4: Group Mean Litter and Pup Weight (g) ± Standard Deviation

Day of Lactation	Dose Level (mg/kg/day)
	0	100	300	1000
Litter Day 1 Day 4	86 ± 7 123 ± 19	83 ± 7 125 ± 13	88 ± 15 128 ± 23	88 ± 16 125 ± 24
Mean of Litter Mean Pup Weight
Males Day 1 Day 4	6.6 ± 1.0 9.5 ± 1.8	6.5 ± 0.5 9.6 ± 1.1	6.9 ± 0.8 10.0 ± 1.5	7.0 ± 1.0 10.1 ± 1.6
Females Day 1 Day 4	6.3 ± 0.9 9.0 ± 1.8	6.0 ± 0.2 9.0 ± 0.7	6.5 ± 0.7 9.6 ± 1.5	6.6 ± 0.9 9.5 ± 1.6

Conclusions:

Under the conditions of this study in Sprague-Dawley strain rats the No Observed Adverse Effect Level (NOAEL) for developmental effects was considered to be 1000 mg/kg/day.

Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the test material in accordance with the standardised guideline OECD 422.

Three groups of 10 male and 10 female Sprague-Dawley rats of the Crl:CD(SD) strain were dosed once daily, by oral gavage, with the test material at dose levels of 100, 300, or 1000 mg/kg/day. Another group of 10 male and 10 female rats were dosed with the vehicle (0.5 % w/v methylcellulose) following the same dosing regimen as the treated animals and were used as controls.

The males were treated for 2 weeks prior to mating, then through mating, until the day prior to necropsy (ca. 4 weeks of treatment). Females were treated for 2 weeks prior to mating, then through mating, gestation and until at least Day 4 of lactation (ca. 6 weeks of treatment). 

The following parameters and end points were evaluated: clinical signs, bodyweights, bodyweight changes, food consumption, ophthalmology, detailed functional tests and observations, clinical pathology parameters (haematology, coagulation and clinical chemistry), gross necropsy findings, organ weights, and histopathological examinations, mating and pregnancy performance, fertility, maternal care and pup performance (litter survival and pup weights).

Mating performance, fertility indices, corpora lutea and implantation counts, duration of gestation, and the mean number of live pups born per litter were similar between control and treated females. Litter survival, litter weights and mean pup weights were similar between litters derived from control and treated females.

In conclusion, under the conditions of this study the No Observed Adverse Effect Level (NOAEL) for systemic toxicity (parents) was considered to be 1000 mg/kg/day and the reproductive/developmental No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day, for both males and females.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Due to the absence of adverse effects on developmental toxicity in an OECD 422 screening reproductive / developmental toxicity study, the Pre-natal Development Toxicity study is considered scientifically unjustified and hence no further testing is proposed for this endpoint.

Justification for classification or non-classification

No adverse effects on fertility or developmental toxicity were observed in an OECD 422 screening reproductive / developmental toxicity study. Hence, classification is not required.