Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 July 2012 to 2 October 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.

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Appearance: dark brown, viscous liquid
- Storage conditions: room temperature in the dark
- Stability under storage conditions: stable

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 weeks.
- Weight at study initiation: The average weight of the males was 326 g and the average weight of the females was 206 g on Day 1 of the study.
- 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, males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in plastic cages (height 18 cm). During lactation, pups were kept with the dam until termination in plastic cages (height 18 cm).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet.
- Water (e.g. ad libitum): Water Free access to tap-water.
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24 °C
- Humidity (%): 40 to 70 % relative
- Air changes (per hr): approximately 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark cycle

IN-LIFE DATES: From: 9 August 2012 To: 2 October 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the testing laboratory.
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
- Specific gravity: 1.036
- Method of formulation: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenised to a visually acceptable level. No correction was made for the purity of the test material. Adjustment was made for specific gravity of the vehicle. Formulations were placed on a magnetic stirrer during dosing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The purpose of the analytical phase was to determine the accuracy of preparation, homogeneity and stability of the test material in formulations.
Samples of dose preparations were taken on a single occasion during the treatment phase and were stored and dispatched on dry ice to the test site for formulation analysis.
Formulations were analysed to check homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in the vehicle over 5 hours was also determined (highest and lowest concentration).

Specific sampling instructions:
− Appearance of the formulations was recorded prior to sampling
− Formulations were placed on a magnetic stirrer during sampling
− Sampling was conducted with a clean pipette tip used for every group
− Duplicate samples were taken from each sampling height, with a sample volume of 0.5 mL
− Samples were weighed (grams) on an analytical balance at a precision of 4 decimals
− T, M and B samples were stored on dry ice immediately after sampling
− S (Stability) samples were kept at room temperature under normal laboratory light conditions for
5 hours, and then placed on dry ice
− All samples were transferred to the weighing room at once for dispatch

SAMPLES
In total, 20 samples were included, randomised over 4 formulation groups. Group 1 was the control group. Group 2, 3 and 4 were dosed at 75, 250 and 750 mg/kg bw/day, respectively. The samples of the control group and group 3 were taken in duplicate from the middle position of the container and immediately stored on dry ice.
Samples of treatment groups 2 and 4 were taken in duplicate from the top, middle and bottom position of the container. In addition, stability samples were taken from formulation groups 2 and 4, in duplicate at the middle position of the container, stored for 5 hours at room temperature under normal laboratory light conditions and then placed on dry ice.
The samples were stored at the analytical facility at a target temperature ≤-70 °C.

ANALYTICAL METHOD
The determination was performed using LCMS/MS.

SAMPLE INJECTIONS
The analytical run was acquired in the following order:
- Analytical blank sample
- Calibration curve
- Analytical blank sample
- Analytical blank sample
- Procedural recovery sample low and high (replicate 1)
- Analytical samples
- Stability at storage conditions low and high (replicate 1 and 2)
- Procedural recovery samples low and high (replicate 2)
Calibration solutions were injected in duplicate. Test samples and procedural recovery samples were
analysed by single injection.

ELECTRONIC DATA CAPTURE
System control, data acquisition and data processing were performed using the program Analyst version 1.5.2 (Applied Biosystems, Burlington, Canada).
Electronic raw data was stored in the document management system using KnowledgeTree version 3.5.2c (Raleigh, USA).

FORMULAS
Response (Y)
Peak area test substance [cps]

Calibration curve
Y = bX + a
where:
X = nominal concentration [mg/L]
b = slope [cps × L/mg]
a = intercept [cps]

Analysed concentration (X)
X = ((Y - a) / b) x ((V x d) / w [mg/g]
where:
w = weight sample [mg]
V = volume volumetric flask [mL]
d = dilution factor
(Y - a) / b values are obtained from Analyst

Recovery
(Analysed concentration [mg/g] / Nominal concentration [mg/g]) x 100 [%]

Accuracy
(Analysed concentration [mg/g] / Target concentration [mg/g]) x 100 [%]

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]

SPECIFICATIONS
Run Acceptance
Calibration curves with a coefficient of correlation (r) of >0.99 and accuracies of the back calculated values of the calibration standard solutions in the range 85 to 115 % were accepted.
The mean recovery of the procedural recovery samples at each level had to be in the range 90 to 110 %.
The response in all analytical blank samples at the retention time of the test substance should be less than or equal to 30 % compared to the response of the lowest calibration standard (mean response of the duplicate injections).

Acceptance criteria formulations
Preparation of formulations was considered acceptable if the mean accuracy was in the range 90 to 110 % of the target concentration and if the coefficient of variation was ≤ 10 %. Formulations were considered stable if the absolute relative difference between the stored and freshly taken samples was ≤ 10 %.

RESULTS
Calibration curves
Calibration curves were constructed using six concentrations. For each concentration, two responses were acquired. Linear regression analysis was performed using the least squares linear regression with a 1/concentration² weighting factor.
The coefficient of correlation (r) of the calibration curve was 0.9998.
Additionally, there was no response in all analytical blank samples at the retention time of the test material.

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

Test samples
In the Group 1 formulation, no test material was detected. The concentrations analysed in the formulations of Group 2, 3 and 4 were in agreement with the target concentrations (i.e. mean accuracies between 90 and 110 %).
The formulations of Group 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10 %). Analysis of Group 2 and Group 4 formulations after storage yielded an absolute relative difference ≤ 10 %. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 5 hours.

Stability at storage conditions
It was observed that the mean accuracy was 94.9 and 98.2 % at the levels high and low, respectively, which is within the criteria set (mean accuracy is in the range 90 to 110 %). It was therefore concluded that the test material was stable in propylene glycol over a storage period of 29 days at a temperature of ≤-70 °C.
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. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.
Duration of treatment / exposure:
Males were exposed for 28 days: 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 42 to 55 days: 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).
Frequency of treatment:
Once daily
Duration of test:
28 days for males, 42 to 55 days for females.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 75, 250 and 750 mg/kg
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:
- Dose selection rationale: Based on the results of a 10-day dose range finding study conducted at the testing laboratory.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Parental animals were observed for mortality / viability at least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily, detailed clinical observations were conducted for all animals after dosing (at no specific time point, but around the same time point for all animals). 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. The time of onset, grade and duration of any observed sign was recorded.

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 postcoitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY: No

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.

OTHER: Observations were carried out for haematology, clotting potential, clinical chemistry and neurobehavioural examinations.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes. The numbers of former implantation sites and corpora lutea were recorded for all paired females.
Fetal examinations:
Each litter was examined to determine the following, if practically possible:
- Mortality/viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were conducted for all animals.
- Bodyweights: 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 following additional methods of statistical analysis were used:
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.
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 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:yes

Details on maternal toxic effects:
MORTALITY
One female at 250 mg/kg was euthanised in extremis after 26 days of treatment; no definitive cause of death could be determined.
One female at 750 mg/kg was found dead after 33 days of treatment. Macroscopic examination confirmed that her death was secondary to a gavage error, and was in no way related to treatment.

CLINICAL SIGNS
Clinical signs noted for the female at 250 mg/kg that was euthanised in extremis included lethargy, hunched posture, laboured or shallow respiration, rales, piloerection, lean appearance and salivation. These were noted for several days prior to her death.
There were no other toxicology relevant observations noted up to 750 mg/kg.

CLINICAL CHEMISTRY
At 750 mg/kg the following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from controls:
-Increased alanine aminotransferase
-Increased alkaline phosphatase

MACROSCOPIC EXAMINATION
Macroscopic findings noted for the female at 250 mg/kg that was euthanised in extremis included enlarged mandibular lymph node, thymus reduced in size and GI tract distended with gas.
Findings noted for the female at 750 mg/kg that died due to a gavage error included fluid in the pericardium, perforation of the oesophagus, both adrenals enlarged and many dark red foci on the thymus. In the uterus, 1 foetus and 4 early resorptions were seen in the right uterine horn and 7 foetuses and 1 early resorption were seen in the left uterine horn.
At 750 mg/kg 7/10 females had visibly enlarged livers at the macroscopic examination.
Enlarged liver was also noted for 2 females at 250 mg/kg. This was considered toxicologically relevant.
One female at 250 mg/kg was found with 1 mummified foetus and dark red and watery clear fluid in the uterus. This female did not deliver any live pups.
Incidental findings noted for control and treated animals included enlarged mandibular lymph nodes, pelvic dilation of one or both kidneys, enlarged spleen, ectopic splenic tissue, reddish or dark red discoloration of the mandibular or renal lymph nodes, tan focus on the left clitoral gland, reduced size of the thymus, and alopecia on various body regions. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain and did not show a dose-related incidence trend. As such, these were not considered to be toxicologically relevant.

ORGAN WEIGHTS
At 750 mg/kg significantly increased absolute and relative liver weights (both sexes) were seen. Absolute and relative liver weights were significantly higher at 250 mg/kg as well.

MICROSCOPIC EXAMINATION
There were no morphological findings in the reproductive organs which could be attributed to the test material.
There were treatment-related microscopic findings present in the liver and thyroid gland.

Liver
-Centrilobular hypertrophy of the hepatocytes (above background) was present in female rats treated at 250 mg/kg (4/7 rats) up to a slight degree and in female rats treated at 750 mg/kg (9/9 rats) up to marked degree.

Thyroid glands
-Hyperplasia and hypertrophy, follicular was present at increased incidence and severity in females treated at 750 mg/kg (5/6) compared to control (1/5), 75 mg/kg (1/5) and 250 mg/kg (0/5) treated rats.


REPRODUCTIVE PARAMETERS
The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

DEVELOPMENTAL DATA
No toxicologically relevant effects on the gestation index and duration, parturition, maternal care and most aspects of early postnatal pup development (clinical signs, body weight and macroscopy) were observed.

Gestation
The gestation index and duration of gestation were unaffected by treatment up to 750 mg/kg. The gestation index was 100 % for controls and animals at 75 mg/kg, and was 80 % for animals at 250 and 750 mg/kg. There was one female each at 250 and 750 mg/kg with implantation sites only.

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 and no deficiencies in maternal care were observed.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
The postnatal loss and viability index were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

VIABILITY (OFFSPRING)
Three pups of the control group and five, seven and eleven pups in the 75, 250 and 750 mg/kg groups were found dead or went missing during the first days of lactation. Missing pups were most likely cannibalised. At 250 mg/kg, 5 dead pups came from a single litter. At 750 mg/kg 10 of the 11 dead pups came from two litters, which had 7 and 3 dead pups, respectively. A relationship to treatment could not be excluded at either dose level.

CLINICAL SIGNS (OFFSPRING)
All clinical signs noted for pups were incidental in nature and included scabbing on the hindleg or abdomen, blue spot on the neck or snout, laboured respiration, cold to the touch, pale appearance and lean. 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.

BODY WEIGHT (OFFSPRING)
Body weights of pups were unaffected by treatment.

GROSS PATHOLOGY (OFFSPRING)
Incidental macroscopic findings of pups that were found dead included no milk in the stomach, insufficient milk in the stomach, partial cannibalism (only the head was present) and/or beginning autolysis. Incidental macroscopic findings noted for surviving pups included scabbing on the right hindleg or abdomen, and blue spot on the neck. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore not considered toxicologically relevant.

OTHER FINDINGS (OFFSPRING)
Mean litter sizes were significantly smaller at 750 mg/kg then compared to controls. A high number of dead pups at first litter check (11, compared to 1 for controls) contributed to this.
The ratio of males/females was significantly lower for litters at 250 and 750 mg/kg. The pup deaths in these groups did not contribute to this as the pup deaths were predominantly of females, and as such this was considered a chance finding.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1 Summary of Developmental Data (F0 - Generation - Lactation)

 

Dose group (mg/kg)

Control

75

250

750

Total number of litters

10

10

8

8

Duration of gestation

Mean*

SD

N

 

21.2

0.4

10

 

21.2

0.4

10

 

21.4

0.5

8

 

21.4

0.5

8

Dead pups at first litter check

Litters affected

Total

Mean*

SD

N

 

1

1

0.1

0.3

10

 

1

1

0.1

0.3

10

 

2

4

0.5

1.1

8

 

3

11

1.4

2.5

8

Living pups at first litter check

% of males/females

Total

Mean*

SD

N

 

55/45

119

11.9

2.4

10

 

48/53

120

12.0

2.4

10

 

38/62

89

11.1

2.6

8

 

39/61

75

9.4*

1.8

8

Postnatal loss

% of living pups

Litters affected

Total

Mean*

SD

N

 

1.7

2

2

0.2

0.4

10

 

3.3

2

4

0.4

0.8

10

 

3.4

2

3

0.4

0.7

8

 

0.0

0

0

0.0

0.0

8

Viability index

98.3

96.7

96.6

100.0

*Significant at 5 % using the Steel-test

Significant at 5 % using Fisher’s Exact test

SD = Standard deviation

 

Table 2 Summary of Pup Bodyweights in g (F0 - Generation - Lactation)

Day

Sex

 

Dose group (mg/kg)

Control

75

250

750

 

 

 

 

1

Male

Mean

SD

N

6.0

0.3

10

5.9

0.8

10

5.9

0.6

8

5.9

0.8

8

Female

Mean

SD

N

5.6

0.4

10

5.7

0.7

10

5.8

0.6

8

5.6

0.8

8

Male + Female

Mean

SD

N

5.8

0.3

10

5.8

0.7

10

5.9

0.6

8

5.7

0.8

8

 

 

 

 

4

 

Male

Mean

SD

N

8.9

0.6

10

8.9

1.4

10

8.9

1.2

8

9.3

1.4

8

Female

Mean

SD

N

8.6

0.9

10

8.6

1.2

10

8.7

1.2

8

8.6

1.4

8

Male + Female

Mean

SD

N

8.8

0.8

10

8.8

1.3

10

8.8

1.2

8

8.9

1.5

8

SD = Standard deviation

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the NOAEL for developmental parameters is considered to be 75 mg/kg/day. The test material therefore requires classification in accordance with EU criteria for developmental toxicity as Category 2.
Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was carried out in order to assess the test material in accordance with the standardised guidelines OECD 422 and EPA OPPTS 870.3650 under GLP conditions.

Four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test material at 0, 75, 250 and 750 mg/kg/day in propylene glycol. Males were exposed for 28 days (2 weeks prior to mating, during mating, and up to termination) and females were exposed for 41 to 55 days (2 weeks prior to mating, during mating, during postcoitum and during at least 4 days of lactation).

Animals were evaluated for mortality/viability, clinical signs, functional observations and locomotor activity, body weight and food consumption, clinical pathology, macroscopy at termination, organ weights and histopathology on a selection of tissues and reproduction/developmental parameters.

Under the conditions of this study, the NOAEL for parental repeated dose toxicity was determined to be 75 mg/kg bw/day.

The mean litter size was smaller at 750 mg/kg than controls and higher pup mortality was evident at both 250 and 750 mg/kg. No toxicologically significant changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and clinical signs, bodyweights and macroscopy of pups).

Under the conditions of this study, the NOAEL for developmental parameters is considered to be 75 mg/kg/day. The test material therefore requires classification in accordance with EU criteria for developmental toxicity as Category 2.