Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 August 2012 to 03 December 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:
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:
other: Crl:CD (SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8-9 weeks (initiation of dosing).
- Weight at study initiation: 329 - 379 g (males) and 202 - 258 g (females) at initiation of dosing.
- Housing: 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. 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: rat and mouse breeder diet ad libitum
- Water: water from the public supply, ad libitum
- Acclimation period: 13 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23 °C
- Humidity (%): 38 - 74 %
- Air changes (per hr): 10 air changes per hour (minimum).
- Photoperiod (hrs dark / hrs light): a 12 hour light/dark cycle was maintained.
- Environmental enrichment: Chewing objects and hiding devices were provided to the animals as appropriate for psychological/environmental enrichment.

IN-LIFE DATES: From 03 September 2012 to 19 October 2012
Route of administration:
oral: gavage
Vehicle:
other: 1 % (w/v) Carboxymethylcellulose in Milli-Q water
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 a high shear mixer and magnetic stirring until a visibly homogenous formulation 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 had been previously established.
Dose volume: 10 mL per kg body weight.
Concentration in vehicle: 10, 30 and 100 mg/mL for the 100, 300 and 1000 mg/kg dose levels, respectively.
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: 1:1
- 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:
From each formulation, duplicate sets of top, middle and bottom samples (ca 0.5 mL) were taken at each sampling time point and analysed by ICP-OES (duplicate middle samples only obtained from control formulations). An additional sample 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.

- Preparation of test solutions: Samples were digested in approximately 10 mL of 69 % nitric acid in a microwave. Once digested, the samples were transferred to 50 mL volumetric flasks with washings and made to volume in 2 % nitric acid. The samples were further diluted in 2 % nitric acid to give sample solution concentrations within the range of the calibration standards used. Samples and standards were analysed by ICP-OES.

- Preparation of calibration standards: 20 mg of test material was added to a digestion vessel and digested in a microwave in approximately 10 mL of 69 % nitric acid. Once digested the solution was transferred to a 100 mL volumetric flask with washings and made to volume in 2 % nitric acid to obtain a stock solution with a concentration of 0.200 mg/mL dierbium trioxide. The stock solution was diluted volumetrically in 2 % nitric acid to obtain target concentrations of approximately 0.00100, 0.00200, 0.00400, 0.00600, 0.00800 and 0.0100 mg/mL dierbium trioxide.

- ICP-OES conditions:
> Instrument: Perkin Elmer Optima 8000 ICP-OES
> Element and wavelength: Er (369.265 nm)
> No. of replicates: 3
> Delay time: 60 seconds
> Read time: set to auto
> Minimum read time: 2 seconds
> Maximum read time: 10 seconds
> Points per peak: 7
> Plasma conditions:
Plasma: 8 L/min
Auxiliary: 0.2 L/min
Nebuliser: 0.7 L/min
RF power: 1300 Watts
Plasma viewing mode: radial
> Peristaltic pump sample flow rate: 1.50 mL/min
> Wash:
Frequency: between samples
Flow rate: 1.50 mL/min
Normal time: 30 seconds
Wash solvent: Milli-Q water
> Diluent: 2 % nitric acid
> Microwave digestion:
Ramp: 12 min to 150 °C
Hold: 10 min at 150 °C
Cool down: 30 min

- Calculations: a calibration curve was constructed by plotting the mean peak areas of the standards (including the calibration blank) against the relevant dierbium trioxide concentration in mg/mL. The concentration of dierbium trioxide in the test samples was determined by interpolation of this line.

Concentration of dierbium trioxide in formulations (mg/mL) = (C x D x P) / W
Where
C = concentration of the sample solution obtained by interpolation from the calibration line (mg/mL)
D = dilution factor
P = density of the formulation (g/mL)
W = weight of aliquot (g)

- The limit of detection (LOD): 0.00000269 mg/mL dierbium trioxide, estimated as 3.3 times the ratio of the standard deviation of the calibration blank to the slope of the calibration curve.
- The limit of quantification (LOQ): 0.00000814 mg/mL dierbium trioxide, estimated as 10 times the ratio of the standard deviation of the calibration blank to the slope of the calibration curve.
Duration of treatment / exposure:
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). 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 that day.
Frequency of treatment:
Animals were dosed once daily.
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 males and 10 females per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected after a review of existing relevant toxicological data, including a separate 14-day dose range finding study conducted at the testing facility, where dose levels up to 1000 mg/kg/day produced no adverse reaction to treatment.
- Animal assignment: On arrival from the suppliers, the animals were housed in cages. The cages were suspended on a series of racks. Male and female cages were racked separately. Cages were allocated to treatment group by the use of randomly sequenced numbers in such a way that each complete rack contained representatives from all treatment groups. During pre-trial Animal 66 (Group 3 female) was replaced with spare Animal 83 due to findings in the pre-trial ophthalmic examinations.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked for viability 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. Detailed functional observations took place weekly from pre-treatment (week -1).

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

FOOD CONSUMPTION: Yes
- Time schedule: 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 measurement did not recommence after pairing for mating.

OTHER: Haematology, coagulation and clinical chemistry parameters were also evaluated.
Oestrous cyclicity (parental animals):
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.
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations: testis weight 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
The males were killed when mating was completed and the animals had been dosed for at least 4 weeks (Study Day 30).
The females were killed between Day 5 and 7 of lactation (Study Days 43, 45 and 47).
Animals were killed by exposure to carbon dioxide followed by exsanguination.

GROSS PATHOLOGY: Yes
All adult animals surviving to scheduled euthanasia 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 for signs of implantation, the number of any implantation sites being recorded and the total numbers of corpora lutea graviditatis were counted.

ORGAN WEIGHTS: Yes
The following organs were weighed at necropsy for all adult animals surviving to terminal kill: brain, epididymis, adrenal gland, pituitary gland, prostate gland, thyroid gland, heart, kidney, liver, lung, ovary, spleen, testis, thymus and uterus.

HISTOPATHOLOGY: Yes
Representative samples of the following tissues were collected from all adult animals and preserved in 10 % neutral buffered formalin, unless otherwise indicated:
Aortic artery, blood smear* (animals sacrificed prematurely only), bone marrow smear# , bone marrow (femur, sternum), bone (femur, rib and sternum), brain, cervix, epididymides, eyes~, adrenal glands, harderian glands~ , lacrimal glands, mammary gland, parathyroid glands, pituitary gland, prostate gland, salivary glands, seminal vesicle gland, thyroid glands, gross lesions/masses, gut-associated lymphoid tissue, heart, kidneys, large intestine (caecum, colon, rectum), larynx, liver, lung, mandibular lymph node, mesenteric lymph node, skeletal muscle, nasal cavity, optic nerve~, sciatic nerve, oesophagus, ovaries, oviducts, pancreas, pharynx, skin, small intestine (duodenum, ileum, jejunum), spinal cord, spleen, stomach, testes+, thymus, tongue, trachea, ureters, urinary bladder, uterus and vagina.
* Air dried
# Fixed in methanol and then air dried
~ Preserved in Davidson’s fixative
+ Preserved in Modified Davidson’s fixative

- Bone Marrow Smears
Duplicate bone marrow smears were taken at necropsy from all adult animals. Both bone marrow smears were stained using May-Grunwald-Giemsa as the Romanowsky stain. Bone marrow smears were not evaluated.

- Histopathology
The tissues, as listed above (except nasal cavity, blood smears and bone marrow smears), were processed to paraffin wax block from selected animals. Sections were cut 4-6 µm thick, stained with haematoxylin and eosin from 5 males and 5 females in the control and high dose groups (the same animals that were used for laboratory investigations). The testes of all male animals were processed to wax impregnation. This was done in order to maintain tissue integrity as the testes must be removed from fixative and processed to wax impregnation within 72 hrs. A PAS-Haematoxylin stained slide was prepared from the testes and epididymides of the males.
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:
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 body weight and food consumption, 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.
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)" for information.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See "Details on results (parental animals)" for information.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See "Details on results (parental animals)" for information.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment related effects.
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
See "Details on results (parental animals)" for information.
CLINICAL SIGNS AND MORTALITY
Animal 1 (Group 1 male) was killed prematurely on study Day 19 due to adverse clinical observations including enlarged and discoloured hind feet and dragging hind limbs continuously. The death of this animal was not related to treatment.
Treatment at levels up to 1000 mg/kg/day in males produced no clinical observations that were considered related to treatment with test material.
In females dosed at 1000 mg/kg/day there was a slight increase in incidence in several clinical observations such as body hunched, piloerection and walking on tip toes. These clinical observations were observed at this dose level only and were evident from around the time of parturition onwards. These observations are typical of females giving birth however, the increased incidence of these signs indicate that these clinical observations are related to treatment with the test material. In females at levels up to 300 mg/kg/day there were no clinical observations that were considered to be related to treatment with test material.

BODY WEIGHT AND WEIGHT GAIN
In males dosed up to 1000 mg/kg/day the group mean body weight gains were similar to the control for the duration of the study.
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 the control. At 1000 mg/kg/day there was a 36 % reduction in female group mean body weight gain over Days 1-4 of lactation, when compared to the control. At levels up to 300 mg/kg/day the group mean body weight gain during lactation was similar to the control. Bodyweight measurements can be seen in Tables 3 and 4.

FOOD CONSUMPTION
Group mean food consumption in all treated males was similar to the control. During pre-trial the group mean food consumption in the females from treated groups was slightly lower than the control, however, was similar to the control from Day 1 of dosing until pairing for mating. The group mean food consumption for females throughout gestation and lactation was also similar to controls.

REPRODUCTIVE PERFORMANCE, FERTILITY AND DURATION OF GESTATION AND LITTER SIZE
In all treated groups the mating performance, fertility indices and duration of gestation were similar to controls.
There was a slight reduction in gestation index at 300 and 1000 mg/kg/day due to 1 female in each of these groups giving birth to dead pups only.
In all treated groups the mean number of corpora lutea and the mean number of implantation sites were similar to controls.
The mean number of pups born per litter was similar to controls in all treatment groups.

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 and of the nature commonly observed in this strain and age of rat. The incidence of gross findings was similar in the control and treated animals and, therefore, was considered unrelated to administration of the test material.

HISTOPATHOLOGY
No test material-related microscopic findings were noted. The microscopic findings observed were considered incidental and of the nature commonly observed in this strain and age of rat. The incidence and severity of microscopic findings were similar in control and treated animals and, therefore, were considered unrelated to administration of the test material.
Dose descriptor:
NOEL
Remarks:
reproduction
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No effects on fertility noted at the highest dose tested in males.
Dose descriptor:
NOEL
Remarks:
reproduction
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
other: Results are still under consideration
Remarks:
Effect level currently unavailable
Effect level:
10 other: Results are still under consideration
Based on:
other: Results are still under consideration
Remarks:
Effect level currently unavailable
Sex:
not specified
Basis for effect level:
other: Results are still under consideration
Remarks on result:
other: Results are still under consideration
Remarks:
Results are still under consideration
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment related effects.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
See "Details on results (offspring)" for information.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No treatment related effects.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
In all treated groups the birth index was similar to controls. At 300 mg/kg/day and above there was a reduction in live birth index compared to controls. The live birth index at 300 mg/kg/day was 85 % and at 1000 mg/kg/day was 89 %, compared to 100 % in controls. The mean live birth index in the current historical control data for this species and strain is 98 % with the lowest value recorded in a single study being 88 %.
At 300 mg/kg/day and above there was also a notable decrease in pup viability over Days 1-4 of lactation. While a single control dam had a total litter loss, at 300 mg/kg/day 1/9 animals had a total litter loss and 1 animal lost all but 1 of its pups, resulting in a viability index of 72 %. At 1000 mg/kg/day 3/9 animals had a total litter loss and the overall group mean viability index was reduced to 65 %, compared to 86 % in controls. The mean viability index in the current historical control data for this species and strain is 95 % with the lowest value recorded in a single study being 79 %.
At 1000 mg/kg/day there was an increased incidence of dams with few teats evident and this finding was correlated to the dams who incurred a total litter loss.
With the exception of the dams that lost their entire litter, the group mean litter size remained comparable to controls throughout lactation.

BODY WEIGHT (OFFSPRING)
In all treated groups the group mean litter weight and the mean of litter mean pup weight were similar to controls throughout lactation.

GROSS PATHOLOGY (OFFSPRING)
The type and distribution of observations amongst pups did not indicate any association with treatment.
Dose descriptor:
other: Results are still under consideration
Remarks:
Effect level currently unavailable
Generation:
other: Results are still under consideration
Effect level:
10 other: Results are still under consideration
Based on:
other: Results are still under consideration
Remarks:
Effect level currently unavailable
Sex:
not specified
Basis for effect level:
other: Results are still under consideration.
Remarks on result:
other: Results are still under consideration
Remarks:
Effect level currently unavailable
Reproductive effects observed:
not specified

Dose formulation Analysis

The results of the analyses of the group 2-4 dosing formulations prepared for use on day 1 of dosing were found to be below the acceptance criteria (% difference from nominal: group 2, -48.3 %, group 3, -53 % and group 4, -11.9 %). An Out Of Specification (OSS) investigation was carried out to try and source any issue/error that may have led to these spurious results. The investigation was inconclusive and so the system was cleaned out and performance checks were conducted on the instrumentation prior to the original samples being re-run along with the back-up samples. The analysis of back-up and original samples showed improved results but many were still out of specification. Following further investigation it was concluded that there was evidence that the analytical samples were not prepared appropriately prior to analysis causing inadequate sample digestion in all of the day 1 samples.

A further analysis time point was added to formulations prepared for use on week 2 to confirm the investigation conclusions and confirm that all formulations are accurate and homogenous. 

 

Results

The results of the analysis of dosing formulations prepared for use on week 2 and week 4 of dosing were all found to be within the ± 10 % acceptance criteria specified in the analytical method. This indicates an acceptable accuracy of formulation for the duration of the study and the spurious results obtained on day 1 samples were due to an analytical error and not an error in the formulation procedure. The low coefficient of variation (< 5 %) indicated that the formulations were homogenous.

Test solutions were also found to be stable over at least 8 days, when stored at ambient temperature or 2-8 °C in the dark.

Results can be seen in Table 2.

Table 2: Formulation Analysis Results

Day 1

Dose

group

Nominal conc.

(mg/mL)

Mean found

(mg/mL)

% Difference

from nominal

1

0

0

n/a

2

10

5.17

-48.3

3

30

14.1

-53.0

4

100

88.1

-11.9

Day 1 (re-run of original samples and back-up samples)

Dose

group

Nominal conc.

(mg/mL)

Mean found

(mg/mL)

% Difference

from nominal

2

10

9.47

-5.3

3

30

18.8

-37.3

4

100

96.6

-3.4

2 (back-up)

10

10.3

3.0

3 (back-up)

30

10.3

-65.7

4 (back-up)

100

88.0

-12.0

Week 2

Dose

group

Nominal conc.

(mg/mL)

Mean found

(mg/mL)

% Difference

from nominal

1

0

0

n/a

2

10

9.78

-2.2

3

30

29.5

-1.7

4

100

103

3.0

Week 4

Dose

group

Nominal conc.

(mg/mL)

Mean found

(mg/mL)

% Difference

from nominal

1

0

0

n/a

2

10

10.1

1.0

3

30

29.6

-1.3

4

100

105

5.0

 

Table 3: Body weights (g): Group Mean Values (Males)

Group/sex

Day

Change

-7

0

7

14

21

28

0-28

1M

291

358

402

430

455

484

126

2M

292

356

396

428

442

472

115

3M

287

349

394

424

444

472

123

4M

286

354

397

427

448

479

126

 

Table 4: Body weights (g): Group Mean Values (Females)

Group/sex

Day (prior to mating)

Change

Day (during gestation)

Change

Day (during lactation)

-7

0

7

14

0 - 14

0

7

14

16

20

0 - 20

1

4

1F

210

240

260

275

35

284

320

362

386

437

153

304

334

2F

204

232

249

267

35

275

316

363

385

431

156

294

322

3F

203

227

248

265

38

271

308

356

376

425

154

296

323

4F

203

234

252

269

35

275

313

360

381

441

166

307

324

 

Table 5: Mating Performance and Fertility Indices

No. of nights to positive mating sign

Group / Dose level (mg/kg/day)

1 / 0

2 / 100

3 / 300

4 / 1000

1

Number of animals (not becoming pregnant)

1

2

3

2

2

1

3

2

1

3

4

0

0

2

4

3

5

4

5

5

1

0

0

0

No clear indication of mating

1

0

0

0

No indication of mating

0

0

1

0

Median no. of nights to positive mating sign

3

3

2

3.5

No. passing one oestrous

0

0

0

0

No. of males paired

10

10

10

10

No. of siring males

10

10

10

10

Male fertility index (%)

100

100

100

100

No. of females paired

10

10

10

10

No. pregnant

10

10

10

10

Female fertility index (%)

100

100

100

100

 

Table 6: Group Mean Duration of Gestation and Overall Litter Performance

Group / Dose level (mg/kg/day)

1 / 0

2 / 100

3 / 300

4 / 1000

No. pregnant

10

10

10

10

Duration of gestation (days)

20

1

0

0

0

21

6

3

3

2

22

3

7

6

7

23

0

0

0

1

Gestation length unclear

0

0

1

0

Mean duration

21.2

21.7

21.7

21.9

No. of females producing a live litter

10

10

9

9

Gestation index (%)

100

100

90

90

Mean no. of implant sites* per pregnancy ± SD

16.6 ± 2.5

16.7 ± 2.2

17.5 ± 1.8

16.8 ± 1.9

Mean no. of corpora lutea sites* per pregnancy ± SD

20.0 ± 3.2

20.4 ± 3.2

20.9 ± 2.9

19.7 ± 3.4

Mean total no. of pups born* per litter ± SD

15.4 ± 2.8

15.2 ± 2.3

15.5 ± 2.4

14.8 ± 2.7

Mean no. of live pups* per litter ± SD born on:

Day 0 of lactation

15.4 ± 2.8

15.0 ± 2.4

14.5 ± 1.9

14.7 ± 2.5

Day 1 of lactation

15.0 ± 3.0

14.5 ± 2.2

12.5 ± 3.8

14.7 ± 2.5

Day 4 of lactation

14.7 ± 2.9

14.2 ± 2.2

11.9 ± 5.0

14.2 ± 2.5

Total no. of males* on day 1 of lactation (%)

65 (48)

71 (49)

52 (52)

45 (51)

Total no. of females* on day 1 of lactation (%)

70 (52)

74 (51)

48 (48)

43 (49)

* excludes litters where all pups died

 

Table 7: Group Mean F1 Survival Indices

Group / Dose level (mg/kg/day)

1 / 0

2 / 100

3 / 300

4 / 1000

Birth index

Mean litter index (%)

93

91

91

91

No. losing > 2 pups

2

2

3

2

No. of litters

10

10

10

10

Live birth index

Mean litter index (%)

100

99

85

89

No. losing > 1 pup

0

0

3

1

No. of litters

10

10

10

10

Viability index (Days 0-4)

Mean litter index (%)

86

95

72

65

No. losing > 3 pups

1

0

3

3

No. of litters

10

10

9

9

 

Conclusions:
Treatment in females at 300 mg/kg/day and above was associated with a slight reduction in gestation index, a reduced live birth index and a decrease in pup viability over Days 1-4 of lactation. At 1000 mg/kg/day there was an increased incidence of clinical observations around the time parturition and an increase in the number of dams with no teats evident in early lactation (dams which subsequently lost their entire litters). A 36.7 % decrease in group mean bodyweight gain over Days 1-4 of lactation was also noted at 1000 mg/kg/day.
Treatment at 100 mg/kg/day in both sexes resulted in no significant changes in any of the parameters assessed that were considered to be indicative of a reaction to treatment.
In conclusion, under the conditions of the study, the male reproductive No Observed Effect Level (NOEL) was considered to be 1000 mg/kg/day.
There were effects on the reproductive performance/survival of the pups at 300 mg/kg/day and above. Although the cause of reduced pup survival at these dose levels could not be determined from this study, the female reproductive No Observed Effect Level (NOEL) was considered to be 100 mg/kg/day.
Executive summary:

The toxicity of the test material to reproduction was investigated in a GLP study which was conducted in accordance with the standardised guideline OECD 422.

During the study groups of 10 male and 10 female Sprague-Dawley Crl:CD (SD) strain rats were administered test material, by oral gavage, at dose levels of 0 (vehicle control), 100, 300 and 1000 mg/kg bw/day. Another group of 10 male and 10 female rats was dosed with the vehicle (1 % w/v carboxymethylcellulose in Milli-Q water) 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 endpoints were evaluated: clinical signs, bodyweight, bodyweight changes, food consumption, gross necropsy findings, organ weights, histopathological examinations, mating and pregnancy performance, fertility, maternal care, pup performance (litter survival and pup weights). 

Treatment in females at 300 mg/kg/day and above was associated with a slight reduction in gestation index, a reduced live birth index and a decrease in pup viability over Days 1-4 of lactation. At 1000 mg/kg/day there was an increased incidence of clinical observations around the time parturition and an increase in the number of dams with no teats evident in early lactation (dams which subsequently lost their entire litters). A 36.7 % decrease in group mean bodyweight gain over Days 1-4 of lactation was also noted at 1000 mg/kg/day.

Treatment at 100 mg/kg/day in both sexes resulted in no significant changes in any of the parameters assessed that were considered to be indicative of a reaction to treatment.

In conclusion, under the conditions of the study, the male reproductive No Observed Effect Level (NOEL) was considered to be 1000 mg/kg/day.

There were effects on the reproductive performance/survival of the pups at 300 mg/kg/day and above. Although the cause of reduced pup survival at these dose levels could not be determined from this study, the female reproductive No Observed Effect Level (NOEL) was considered to be 100 mg/kg/day.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Study duration:
subacute
Species:
rat
Quality of whole database:
The study was conducted to GLP and in accordance with the standardised guideline OECD 422. The study was assigned a reliability score of 1 according to the criteria of Klimisch (1997). The quality of the database is good.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The toxicity of the test material to reproduction was investigated in a GLP study which was conducted in accordance with the standardised guideline OECD 422.

During the study groups of 10 male and 10 female Sprague-Dawley Crl:CD (SD) strain rats were administered test material, by oral gavage, at dose levels of 0 (vehicle control), 100, 300 and 1000 mg/kg bw/day. Another group of 10 male and 10 female rats was dosed with the vehicle (1 % w/v carboxymethylcellulose in Milli-Q water) 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 endpoints were evaluated: clinical signs, bodyweight, bodyweight changes, food consumption, gross necropsy findings, organ weights, histopathological examinations, mating and pregnancy performance, fertility, maternal care, pup performance (litter survival and pup weights). 

Treatment in females at 300 mg/kg/day and above was associated with a slight reduction in gestation index, a reduced live birth index and a decrease in pup viability over Days 1-4 of lactation. At 1000 mg/kg/day there was an increased incidence of clinical observations around the time parturition and an increase in the number of dams with no teats evident in early lactation (dams which subsequently lost their entire litters). A 36.7 % decrease in group mean bodyweight gain over Days 1-4 of lactation was also noted at 1000 mg/kg/day.

Treatment at 100 mg/kg/day in both sexes resulted in no significant changes in any of the parameters assessed that were considered to be indicative of a reaction to treatment.

In conclusion, under the conditions of the study, the male reproductive No Observed Effect Level (NOEL) was considered to be 1000 mg/kg/day.

There were effects on the reproductive performance/survival of the pups at 300 mg/kg/day and above. Although the cause of reduced pup survival at these dose levels could not be determined from this study, the female reproductive No Observed Effect Level (NOEL) was considered to be 100 mg/kg/day.


Short description of key information:
REPRODUCTIVE/DEVELOPMENTAL TOXICITY SCREENING STUDY
NOEL (reproduction) 1000 mg/kg bw/day, male rats (no effects on fertility noted at the highest dose tested in males) , OECD 422, Charles River (2013).
NOEL (reproduction) 100 mg/kg bw/day, female rats (reduced gestation index, live birth index and a decrease in pup viability during days 1-4 of lactation), OECD 422, Charles River (2013)

TWO GENERATION REPRODUCTIVE TOXICITY STUDY
No study available, a data waiver has been submitted to address this endpoint.

Justification for selection of Effect on fertility via oral route:
A single repeated dose and reproductive/developmental screening study was available for assessment. The study was performed and reported to a high standard, in line with a standardised guideline (OECD 422) and in compliance with GLP.

Effects on developmental toxicity

Description of key information
REPRODUCTIVE/DEVELOPMENTAL TOXICITY SCREENING STUDY
NOEL (maternal toxicity) 300 mg/kg bw/day, rats (increase in clinical effects and a decrease in bodyweight gain during lactation), OECD 422, Charles River (2013)
NOEL (developmental toxicity) 100 mg/kg bw/day, rats (reduction in gestation index, live birth index and a decrease in pup viability during lactation), OECD 422, Charles River (2013)
PRENATAL DEVELOPMENTAL TOXICITY STUDY
No study available, a data waiver has been submitted to address this endpoint.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 August 2012 to 03 December 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:
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:
other: Crl:CD (SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8-9 weeks (initiation of dosing).
- Weight at study initiation: 329 - 379 g (males) and 202 - 258 g (females) at initiation of dosing.
- Housing: 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. 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: rat and mouse breeder diet ad libitum
- Water: water from the public supply, ad libitum
- Acclimation period: 13 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23 °C
- Humidity (%): 38 - 74 %
- Air changes (per hr): 10 air changes per hour (minimum).
- Photoperiod (hrs dark / hrs light): a 12 hour light/dark cycle was maintained.
- Environmental enrichment: Chewing objects and hiding devices were provided to the animals as appropriate for psychological/environmental enrichment.

IN-LIFE DATES: From 03 September 2012 to 19 October 2012
Route of administration:
oral: gavage
Vehicle:
other: 1 % (w/v) Carboxymethylcellulose in Milli-Q water
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 a high shear mixer and magnetic stirring until a visibly homogenous formulation 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 had been previously established.
Dose volume: 10 mL per kg body weight.
Concentration in vehicle: 10, 30 and 100 mg/mL for the 100, 300 and 1000 mg/kg dose levels, respectively.
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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
From each formulation, duplicate sets of top, middle and bottom samples (ca 0.5 mL) were taken at each sampling time point and analysed by ICP-OES (duplicate middle samples only obtained from control formulations). An additional sample 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.

- Preparation of test solutions: Samples were digested in approximately 10 mL of 69 % nitric acid in a microwave. Once digested, the samples were transferred to 50 mL volumetric flasks with washings and made to volume in 2 % nitric acid. The samples were further diluted in 2 % nitric acid to give sample solution concentrations within the range of the calibration standards used. Samples and standards were analysed by ICP-OES.

- Preparation of calibration standards: 20 mg of test material was added to a digestion vessel and digested in a microwave in approximately 10 mL of 69 % nitric acid. Once digested the solution was transferred to a 100 mL volumetric flask with washings and made to volume in 2 % nitric acid to obtain a stock solution with a concentration of 0.200 mg/mL dierbium trioxide. The stock solution was diluted volumetrically in 2 % nitric acid to obtain target concentrations of approximately 0.00100, 0.00200, 0.00400, 0.00600, 0.00800 and 0.0100 mg/mL dierbium trioxide.

- ICP-OES conditions:
> Instrument: Perkin Elmer Optima 8000 ICP-OES
> Element and wavelength: Er (369.265 nm)
> No. of replicates: 3
> Delay time: 60 seconds
> Read time: set to auto
> Minimum read time: 2 seconds
> Maximum read time: 10 seconds
> Points per peak: 7
> Plasma conditions:
Plasma: 8 L/min
Auxiliary: 0.2 L/min
Nebuliser: 0.7 L/min
RF power: 1300 Watts
Plasma viewing mode: radial
> Peristaltic pump sample flow rate: 1.50 mL/min
> Wash:
Frequency: between samples
Flow rate: 1.50 mL/min
Normal time: 30 seconds
Wash solvent: Milli-Q water
> Diluent: 2 % nitric acid
> Microwave digestion:
Ramp: 12 min to 150 °C
Hold: 10 min at 150 °C
Cool down: 30 min

- Calculations: a calibration curve was constructed by plotting the mean peak areas of the standards (including the calibration blank) against the relevant dierbium trioxide concentration in mg/mL. The concentration of dierbium trioxide in the test samples was determined by interpolation of this line.

Concentration of dierbium trioxide in formulations (mg/mL) = (C x D x P) / W
Where
C = concentration of the sample solution obtained by interpolation from the calibration line (mg/mL)
D = dilution factor
P = density of the formulation (g/mL)
W = weight of aliquot (g)

- The limit of detection (LOD): 0.00000269 mg/mL dierbium trioxide, estimated as 3.3 times the ratio of the standard deviation of the calibration blank to the slope of the calibration curve.
- The limit of quantification (LOQ): 0.00000814 mg/mL dierbium trioxide, estimated as 10 times the ratio of the standard deviation of the calibration blank to the slope of the calibration curve.
Details on mating procedure:
- M/F ratio per cage: 1:1
- 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 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). 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 that day.
Frequency of treatment:
Animals were dosed once daily.
Duration of test:
The males were killed when mating was completed and the animals had been dosed for at least 4 weeks (Study Day 30).
The females and litters were killed between Day 5 and 7 of lactation (Study Days 43, 45 and 47).
No. of animals per sex per dose:
10 males and 10 females per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected after a review of existing relevant toxicological data, including a separate 14-day dose range finding study conducted at the testing facility, where dose levels up to 1000 mg/kg/day produced no adverse reaction to treatment.
- Animal assignment: On arrival from the suppliers, the animals were housed in cages. The cages were suspended on a series of racks. Male and female cages were racked separately. Cages were allocated to treatment group by the use of randomly sequenced numbers in such a way that each complete rack contained representatives from all treatment groups. During pre-trial Animal 66 (Group 3 female) was replaced with spare Animal 83 due to findings in the pre-trial ophthalmic examinations.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked for viability 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. Detailed functional observations took place weekly from pre-treatment (week -1).

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

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption was measured 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.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice between lactation days 5 and 7. Animals were killed by exposure to carbon dioxide followed by exsanguination.

GROSS PATHOLOGY: Yes
Dams surviving to scheduled euthanasia 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 for signs of implantation, the number of any implantation sites being recorded and the total numbers of corpora lutea graviditatis were counted.

ORGAN WEIGHTS: Yes
The following organs were weighed at necropsy for all adult animals surviving to terminal kill: brain, adrenal gland, pituitary gland, thyroid gland, heart, kidneys, liver, lung, ovaries, spleen, thymus and uterus.

HISTOPATHOLOGY: Yes
Representative samples of the following tissues were collected from all adult animals and preserved in 10 % neutral buffered formalin, unless otherwise indicated:
Aortic artery, blood smear* (animals sacrificed prematurely only), bone marrow smear# , bone marrow (femur, sternum), bone (femur, rib and sternum), brain, cervix, eyes~, adrenal glands, harderian glands~ , lacrimal glands, mammary gland, parathyroid glands, pituitary gland, salivary glands, thyroid glands, gross lesions/masses, gut-associated lymphoid tissue, heart, kidneys, large intestine (caecum, colon, rectum), larynx, liver, lung, mandibular lymph node, mesenteric lymph node, skeletal muscle, nasal cavity, optic nerve~, sciatic nerve, oesophagus, ovaries, oviducts, pancreas, pharynx, skin, small intestine (duodenum, ileum, jejunum), spinal cord, spleen, stomach, thymus, tongue, trachea, ureter, urinary bladder, uterus and vagina.
* Air dried
# Fixed in methanol and then air dried
~ Preserved in Davidson’s fixative

- Bone Marrow Smears
Duplicate bone marrow smears were taken at necropsy from all adult animals. Both bone marrow smears were stained using May-Grunwald-Giemsa as the Romanowsky stain. Bone marrow smears were not evaluated.

- Histopathology
The tissues, as listed above (except animal identification, nasal cavity, blood smears and bone marrow smears), were processed to paraffin wax block from selected animals. Sections were cut 4-6 µm thick, stained with haematoxylin and eosin from 5 males and 5 females in the control and high dose groups (the same animals that were used for laboratory investigations).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
Fetal examinations:
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.

SACRIFICE
- The F1 offspring were killed by intra-peritoneal injection of sodium pentobarbitone between Days 5 and 7 of lactation.
Statistics:
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 body weight and food consumption, 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.
Indices:
For each group:
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:yes. Remark: An increase in clinical signs was observed in top dose females during parturition.

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
At 1000 mg/kg/day there was a slight increase in incidence in several clinical observations such as body hunched, piloerection and walking on tip toes. These clinical observations were observed only at this level only and were evident from around the time of parturition onwards. These observations are typical of females giving birth; however, the increased incidence of these signs indicates that these clinical observations are related to treatment with the test material. In females at levels up to 300 mg/kg/day there were no clinical observations that were considered to be related to treatment with test material.

BODY WEIGHT AND WEIGHT GAIN
At levels up to 1000 mg/kg/day, group mean body weight gains were similar to those of the controls, for females prior to mating and throughout gestation.
At 1000 mg/kg/day there was a 27 % reduction in female group mean body weight gain over Days 1-4 of lactation, when compared to control. At levels up to 300 mg/kg/day the group mean body weight gain during lactation was similar to control.

FOOD CONSUMPTION
During pre-trial the group mean food consumption in females from treated groups was slightly lower than control, however, was similar to control from Day 1 of dosing until pairing for mating. The group mean food consumption for females throughout gestation and lactation was also similar to controls.

MATING PERFORMANCE, FERTILITY AND DURATION OF GESTATION AND LITTER SIZE
In all treated groups the mating performance, fertility indices and duration of gestation were similar to controls.
There was a slight reduction in gestation index at 300 and 1000 mg/kg/day due to 1 female in each of these groups giving birth to dead pups only.
In all treated groups the mean number of corpora lutea and the mean number of implantation sites were similar to controls.
The mean number of pups born per litter was similar to controls in all treatment groups.

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 and of the nature commonly observed in this strain and age of rat. The incidence of gross findings was similar in the control and treated animals and, therefore, was considered unrelated to administration of the test material.

HISTOPATHOLOGY
No test material-related microscopic findings were noted. The microscopic findings observed were considered incidental and of the nature commonly observed in this strain and age of rat. The incidence and severity of microscopic findings were similar in control and treated animals and, therefore, were considered unrelated to administration of the test material.
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: Reduction in live birth index and pup viability.

Details on embryotoxic / teratogenic effects:
LITTER SURVIVAL, LITTER AND PUP WEIGHTS
In all treated groups the birth index was similar to controls. At 300 mg/kg/day and above there was a reduction in live birth index compared to controls. The live birth index at 300 mg/kg/day was 85 % and at 1000 mg/kg/day was 89 %, compared to 100 % in controls. The mean live birth index in the current historical control data for this species and strain is 98 % with the lowest value recorded in a single study being 88 %.
At 300 mg/kg/day and above, there was also a notable decrease in pup viability over Days 1-4 of lactation. While a single control dam had a total litter loss, at 300 mg/kg/day 1/9 animals had a total litter loss and 1 animal lost all but 1 of its pups, resulting in a viability index of 72 %. At 1000 mg/kg/day 3/9 animals had a total litter loss and the overall group mean viability index was reduced to 65 %, compared to 86 % in controls. The mean viability index in the current historical control data for this species and strain is 95 % with the lowest value recorded in a single study being 79 %.
With the exception of the dams that lost their entire litter, the group mean litter size remained comparable to controls throughout lactation.
In all treated groups the group mean litter weight and the mean of litter mean pup weight were similar to controls throughout lactation.

OBSERVATIONS AMONG DAMS/PUPS
The type and distribution of observations amongst pups did not indicate any association with treatment. At 1000 mg/kg/day there was an increased incidence of dams with few teats evident and this finding was correlated to the dams who incurred a total litter loss.
Dose descriptor:
other: Results are still under consideration
Remarks:
Effect level currently unavailable
Effect level:
10 other: Results are still under consideration
Based on:
other: Results are still under consideration
Remarks:
Effect level currently unavailable
Sex:
not specified
Basis for effect level:
other: Results are still under consideration
Remarks on result:
other: Results are still under consideration
Remarks:
Effect level currently unavailable
Abnormalities:
not specified
Developmental effects observed:
not specified

Dose formulation Analysis

The results of the analyses of the group 2-4 dosing formulations prepared for use on day 1 of dosing were found to be below the acceptance criteria (% difference from nominal: group 2, -48.3 %, group 3, -53 % and group 4, -11.9 %). An Out Of Specification (OSS) investigation was carried out to try and source any issue/error that may have led to these spurious results. The investigation was inconclusive and so the system was cleaned out and performance checks were conducted on the instrumentation prior to the original samples being re-run along with the back-up samples. The analysis of back-up and original samples showed improved results but many were still out of specification. Following further investigation it was concluded that there was evidence that the analytical samples were not prepared appropriately prior to analysis causing inadequate sample digestion in all of the day 1 samples.

A further analysis time point was added to formulations prepared for use on week 2 to confirm the investigation conclusions and confirm that all formulations are accurate and homogenous. 

 

Results

The results of the analysis of dosing formulations prepared for use on week 2 and week 4 of dosing were all found to be within the ± 10 % acceptance criteria specified in the analytical method. This indicates an acceptable accuracy of formulation for the duration of the study and the spurious results obtained on day 1 samples were due to an analytical error and not an error in the formulation procedure. The low coefficient of variation (< 5 %) indicated that the formulations were homogenous.

Test solutions were also found to be stable over at least 8 days, when stored at ambient temperature or 2-8 °C in the dark.

Results can be seen in Table 2.

Table 2: Formulation Analysis Results

Day 1

Dose

group

Nominal conc.

(mg/mL)

Mean found

(mg/mL)

% Difference

from nominal

1

0

0

n/a

2

10

5.17

-48.3

3

30

14.1

-53.0

4

100

88.1

-11.9

Day 1 (re-run of original samples and back-up samples)

Dose

group

Nominal conc.

(mg/mL)

Mean found

(mg/mL)

% Difference

from nominal

2

10

9.47

-5.3

3

30

18.8

-37.3

4

100

96.6

-3.4

2 (back-up)

10

10.3

3.0

3 (back-up)

30

10.3

-65.7

4 (back-up)

100

88.0

-12.0

Week 2

Dose

group

Nominal conc.

(mg/mL)

Mean found

(mg/mL)

% Difference

from nominal

1

0

0

n/a

2

10

9.78

-2.2

3

30

29.5

-1.7

4

100

103

3.0

Week 4

Dose

group

Nominal conc.

(mg/mL)

Mean found

(mg/mL)

% Difference

from nominal

1

0

0

n/a

2

10

10.1

1.0

3

30

29.6

-1.3

4

100

105

5.0

Table 3: Group Mean Litter and Pup Weight (g)

Day of lactation

Group / Dose level (mg/kg/day)

1 / 0

2 / 100

3 / 300

4 / 1000

LITTER

Day 1

95

92

77

98

Day 4

136

134

113

143

Mean of litter mean pup weight

MALES

Day 1

6.6

6.4

6.3

6.9

Day 4

9.6

9.7

9.5

10.3

FEMALES

Day 1

6.2

6.2

5.9

6.5

Day 4

9.1

9.4

9.3

9.9

 

Table 4: Group Mean Duration of Gestation and Overall Litter Performance

Group / Dose level (mg/kg/day)

1 / 0

2 / 100

3 / 300

4 / 1000

No. pregnant

10

10

10

10

Duration of gestation (days)

20

1

0

0

0

21

6

3

3

2

22

3

7

6

7

23

0

0

0

1

Gestation length unclear

0

0

1

0

Mean duration

21.2

21.7

21.7

21.9

No. of females producing a live litter

10

10

9

9

Gestation index (%)

100

100

90

90

Mean no. of implant sites* per pregnancy ± SD

16.6 ± 2.5

16.7 ± 2.2

17.5 ± 1.8

16.8 ± 1.9

Mean no. of corpora lutea sites* per pregnancy ± SD

20.0 ± 3.2

20.4 ± 3.2

20.9 ± 2.9

19.7 ± 3.4

Mean total no. of pups born* per litter ± SD

15.4 ± 2.8

15.2 ± 2.3

15.5 ± 2.4

14.8 ± 2.7

Mean no. of live pups* per litter ± SD born on:

Day 0 of lactation

15.4 ± 2.8

15.0 ± 2.4

14.5 ± 1.9

14.7 ± 2.5

Day 1 of lactation

15.0 ± 3.0

14.5 ± 2.2

12.5 ± 3.8

14.7 ± 2.5

Day 4 of lactation

14.7 ± 2.9

14.2 ± 2.2

11.9 ± 5.0

14.2 ± 2.5

Total no. of males* on day 1 of lactation (%)

65 (48)

71 (49)

52 (52)

45 (51)

Total no. of females* on day 1 of lactation (%)

70 (52)

74 (51)

48 (48)

43 (49)

* excludes litters where all pups died

 

Table 5: Group Mean F1 Survival Indices

Group / Dose level (mg/kg/day)

1 / 0

2 / 100

3 / 300

4 / 1000

Birth index

Mean litter index (%)

93

91

91

91

No. losing > 2 pups

2

2

3

2

No. of litters

10

10

10

10

Live birth index

Mean litter index (%)

100

99

85

89

No. losing > 1 pup

0

0

3

1

No. of litters

10

10

10

10

Viability index (Days 0-4)

Mean litter index (%)

86

95

72

65

No. losing > 3 pups

1

0

3

3

No. of litters

10

10

9

9

 

Conclusions:
Treatment in females at 300 mg/kg/day and above was associated with a slight reduction in gestation index, a reduced live birth index and a decrease in pup viability over Days 1-4 of lactation. At 1000 mg/kg/day there was an increased incidence of clinical observations around the time of parturition and an increase in the number of dams with no teats evident in early lactation (dams which subsequently lost their entire litters). A 36.7 % decrease in group mean bodyweight gain over Days 1-4 of lactation was also noted at 1000 mg/kg/day.
In conclusion, under the conditions of the study, the maternal systemic No Observed Effect Level (NOEL) was considered to be 300 mg/kg/day.
There were effects on the reproductive performance/survival of the pups at 300 mg/kg/day and above. Although the cause of reduced pup survival at these dose levels could not be determined from this study, the developmental No Observed Effect Level (NOEL) was considered to be 100 mg/kg/day.
Executive summary:

The developmental toxicity of the test material was investigated in a GLP study which was conducted in accordance with the standardised guideline OECD 422.

During the study, groups of 10 male and 10 female Sprague-Dawley Crl:CD (SD) strain rats were administered test material, by oral gavage, at dose levels of 0 (vehicle control), 100, 300 and 1000 mg/kg bw/day. Another group of 10 male and 10 female rats was dosed with the vehicle (1 % w/v carboxymethylcellulose in Milli-Q water) 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 endpoints were evaluated: clinical signs, bodyweight, bodyweight changes, food consumption, gross necropsy findings, organ weights, histopathological examinations, mating and pregnancy performance, fertility, maternal care, pup performance (litter survival and pup weights).

Treatment in females at 300 mg/kg/day and above was associated with a slight reduction in gestation index, a reduced live birth index and a decrease in pup viability over Days 1-4 of lactation. At 1000 mg/kg/day there was an increased incidence of clinical observations around the time of parturition and an increase in the number of dams with no teats evident in early lactation (dams which subsequently lost their entire litters). A 36.7 % decrease in group mean bodyweight gain over Days 1-4 of lactation was also noted at 1000 mg/kg/day.

In conclusion, under the conditions of the study, the maternal systemic No Observed Effect Level (NOEL) was considered to be 300 mg/kg/day.

There were effects on the reproductive performance/survival of the pups at 300 mg/kg/day and above. Although the cause of reduced pup survival at these dose levels could not be determined from this study, the developmental No Observed Effect Level (NOEL) was considered to be 100 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Study duration:
subacute
Species:
rat
Quality of whole database:
The study was conducted to GLP and in accordance with the standardised guideline OECD 422. The study was assigned a reliability score of 1 according to the criteria of Klimisch (1997). The quality of the database is good.
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

The developmental toxicity of the test material was investigated in a GLP study which was conducted in accordance with the standardised guideline OECD 422.

During the study, groups of 10 male and 10 female Sprague-Dawley Crl:CD (SD) strain rats were administered test material, by oral gavage, at dose levels of 0 (vehicle control), 100, 300 and 1000 mg/kg bw/day. Another group of 10 male and 10 female rats was dosed with the vehicle (1 % w/v carboxymethylcellulose in Milli-Q water) 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 endpoints were evaluated: clinical signs, bodyweight, bodyweight changes, food consumption, gross necropsy findings, organ weights, histopathological examinations, mating and pregnancy performance, fertility, maternal care, pup performance (litter survival and pup weights).

Treatment in females at 300 mg/kg/day and above was associated with a slight reduction in gestation index, a reduced live birth index and a decrease in pup viability over Days 1-4 of lactation. At 1000 mg/kg/day there was an increased incidence of clinical observations around the time of parturition and an increase in the number of dams with no teats evident in early lactation (dams which subsequently lost their entire litters). A 36.7 % decrease in group mean bodyweight gain over Days 1-4 of lactation was also noted at 1000 mg/kg/day.

In conclusion, under the conditions of the study, the maternal systemic No Observed Effect Level (NOEL) was considered to be 300 mg/kg/day.

There were effects on the reproductive performance/survival of the pups at 300 mg/kg/day and above. Although the cause of reduced pup survival at these dose levels could not be determined from this study, the developmental No Observed Effect Level (NOEL) was considered to be 100 mg/kg/day.


Justification for selection of Effect on developmental toxicity: via oral route:
A single repeated dose and reproductive/developmental screening study was available for assessment. The study was performed and reported to a high standard, in line with a standardised guideline (OECD 422) and in compliance with GLP.

Justification for classification or non-classification

In accordance with the Regulation (EC) No. 1272/2008, no classification for toxicity to reproduction or developmental toxicity is required based on the results of the repeated dose toxicity and reproductive/developmental toxicity screening study.