Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 May 2015 to 02 September 2015
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:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD 422 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: US EPA OPPTS 870.3650
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Qualifier:
equivalent or similar to
Guideline:
other: US EPA OPPTS 870.3550
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Appearance: Slightly yellow powder with lumps
- Storage conditions of test material: Room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl:WI(Han), outbred, SPF-Quality
- Age at study initiation: Approximately 11-12 weeks
- Weight at study initiation: Females (mean): 217 g
- Fasting period before study: No
- Housing: Pre-mating, animals were housed in groups of 5 animals/sex/cage in plastic cages (height 18 cm); during mating, females were caged together with males on a one-to-one-basis in plastic cages (height 18 cm). Post-mating, females were individually housed in plastic cages (height 18 cm). Sterilised sawdust as bedding material and paper as cage-enrichment/nesting material were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet: Free access to pelleted rodent diet
- Water: Free access to tap-water
- Acclimation period: At least 5 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24 °C
- Humidity: 40 to 70 % (relative)
- Air changes: at least 10 room air changes/hour
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES
- From: Experimental starting date: 30 May 2015 (allocation dose range finding study). Dosing for the main study began on the 08 July 2015.
- To: 18, 19, 20, 21, 24 and 25 August 2015, and 02 September 2015 (females and/or pups)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Formulations were heated up to a maximum temperature between 56.7 to 61.8 °C, for a maximum of 36 minutes to obtain visual homogeneity.
Formulations were released for dosing when they obtained a temperature below 40 °C. No adjustment was made for specific gravity/density of the test material and formulation. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test material.
The solutions were stored at room temperature.

VEHICLE
- Specific gravity: 0.92
- Amount of vehicle (if gavage): Dose volume was 5 mL/kg. Actual dose volumes were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
CHEMICAL ANALYSIS OF DOSE PREPARATIONS
Analyses were conducted on a single occasion during the treatment phase (day 2 of dosing), according to a validated method. Samples of formulations were analysed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in the vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90 to 110 % of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10 %. Formulations were considered stable if the relative difference before and after storage was maximally 10 %.

ANALYTICAL CONDITIONS
Quantitative analysis was based on the analytical method validated for the test material.
- Instrument: Acquity UPLC system (Waters, Milford, MA, USA)
- Detector: Acquity UPLC TUV detector (Waters)
- Column: Acquity UPLC BEH Shield RP-18, 100 mm x 2.1 mm i.d.,dp = 1.7 μm (Waters)
- Column temperature: 40 ± 1°C
- Injection volume: 10 μL
- Mobile phase: 80/20 (v/v) acetonitrile/water
- Flow: 0.6 mL/min
- UV detection: 220 nm

SAMPLES
Accuracy, homogeneity and stability were determined for formulations prepared for use during treatment.
Duplicate accurately weighed samples of approximately 500 mg were taken from the formulations using a pipette and were transferred into volumetric flasks of 10 mL. For determination of accuracy, samples were taken at middle position (50 % height) or at top, middle and bottom position (90, 50 and 10 % height). The samples taken at 90, 50 and 10 % height were also used for the determination of the homogeneity of the formulations. For determination of stability, additional samples were taken at 50 % height and stored at room temperature under normal laboratory light for 6 hours.
The volumetric flasks were filled up to the mark with acetonitrile. The samples were extracted by shaking for 30 seconds. The solutions were further diluted to obtain an end solution of 50/50 (v/v) acetonitrile/water and concentrations within the calibration range.

PREPARATION OF SOLUTIONS
- Stock and spiking solutions
Stock and spiking solutions of the test material were prepared in acetonitrile at concentrations of 1000 to 4000 mg/L.

- Calibration solutions
Calibration solutions in the concentration range of 0.1 to 20 mg/L were prepared from two stock solutions. The end solution of the calibration solutions was 50/50 (v/v) acetonitrile/water.

-Procedural recovery samples
Approximately 500 mg blank vehicle was spiked with the test material at a target concentration of 0.4 or 10 mg/g. The accuracy samples were treated similarly as the test samples.

SAMPLE INJECTIONS
Calibration solutions were injected in duplicate. Test samples and procedural recovery samples were analysed by single injection.

FORMULAE
Response (R): Peak area test substance [units]

Calibration curve: R = a CN + b
where:
CN = nominal concentration [mg/L]
a = slope [units x L/mg]
b = intercept [units]

Analysed concentration (CA): Ca = [(R – b) / a] x [(V x d) / w] [mg/g]
where:
w = weight sample [mg]
V = volume volumetric flask [mL]
d = dilution factor

Recovery: (CA / CN) x 100 [%]
where:
CN = nominal concentration [mg/g]

Accuracy: (CA / CT) x 100 [%]
where:
CT = target concentration [mg/g]

Relative difference (relative diff.): [(Ct – C0) / C0] x 100 [%]
where:
Ct = mean concentration of stored samples [mg/g]
C0 = mean concentration of non-stored samples [mg/g]

RESULTS
CALIBRATION CURVES
A calibration curve was constructed using six concentrations. For each concentration, two responses were used. Linear regression analysis was performed using the least squares method with a 1/concentration² weighting factor. The coefficient of correlation (r) was > 0.99.

SAMPLES
- Procedural recovery samples
The mean recoveries of the procedural recovery samples fell within the criterion of 90 – 110 %. It demonstrated that the analytical method was adequate for the determination of the test material in the test samples.

- Test samples
Accuracy of preparation
In the control group formulations, no test material was detected. The concentrations analysed in the formulations of the 10 and 30 mg/kg dose groups were in agreement with target concentrations (i.e. mean accuracies between 90 and 110 %).
For the formulation of the 3 mg/kg dose group prepared for use, the mean accuracy was slightly below the target concentration (i.e. 89 % of target). Since the deviation was small, it was considered to have no effect on the integrity of the study and results were accepted.
Homogeneity
The formulations of the 3 and 30 mg/kg dose groups were homogeneous (i.e. coefficient of variation ≤ 10 %).
Stability
Analysis of the 3 and 30 mg/kg dose group formulations after storage yielded a relative difference of ≤ 10 %. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 6 hours.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- After successful mating each pregnant female was caged (how): Once mating occurred, the males and females were separated. Females were individually housed in plastic cages.
Duration of treatment / exposure:
- Females were exposed for 41 to 56 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).
- Pups were not dosed directly but could have potentially been exposed to the test material in utero, via maternal milk or from exposure to maternal urine/faeces.
Frequency of treatment:
Once daily, 7 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 3, 10 and 30 mg/kg bw/day (dose Groups 1, 2, 3 and 4 respectively).
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for dose levels: Based on the results of a 10-day dose range finding study in which 3 females per dose were treated with the test material at 50, 100 and 250 mg/kg body weight, the dose levels were selected to be 3, 10 and 30 mg/kg.
- Rationale for animal assignment: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20 % of the sex mean.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were examined for mortality/viability at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals, at least immediately after dosing. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION: Yes
- Time schedule: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected animals. Blood samples were drawn from the retro-orbital sinus and collected into tubes prepared with K3-EDTA for haematological parameters (0.5 mL) and with citrate for clotting tests (0.45 mL).
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available.
- How many animals: 5 animals per sex per dose group were evaluated
- The following parameters were examined: White blood cells (WBC; differential leucocyte count: neutrophils, lymphocytes, monocytes, eosinophils and basophils), red blood cells, reticulocytes, red blood cell distribution width (RDW), haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) and platelets. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were also determined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected animals. Blood samples were drawn from the retro-orbital sinus and collected into tubes treated with Li-heparin for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids.
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available.
- How many animals: 5 animals per sex per dose group were evaluated
- The following parameters were examined: Alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium and inorganic phosphate (Inorg. Phos). All parameters were determined in plasma, except for bile acids which were determined in serum.

OTHER: Five animals per sex per group were subjected to functional observations and observations of locomotor activity (the same animals selected for haematology and clinical chemistry evaluations).

General reproduction data were also recorded; mating date, confirmation of pregnancy and delivery day were recorded. Palpation was used to aid in confirmation of pregnancy. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Following completion of dosing, all surviving animals were sacrificed and subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
The numbers of former implantation sites and corpora lutea were recorded for all paired females. Samples of selected tissues and organs were collected and fixed in 10 % buffered formalin (neutral phosphate buffered 4 % formaldehyde solution) for 5 selected animals/group. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
Terminal body weights and selected organ weights were recorded from the selected 5 females/group. All organ and tissue samples were processed, embedded and cut at a thickness of 2 to 4 micrometres. The slides were stained with haematoxylin and eosin.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
Fetal examinations:
PARTURITION
The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring, if practically possible:
- Mortality / Viability: The numbers of live and dead pups were determined on Day 1 of lactation and daily thereafter. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made for all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation.
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.
Indices:
For each group, the following calculations were performed:
- Mating index (%) = (Number of females mated / Number of females paired) x 100
- Fertility index (%) = (Number of pregnant females / Number of females paired) x 100
- Conception index (%) = (Number of pregnant females / Number of females mated) x 100
- Gestation index (%) = (Number of females bearing live pups / Number of pregnant females) x 100
- Duration of gestation = Number of days between confirmation of mating and the beginning of parturition
- Percentage live males at First Litter Check = (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100
- Percentage live females at First Litter Check = (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100
- Percentage of postnatal loss = (Number of dead pups before planned necropsy / Number of live pups at First Litter Check) x 100
- Viability index = (Number of live pups before planned necropsy / Number of pups born alive) x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortality occurred during the study period. No clinical signs were noted during daily detailed observations and weekly arena observations that were considered toxicologically relevant.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
At 10 and 30 mg/kg, some females showed weight loss during lactation which was notably different to the weight gain observed for control females. At 10 mg/kg, notable weight loss was recorded of 4 or 9 % compared to Day 1 lactation for three females (nos. 63, 64 and 70). At 30 mg/kg, notable weight loss was recorded for three females (nos. 75, 77 and 80) of 5, 7 or 9 %, respectively, compared to Day 1 lactation.
Mean body weight gain of females at 30 mg/kg appeared slightly lower than controls over the post coitum period, which achieved a level of statistical significance on Days 4 and 7. Mean bodyweight of these females on Day 0 of post coitum was statistically significantly higher than controls, as well as on Day 1 of the mating period. None of these changes and other slight statistically significant changes in body weight (gain) were considered toxicologically meaningful, since these remained well within the range considered normal for rats of this age and strain, and showed no clear and consistent trend over time and dose.
Females at 10 and 30 mg/kg showed an apparent lower food consumption before and after correction for body weight over Days 1 to 4 of lactation (not statistically significant; and with a relatively high standard deviation at 30 mg/kg).
Food consumption before or after correction for body weight was considered unaffected by treatment for females at 3 mg/kg.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No toxicologically relevant changes in reproduction parameters were observed up to the highest dose level tested (30 mg/kg). These parameters included mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites and histopathological examination of reproductive organs.
An apparent lower mean number of implantation sites was noted at 30 mg/kg (11.4 vs. 12.7 in the control group; not statistically significant), which was primarily due to a low number of implantation sites for one female (no. 75). Other animals of this dose group showed a number of implantation sites that was within the concurrent control range. Therefore this was not considered to be of toxicological relevance.
Mating, fertility and conception indices, pre-coital time, and number of corpora lutea were unaffected by treatment.
All females showed evidence of mating.
For one control female (no. 49) and one female at 10 mg/kg (no. 68), the number of pups was slightly higher than the number of implantations and/or corpora lutea. This was considered caused by normal resorption of these areas as these enumerations were performed on Day 5 of lactation.

PARTURITION/MATERNAL CARE
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Test material-related higher liver weights (absolute and relative to body weights) were noted in the 10 and 30 mg/kg females. Relative liver weights were increased 63 % in females at 30 mg/kg, and 21 % in females at 10 mg/kg.
Any other statistically significant changes in organ weight at the end of the treatment period were not considered to be treatment-related as they occurred in the absence of a dose-related trend and had no histological correlate.

GROSS PATHOLOGY, HISTOPATHOLOGY AND OTHER FINDINGS (PARENTAL ANIMALS)
Histopathological examination showed hepatocellular hypertrophy up to moderate degree at 10 and 30 mg/kg. This was supported at necropsy by enlargement and/or accentuated lobular pattern of the liver for some animals at 30 mg/kg. At 30 mg/kg, hepatocellular necrosis was present in a single female at minimal degree. The higher liver weights in females at 10 and 30 mg/kg along with the combined occurrence of hepatocellular hypertrophy with necrosis at 30 mg/kg were considered to be adverse in nature.
Decreased splenic haematopoiesis was observed in females treated at 10 and 30 mg/kg, which occurred along with lower reticulocyte counts. However, red blood cell counts showed an increase rather than a decrease at this dose level. Also, since there were no other indicators of toxicity in the spleen, these changes were not considered to represent an adverse effect on red blood cell turn over.
At 30 mg/kg, other changes in blood of females that were considered to be related to treatment consisted of lower relative neutrophil counts, higher relative lymphocyte counts, lower mean corpuscular volume and mean corpuscular haemoglobin, higher total protein, albumin, calcium, cholesterol, potassium and glucose and lower urea and total bilirubin.
At 10 mg/kg, changes in blood consisted of lower mean corpuscular volume, mean corpuscular haemoglobin and total bilirubin, and higher cholesterol.
In the absence of any concurrent morphological lesions, and given the slight nature of these changes (i.e. within or just outside the range considered normal for rats of this age and strain), these were not considered adverse in nature.
Females at 10 and 30 mg/kg showed a lower food consumption during the lactation period, as well as notable weight loss during lactation ranging from 4 to 9 % of Day 1 lactation values. The lower maternal food intake and body weight gain and mean pup body weight gain appeared unrelated on an individual animal basis. Also, these changes were not accompanied by supportive clinical signs or inadequate maternal care. As such, these changes in food intake and body weight gain during lactation were considered not adverse in nature.
No toxicologically relevant clinical signs or changes in functional observation parameters were noted.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
3 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
>= 30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
An increased postnatal loss and lower viability index was noted at 10 and 30 mg/kg. At 10 mg/kg, a total of 3 dams showed postnatal loss (1 each for litters 66 and 67, and 6 for litter 61). At 30 mg/kg, a total of 5 dams showed postnatal loss (1 each for litters 73, 75 and 78, 4 for litter 79 and 5 for litter 80).

The mean number of living pups at first litter check at 30 mg/kg appeared slightly lower than controls (9.4 vs 11.2 in the control group; not statistically significant).

Lower mean body weights were recorded for pups for each sex and for both sexes combined at 10 and 30 mg/kg on Days 1 and 4 of lactation. On Day 4 of lactation mean pup body weight for both sexes combined was approximately 16 and 20 % lower than the control mean at 10 and 30 mg/kg, respectively. Although there were no other developmental changes noted for these pups (macroscopy and clinical signs), the magnitude of changes in pup body weight was considered to represent an adverse effect on pup development. No apparent relationship could be found between maternal food intake and mean pup body weight gain on an individual animal basis.

Mean male pup body weight was also lower at 3 mg/kg on Day 4 lactation (approximately 8 % lower than the control mean). Since this change in body weight was slight this was considered not to represent an adverse effect on pup development.

The occurrence of a single death at first litter check for litter 67 (10 mg/kg) as well as the single control female (no. 42) which did not deliver offspring (implantation sites only), was considered to be within the range considered normal for rats of this age and strain, and hence considered to be unrelated to treatment.

Gestation duration and index, number of dead and living pups at first litter check, sex ratio, parturition, maternal care and other early postnatal pup development parameters (clinical signs and macroscopy) were considered unaffected by treatment.

EARLY POSTNATAL PUP DEVELOPMENT
Number of dead and living pups at first litter check and sex ratio were unaffected by treatment, and clinical signs and external macroscopy did not reveal treatment-related findings.

CLINICAL SIGNS
Incidental clinical symptoms of surviving pups consisted of a blue spot on the snout, head, back or neck, scabs, blue snout, pallor, absence of milk in the stomach, missing tail or tail point, and dehydrated appearance (for the two surviving pups of one Group 4 litter, no. 80). Pallor was noted for two pups at 10 mg/kg found missing (one each for litter 61 and 67). The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

MACROSCOPY
Incidental macroscopic findings among surviving pups included a missing tail or tail point or dehydrated appearance (for the two surviving pups of one Group 4 litter, no. 80). The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1: Reproduction Data Summary

 

Group 1
Control

Group 2
3 mg/kg

Group 3
10 mg/kg

Group 4
30 mg/kg

Females paired

10

10

10

10

Females mated

10

10

10

10

Pregnant females

9

10

10

10

Females with living pups on Day 1

9

10

10

10

Mating index (%)
(Females mated / Females paired) * 100

100

100

100

100

Fertility index (%)
(Pregnant females / Females paired) * 100

90

100

100

100

Conception index (%)
(Pregnant females / Females mated) * 100

90

100

100

100

Gestation index (%)
(Females with living pups on Day 1 / Pregnant females) * 100

100

100

100

100

 

Table 2: Corpora Lutea and Implantation Sites Summary

 

 

Group 1

Control

Group 2

3 mg/kg

Group 3

10 mg/kg

Group 4

30 mg/kg

Corpora lutea

Mean

N

12.9

9

13.8

10

15.2

10

13.7

10

Implantations

Mean

N

12.7

9

13.3

10

12.9

10

11.4

10

Table 3: Mean Body Weights of Pups (g)

 

 

Group 1
Control

Group 2
3 mg/kg

Group 3
10 mg/kg

Group 4
30 mg/kg

 

Sex

 

 

 

 

Day 1

M

6.3

5.9

5.7**

5.7**

F

6.1

5.7

5.4**

5.4*

M+F

6.2

5.8

5.5**

5.6*

Day 4

M

9.6

8.8*

8.2**

7.7**

F

9.4

8.3

7.7**

7.5**

M+F

9.5

8.5

8.0**

7.6**

*/** Dunnett-test based on pooled variance significant at 5 % (*) or 1 % (**) level

Table 4: Summary of Developmental Data

 

Group 1

Control

Group 2

3 mg/kg

Group 3

10 mg/kg

Group 4

30 mg/kg

Number of litters

9

10

10

10

Duration of gestation

Mean

SD

N

 

21.2

0.4

9

 

21.0

0.0

10

 

21.0

0.0

10

 

21.4

0.7

10

Dead pups at first litter check

Litters affected

Total

Mean

SD

N

 

0

0

0.0

0.0

9

 

0

0

0.0

0.0

10

 

1

1

0.01

0.3

10

 

0

0

0.0

0.0

10

Living pups at first litter check

% of males / females

Total

Mean

SD

N

 

50/50

101

11.2

2.0

9

 

50/50

125

12.5

1.6

10

 

50/50

121

12.1

1.9

10

 

55/45

94

9.4

2.5

10

Postnatal loss

% of living pups

Litters affected

Total

Mean

SD

N

 

0.0

0

0

0.0

0.0

9

 

0.0

0

0

0.0

0.0

10

 

6.6

3

8##

0.8

1.9

10

 

12.8

5

12##

1.2

1.8

10

Viability index

100.0

100.0

93.4##

87.2##

## Fisher’s Exact test significant at 1 % Level

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, the developmental NOAEL was determined to be 3 mg/kg (based on lower pup body weight and increased postnatal loss at 10 and 30 mg/kg, and lower mean number of living pups at first litter check at 30 mg/kg).
Executive summary:

The developmental toxicity of the test material was investigated in a combined repeated dose toxicity study with the reproduction / developmental toxicity screening test conducted in accordance with the standardised guidelines OECD 422 and US EPA OPPTS 870.3650 under GLP conditions.

Based on the results of a 10-day dose range finding study, the dose levels for this study were selected to be 3, 10 and 30 mg/kg.

The test material, formulated in corn oil, was administered daily by oral gavage to SPF-bred Wistar Han rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41 to 56 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), clinical pathology (end of treatment), macroscopy at termination, organ weights and histopathology on a selection of tissues. Reproduction/developmental parameters were also evaluated.

Formulations were analysed once during the study to assess accuracy, homogeneity and stability. Analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

Histopathological examination showed hepatocellular hypertrophy up to moderate degree at 10 and 30 mg/kg. This was supported at necropsy by enlargement and/or accentuated lobular pattern of the liver for some animals at 30 mg/kg. At 30 mg/kg, hepatocellular necrosis was present in a single female at minimal degree. Additionally, higher liver weights (absolute and/or relative to body weights) were recorded at 10 and 30 mg/kg; relative liver weights were increased 63 % in females at 30 mg/kg, and 21 % in females at 10 mg/kg. The higher liver weights in females at 10 and 30 mg/kg along with the combined occurrence of hepatocellular hypertrophy with necrosis at 30 mg/kg were considered to be adverse in nature.

At 30 mg/kg, changes in blood of females that were considered to be related to treatment consisted of lower relative neutrophil counts, higher relative lymphocyte counts, lower mean corpuscular volume and mean corpuscular haemoglobin, higher total protein, albumin, calcium, cholesterol, potassium and glucose and lower urea and total bilirubin.

Females at 10 and 30 mg/kg showed lower food consumption during the lactation period, as well as notable weight loss during lactation ranging from 4 to 9 % of Day 1 lactation values. The lower maternal food intake and body weight gain and mean pup body weight gain appeared unrelated on an individual animal basis. Also, these changes were not accompanied by supportive clinical signs or inadequate maternal care. As such, these changes in food intake and body weight gain during lactation were considered not adverse in nature.

No toxicologically relevant clinical signs or changes in functional observation parameters were noted.

No toxicologically relevant changes in reproduction parameters were observed up to the highest dose level tested (30 mg/kg). These parameters included mating, fertility and conception indices, pre-coital time, and numbers of corpora lutea and implantation sites and histopathological examination of reproductive organs.

At 30 mg/kg, the mean number of living pups at first litter check appeared slightly lower than controls. Also, an increased postnatal loss and lower viability index was noted at 10 and 30 mg/kg. At 10 mg/kg, a total of 3 dams showed postnatal loss (1 dead or missing pup for two litters, and 6 missing pups for another litter). At 30 mg/kg, a total of 5 dams showed postnatal loss (1 missing pup for 3 litters, and 4 or 5 missing pups for two litters). The significance of these findings is not clear at this stage and are proposed to be further investigated. The registrant therefore proposes to conduct a full prenatal developmental toxicity study in rats in accordance to the OECD Guideline 414.

Mean pup body weights for both sexes combined on Day 4 of lactation were approximately 16 and 20 % lower than the control mean at 10 and 30 mg/kg, respectively. Although there were no other developmental changes noted for these pups (macroscopy and clinical signs), the magnitude of changes in pup body weight was considered to represent an adverse effect on pup development. No apparent relationship could be found between maternal food intake and mean pup body weight gain on an individual animal basis.

Mean male pup body weight was also lower at 3 mg/kg on Day 4 lactation (approximately 8 % lower than the control mean). Since this change in body weight was slight this was considered not to represent an adverse effect on pup development.

No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of clinical signs and macroscopy).

Under the conditions of the study, the repeated dose NOAEL was determined to be 3 mg/kg (based on higher liver weights at 10 and 30 mg/kg, with hepatocellular hypertrophy and necrosis at 30 mg/kg). The developmental NOAEL was determined to be 3 mg/kg (based on lower pup body weight and increased postnatal loss at 10 and 30 mg/kg, and lower mean number of living pups at first litter check at 30 mg/kg).