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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
04 June 2012 - 27 August 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550 Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
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:
- Physical state: White powder
- Stability under storage conditions: Stable
- Storage condition of test material: At room temperature in the dark.

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl:WI(Han)
- Age at study initiation: (P) approximately 11 weeks.
- Housing:
> Pre-mating Animals were housed in groups of 5 animals/sex/cage in plastic cages.
> Mating Females were caged together with males on a one-to-one-basis in plastic cages.
> Post-mating Males were housed in their home cages with a maximum of 5 animals/cage. Females were individually housed in plastic cages.
> Lactation Pups were kept with the dam until termination in plastic cages.
- Diet: Pelleted rodent diet, ad libitum.
- Water: Tap water, ad libitum.
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 – 24 °C
- Humidity (%): 40 – 70 %
- Air changes (per hr): 15 room air changes/hour.
- Photoperiod (hrs dark / hrs light): A 12 hour light/12 hours dark cycle was maintained.

IN-LIFE DATES: From: 04 June To: 02 August (males) 27 August (females and pups).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity/density of the vehicle. No correction was made for the purity/composition of the test material. Test solutions were stored at ambient temperature.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the test facility.
- Specific gravity: 1.036
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on two occasions during the treatment phase Day 6 (09 July 2012) and Day 10 (13 July 2012) according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).

- Analytical conditions:
Instrument: Alliance Separation Module 2695 (Waters, Milford, MA, USA)
Detector: Dual λ Absorbance Detector 2487 (Waters)
Column: Symmetry C-18, 150 mm × 3 mm i.d., dp = 5 μm (Waters)
Column temperature: 40°C ± 1°C
Injection volume: 100 μl
Mobile phase: 80/20 (v/v) acetonitrile/water
Flow: 1.0 ml/min
UV detection: 235 nm

- Sample Preparation
Duplicate samples (approximately 500 mg), which were taken from the formulations using a pipette, were accurately weighed into volumetric flasks of 25 mL (Day 6) or 50 mL (Day 10).
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 conditions for 6 hours. The volumetric flasks were filled up to the mark and further diluted with acetonitrile. The last step was a dilution in a 1:1 (v/v) ratio with water to obtain a matrix of50/50 (v/v) acetonitrile/water and concentrations within the calibration range.

- Calibration Solutions
> Stock and spiking solutions: prepared in acetonitrile at concentrations of 935 and 1145 mg/L.
> Calibration solutions: prepared in the concentration range of 6.5 - 200 μg/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 mateiral at a target concentration of 20 or 200 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.

- Specification: The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% 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%.
Details on mating procedure:
Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. 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. Once mating occurred, the males and females were separated. Detection of mating was not confirmed for animal no. 61 which did deliver live offspring. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.

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.
Duration of treatment / exposure:
Males were exposed for 29 days (2 weeks prior to mating, during mating, and up to termination). Females were exposed for 39-54 days (during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation).
Frequency of treatment:
Once daily, seven days per week, up to the day before shceduled necropsy.
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Ten per sex per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosing range for the definitive study was based on the results of a 10-day dose range finding study. Three female rats were dosed at 500 and 1000 mg/kg bw/day for 10 consecutive days.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.
- Cage side observations checked: Mortality/viability and clinical signs of toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily within 30 minutes after doing

BODY WEIGHT: Yes
- Time schedule for examinations: Females 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
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
- Calculated 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 postcoitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION: No quantitative investigation was performed; however subjective appraisal was maintained during the study.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: All surviving animals which delivered a litter were scarified on lactation Day 5-7. All surviving animals which failed to deliver a litter were sacrificed approximately 21 days after the last day of the mating period (i.e. females without evidence of mating).
- Organs examined: macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs (Cervix, clitoral gland, coagulation gland, ovaries, preputial gland, uterus, vagina, and all gross lesions.

OTHER: 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.
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
Fetal examinations:
PARAMETERS EXAMINED
Mortality/viability, clinical signs, bodyweights and sex.

GROSS EXAMINATION OF DEAD PUPS:
Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation. All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (manyto-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.

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. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Indices:
- 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 Days 0-4 of lactation:
(Number of dead pups on Day 4 of lactation/ Number of live pups at First Litter Check) x 100

- Viability index:
(Number of live pups on Day 4 post partum/Number of pups born alive) x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period that was considered to be related to treatment with the test material.
One female (no. 67) at 300 mg/kg died during delivery. Subsequent examination could not determine a cause of death that was related to treatment. This was considered to be incidental in nature and not related to treatment.
No clinical signs of toxicity were noted during the observation period.
Alopecia was noted for a single female (no. 52) at 100 mg/kg. This was incidental in nature.

BODY WEIGHT AND FOOD CONSUMPTION
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control animals up to 1000 mg/kg.

PATURITION/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 and no deficiencies in maternal care were observed. Mean litter sizes were 11.8, 12.3, 12.8 and 12.0 pups for the control, 100, 300 and 1000 mg/kg groups, respectively.

REPRODUCTIVE PERFORMANCE
No toxicologically relevant effects on reproductive parameters were noted. There were 10, 9, 9 and 10 pregnant females in the control, 100, 300 and 1000 mg/kg groups, respectively.
The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

ORGAN WEIGHTS
There were no toxicologically relevant effects on terminal body weights up to 1000 mg/kg.

GROSS PATHOLOGY
Necropsy did not reveal any alterations that were attributable to treatment up to 1000 mg/kg.
The female (no. 67) who died during delivery was noted with dilation and a prolapsed vagina, a greenish, soft nodule on the right clitoral gland and 2 fetuses were found in the right uterine horn.
Incidental findings included pelvic dilation of the right kidney, alopecia of the shoulder and abdominal region, reddish hard nodule on the left uterine horn and vagina contains reddish fluid. At the limited incidence observed, these were not considered to be toxicologically relevant.

HISTOPATHOLOGY
There were no treatment related microscopic findings.
A definitive cause of death could not be established for female no. 67. The nodules in the uterus of animal no. 58 seen at the macroscopic examination represented implantation sites.
There were no microscopic findings in any of the animals suspected of infertility which could explain their lack of reproductive performance.
The remaining recorded microscopic findings were within the range of background pathology encountered in Wistar (Han) rats of this age in this type of study and occurred at similar incidences and severity in both control and treated rats.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.

GESTATION
The gestation index and duration of gestation were unaffected by treatment up to 1000 mg/kg.

PATURITION/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 and no deficiencies in maternal care were observed. Mean litter sizes were 11.8, 12.3, 12.8 and 12.0 pups for the control, 100, 300 and 1000 mg/kg groups, respectively.

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

MORTALITY
Three pups of the control group and two, two and eight pups of the 100, 300 and 1000 mg/kg groups, respectively were found dead or went missing during the first days of lactation. Missing pups were most likely cannibalized. Seven of the eight dead pups at 1000 mg/kg were all from female no. 80 who had three living and seven dead pups at first litter check. In the absence of any adverse effects seen for any other female in at this dose level, it was not considered to be treatment related.
No toxicological relevance was attributed to any of these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

CLINICAL SIGNS
Pale or lean appearance, no milk in the stomach and red spot on the head were incidental clinical signs noted. The nature and limited occurrence of these clinical signs remained within the range considered normal for pups of this age, and were not considered toxicologically relevant.

BODYWEIGHTS
Body weights of pups were unaffected by treatment up to 1000 mg/kg.

MACROSCOPY
Incidental macroscopic findings of pups that were found dead included no milk in the stomach and cannibalism of the whole body except the hind legs (noted for three of the seven dead pups from female no. 80). There were no macroscopic findings noted for surviving pups. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

ANALYSIS OF DOSE PREPARATIONS

Samples of Day 6

> Accuracy of preparation:

- No test material was detected in the control group.

- The concentrations analysed in the 100, 300 and 1000 mg/kg bw/day formulations were not in agreement with target concentrations since the mean accuracies were outside the acceptability range of 85-115%; with mean accuracies of 84%, 117% and 130%, respectively.

> Homogeneity:

The 100 and 1000 mg/kg bw/day formulations were not homogeneous since the coefficients of variation were 17% and 42%, respectively.

> Stability:

Analysis of Group 2 and Group 4 formulations after storage yielded a relative difference of 27% and -24%. The relatively large differences are probably caused by an inhomogeneity in the formulations rather than degradation of the test material.

 

Samples of Day 10

> Accuracy of preparation:

- No test material was detected in the control group.

- The concentrations analysed in the 100, 300 and 1000 mg/kg bw/day formulations were in agreement with target concentrations, mean accuracies were between 85% and 115%.

> Homogeneity:

The homogeneity of the 100 and 1000 mg/kg bw/day formulations was still above the criterion of 10%. However, the resulting coefficients of variation were 14% and 11% respectively, were still considered acceptable.

> Stability:

Analysis of 100 and 1000 mg/kg be/day formulations after storage yielded a relative difference of 12% and -9.4%. The formulations are found to be stable when the relative difference is ≤ 10%. Since the relative difference of the 100 mg/kg bw/day formulations of 12% was an increase in concentration, no degradation occurred. The relatively large differences are probably caused by an inhomogeneity in the formulations. The analytical method showed a higher variability at the low concentration level (i.e. a coefficient of variation of 11 and 18% of the procedural recovery samples) which possibly results in a higher variability of the analytical formulation results as well. The formulations are thus considered to be stable during storage at room temperature under normal laboratory light conditions for at least 6 hours.

Table 1: Developmental Data

Parameter

Dose Level mg/kg bw/day

Control (0.0)

100

300

1000

Litters Total

10

9

8

10

Mean duration of gestation

21.4

21.3

21.4

21.6

SD

0.7

0.5

0.5

0.5

N

10

9

8

10

Mean number of dead pups at first litter check

Litters Affected

1

0

0

1

Total

1

0

0

7

Mean

0.1

0.0

0.0

0.7

SD

0.3

0.0

0.0

2.2

N

10

9

8

10

Living Pups at first litter check

 

 

 

 

% of males/females

50/50

44/56

47/53

45/55

Total

118

111

102

120

Mean

11.8

12.3

12.8

12.0

SD

1.1

2.0

2.7

3.4

N

10

9

8

10

Postnatal loss

 

 

 

 

% of living pups

1.7

1.8

2.0

0.8

Litters affected

2

2

2

1

Total

2

2

2

1

Mean

0.2

0.2

0.3

0.1

SD

0.4

0.4

0.5

0.3

N

10

9

8

10

Viability Index

98.3

98.2

98.0

99.2

 

Table 2: Pup Bodyweight

Day

Sex

 

Dose Level mg/kg bw/day

Control (0.0)

100

300

1000

1

M

Mean ± SD

6.6 ± 0.5

6.4 ± 0.6

6.5 ± 0.8

6.6 ± 0.5

N

10

9

8

10

F

Mean ± SD

6.3 ± 0.4

6.1 ± 0.6

6.2 ± 0.6

6.3 ± 0.4

N

10

9

8

9

M+F

Mean ± SD

6.5 ± 0.5

6.2 ± 0.6

6.4 ± 0.6

6.4 ± 0.4

N

10

9

8

10

4

M

Mean ± SD

9.9 ± 0.9

9.6 ± 1.1

9.8 ± 1.3

10.3 ± 1.0

N

10

9

8

10

F

Mean ± SD

9.5 ± 0.8

9.3 ± 1.3

9.4 ± 1.1

9.7 ± 0.7

N

10

9

8

9

M+F

Mean ± SD

9.8 ± 0.9

9.4 ± 1.2

9.6 ± 1.1

10.1 ± 1.0

N

10

9

8

10

 

Table 3: Corpora Lutea and Implantation Sites Summary

Parameter

Dose Level mg/kg bw/day

Control (0.0)

100

300

1000

Corpora Lutea

Mean

15.3

15.3

15.0

15.3

SD

2.1

2.3

1.3

2.3

N

10

9

9

10

Implantations

Mean

12.7

13.7

13.6

13.4

SD

1.7

1.7

2.3

1.3

N

10

9

9

10

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test, no signs of maternal or developmental toxicity were observed at dose levels up to 1000 mg/kg bw/day. Based on these findings the NOAEL for material and developmental toxicity were determined to be the highest dose level, 1000 mg/kg be/day.
Executive summary:

Developmental toxicity was determined in a reproduction/development toxicity screening test, performed under GLP conditions and in line with the standardised guidelines OECD 421 and EPA OPPTS 870.3550. Based on the results of a 10-day dose range finding study, Crl:WI(Han) rats were dosed at 100, 300 and 1000 mg/kg bw/day, formulated in propylene glycol, and administered via oral gavage.

Ten males per dose were exposed for 29 days (2 weeks prior to mating, during mating, and up to termination), and ten females per dose were exposed for 39-54 days (during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation).

The accuracy, homogeneity and stability results were outside the acceptable range during the first formulation analysis. Subsequent analyses revealed the formulations were prepared accurately and were considered stable for at least 6 hours at room temperature, though formulations were not homogeneous. The results were attributable to an interaction between limited sensitivity of the analytical method and the nature of the test material itself and were ultimately accepted. The inhomogeneity of the test material had no negative impact on the study as formulations were stirred constantly during dosing.

No signs of maternal toxicity were noted during exposure or at necropsy up to the maximum dose level tested. Thus the NOAEL for maternal toxicity was considered to be 1000 mg/kg bw/day.

No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed up to the maximum dose level. Based on these findings the NOAEL for developmental toxicity was determined to be 1000 mg/kg bw/day.