Neodymium oxide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 September 2012 - 7 February 2013

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Neodymium oxide

EC Number:

215-214-1

EC Name:

Neodymium oxide

Cas Number:

1313-97-9

Molecular formula:

Nd2O3

IUPAC Name:

dineodymium(3+) trioxidandiide

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Blue powder
- Storage Conditions: Ambient, protected from light

Test animals

Species:

rat

Strain:

other: Crl:CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: On dispatch from the supplier, the animals were all approximately 7 - 8 weeks old. At initiation of dosing, the animals were approximately 9 - 10 weeks old.
- Weight at study initiation: On dispatch from the supplier, a sample of the males weighed 250 - 275 g and a sample of the females weighed 192 - 215 g. At initiation of dosing, the animals weighed 340 - 401 g for males and 212 - 285 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 was provided ad libitum.
- Water (e.g. ad libitum): Water taken from the public supply was available ad libitum.
- Acclimation period: 13 days.

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 beginning at 07:00 h.

IN-LIFE DATES: From: 4 September 2012 To: 2 November 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1 % w/v

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The dosing formulations were prepared weekly, stored in a refrigerator set to maintain 4 °C, and dispensed daily. They were prepared by adding an appropriate amount of vehicle to the required amount of test material and were mixed by magnetic stirring and high shear mixer until a homogenous solution was obtained. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes prior to dosing and continuously during dosing.
All formulations were used within the 8 day stability period that was established previously at the testing laboratory.

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 as measured at the time of administration, 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 and stored in a refrigerator set to maintain 4 °C.

CONCENTRATION AND HOMOGENEITY ANALYSIS
From each formulation, duplicate sets of top, middle and bottom samples (ca 0.5 mL) were taken at each sampling time point (all concentrations on weeks 1 and 4) and sent to the analytical laboratory for analysis (duplicate middle samples only obtained from control formulations). Additional triplicate samples from the top, middle and bottom (middle sample only from control) were also taken 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.

The results of the analyses of dosing formulations prepared for use on Day 1 of dosing and Week 4 of dosing were all found to be within the ± 10 % acceptance criteria. A low coefficient of variation was obtained (<5.2 %), indicating that the formulations were homogenous. These results show an acceptable accuracy of formulation for the duration of the study.

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

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.

Examinations

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, including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta. All animals were examined for reaction to treatment daily during the course of the study. The onset, intensity and duration of any signs were recorded. Furthermore, once during the pre-treatment period (week -1) and weekly thereafter, a more detailed examination was made on all animals.

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: Ophthalmic 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 were 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 (Day 30).
- 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. At necropsy, the reproductive tracts of all females were examined for signs of implantation, the number of any implantation sites being recorded and the numbers of corpora lutea graviditatis on the ovaries were counted.
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) and for the kidneys only from all remaining females and from all males in group 1 and 4.

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.

Selected 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 body weight as the covariate. Organ weights were also expressed as a percentage of terminal body weight and analysed using ANOVA as detailed above.

Incidence data was analysed as proportions in a Kruskal-Wallis analysis, or by categorical methods using contingency tables with the Fisher’s Exact Probability test or the Chi-squared test.

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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:

effects observed, treatment-related

Description (incidence and severity):

see "Details on Results (parental animals)"

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
There were no premature deaths on this study.
Treatment at levels up to 1000 mg/kg/day in males produced no clinical observations that were considered related to administration of the test material.
At 1000 mg/kg/day in females there was an increase in the number of animals walking on tip toes (between days 12-20) and a slight increase in incidence of animals with irregular respiration (between days 12-14) and hunched posture (between days 12-23), when compared to controls. The effects were transient on each occasion lasting up to 4 hours post dose. The incidences were outside of the historical control range on the equivalent study days.
Treatment at levels up to 300 mg/kg/day in females produced no clinical observations that were considered related to administration of the test material.

BODY WEIGHT AND FOOD CONSUMPTION
Food consumption was similar in all treated animals to controls.
At levels up to 1000 mg/kg/day the group mean body weight gain for males was similar to controls.
At levels up to 1000 mg/kg/day the group mean body weight gain for females prior to mating and throughout gestation were similar to controls.
At 300 mg/kg/day and above there was a dose related decrease in female group mean bodyweight gain during the lactation period only. At 1000 mg/kg/day there was a 63 % reduction and at 300 mg/kg/day there was a 26 % reduction in female group mean body weight gain over Days 1 - 4 of lactation, when compared to controls (Table 1). At 300 mg/kg/day the reduced weight gain was still within the mean historical control range and as the group mean bodyweight for animals dosed at 300 mg/kg/day was higher than for other groups, the significance of this reduction is not clear.

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 and treated females. The data are summarised in Tables 2 and 3.

ORGAN WEIGHTS
No test material-related organ weight changes were noted. There were isolated 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/or related to difference of sexual maturity 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 and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of the test material.

HISTOPATHOLOGY
There was a treatment associated finding in the kidney at 1000 mg/kg/day, where marked bilateral cortical tubular necrosis and mild diffuse cortical basophilic tubules (bilateral), were recorded in female 73, and mild diffuse cortical basophilic tubules (bilateral) were noted in female 78. Higher blood levels of creatinine and urea were recorded in female 73, supporting the renal pathology (there were however no microscopic correlates associated with the higher levels of alkaline phosphotase and bilirubin noted in female 73).
The kidney findings are summarised in Table 4.
Otherwise the microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test material.

Effect levels (P0)

Dose descriptor:

NOEL

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.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

MATING PERFORMANCE, FERTILITY AND DURATION OF GESTATION AND LITTER SIZE
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 animals.

LITTER SURVIVAL, LITTER AND PUP WEIGHTS
Litter survival, litter weights and mean pup weights were similar between litters derived from control and treated females. Data is summarised in Tables 5 and 6.
Total litter losses were observed in 4/9 litters at 100 mg/kg/day but not in any other dose group. In the absence of any dose related pattern it is considered that these litter losses were incidental.

OBSERVATIONS AMONG DAMS/PUPS
The nature and incidence of the observations recorded for dams and their pups were similar between control and treated females, with the exception of Group 2 where observations relating to the 4 litter losses were recorded. In the absence of any dose related pattern it is considered that these observations were not related to administration of the test material.

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects were seen on offspring parameters up to the maximum test concentrations.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1 Female Bodyweights (g): Group Mean Values During Gestation and Lactation ± Standard Deviation

Dose Level (mg/kg/day)	Day of Gestation					Weight Gain Days 0 - 20	Weight Gain as % of Control	Day of Lactation*
	0	7	14	16	20			1	4
0	268 ± 20	303 ± 22	340 ± 25	361 ± 25	411 ± 32	143	-	280 ± 27	307 ± 28
100	264 ± 9	296 ± 13	334 ± 10	358 ± 11	416 ± 15	152	106	293 ± 9	321 ± 13
300	283 ± 14	317 ± 16	361 ± 23	383 ± 26	447 ± 30	164	115	313 ± 25	333 ± 22
1000	266 ± 22	306 ± 27	348 ± 28	369 ± 28	429 ± 32	163	114	316 ± 35	326 ± 36

*Animals with litters surviving to Day 4 or after only

 

Table 2 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	2 2 2 4	1 1 3 5(1)	1 3 3 3	3 (1) 2 3 2
No clear indication of mating Median no. of nights to positive mating sign Number passing one oestrus	0 3 0	0 3.5 0	0 3 0	1 2.5 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 9 90 10 9 90	10 10 100 10 10 100	10 9 90 10 9 90

 

Table 3 Group Mean Duration of Gestation and Overall Litter Performance

	Dose Level (mg/kg/day)
	0	100	300	1000
Number Pregnant	10	9	10	9
Duration of Gestation (Days) 21 22 Mean Duration	4 6 21.6	0 9 22.0	2 8 21.8	1 8 21.9
Number of females producing a live litter Gestation index as %	10 100	9 90	10  100	9 90
Mean number of implant sites* per pregnancy ± SD	16.3 ± 1.4	17.4 ± 0.9	16.9 ± 1.7	15.6 ± 2.2
Mean number of corpora lutea sites* per pregnancy ± SD	18.8 ± 3.2	19.2 ± 1.3	18.4 ± 1.8	18.9 ± 2.5
Mean total number of pups born* per litter ± SD	15.1 ± 2.0	16.0 ± 1.9	15.6 ± 2.2	15.0 ± 2.7
Mean number of live pups* per litter ± SD Day 0 of lactation Day 1 of lactation Day 4 of lactation	14.7 ± 2.2 14.6 ± 2.4 14.2 ± 2.7	16.0 ± 1.9 15.0 ± 1.9 14.8 ± 1.5	15.3 ± 2.4 149 ± 2.9 14.7 ± 3.0	14.6 ± 2.6 14.0 ± 2.5 13.8 ± 2.5
Total no. males* on Day 1 of lactation (%) Total no. females* on Day 1 of lactation (%)	61 (46)  72 (54)	28 (37)  47 (63)	79 (53) 70 (47)	71 (56) 55 (44)

* Excludes litters where all pups died

 

Table 4 Summary of Histological Findings in the Kidney

Histological Findings	Group Totals
	Males		Females
	Dose Level (mg/kg/day)
	0	1000	0	100	300	1000
N	10	10	10	10	10	10
No abnormality detected	2	5	6	6	6	6
Necrosis, cortical tubular, bilateral Marked	0	0	0	0	0	1
Necrosis, cortical tubular, bilateral Total Incidence	0	0	0	0	0	1
Basophilic tubules, diffuse, cortical Mild Total Incidence	0 0	0 0	0 0	0 0	0 0	2 2
Chronic progressive nephropathy	0	0	0	1	0	0
Basophilic tubules, focal	7	4	1	2	0	2
Tubular casts	2	1	3	0	0	0
Inflammatory cell foci	1	3	1	1	1	0
Mononuclear cell infiltration, pelvic	0	0	1	0	0	0
Pelvic dilation	0	0	0	2	1	0
Mineral Deposits, medullary	0	0	1	0	2	0

N = the number of animals from which the tissue was examined microscopically

 

Table 5 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	93 2 10	92 2 9	92 2 10	96 0 9
Live Birth Index	Mean Litter Index (%) Number losing >1 pup Number of Litters	97 1 10	98 0 9	98 1 10	97 1 9
Viability Index Days 1 - 4	Mean Litter Index (%) Number losing >3 pups Number of Litters	96 0 10	52 5 9	96 0 10	95 0 9

 

Table 6 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	90 ± 14 129 ± 22	97 ± 7 146 ± 5	96 ± 18 142 ± 24	92 ± 15 136 ± 25
Mean of Litter Mean Pup Weight
Males Day 1 Day 4	6.3 ± 0.5 9.4 ± 1.0	6.8 ± 0.4 10.2 ± 0.7	6.7 ± 0.8 10.0 ± 1.3	6.8 ± 0.6 10.2 ± 1.4
Females Day 1 Day 4	5.9 ± 0.5 9.0 ± 1.2	6.4 ± 0.6 9.8 ± 0.7	6.3 ± 0.8 9.5 ± 1.3	6.5 ± 0.7 9.7 ± 1.4

Applicant's summary and conclusion

Conclusions:

The male reproductive No Observed Effect Level (NOEL) was considered to be 1000 mg/kg/day. The female reproductive/developmental No Observed Effect Level (NOEL) was considered to be 1000 mg/kg/day. Therefore in this study, there was no indication of toxicity to male or female reproduction up to the highest dose tested.

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 was dosed with the vehicle (1 % w/v carboxymethylcellulose) following the same dosing regimen as the treated animals and was used as control.

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: mortality, clinical signs, bodyweights, bodyweight changes, 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.

The male reproductive No Observed Effect Level (NOEL) was considered to be 1000 mg/kg/day. The female reproductive No Observed Effect Level (NOEL) was considered to be 1000 mg/kg/day. Therefore in this study, there was no indication of toxicity to male or female reproduction up to the highest dose tested.