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Toxicity to reproduction: other studies

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Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
12 September 2006 to 1 May 2007
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:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1450: Pubertal Development and Thyroid Function in Intact Juvenile/ Peripubertal Female Rats
Deviations:
no
GLP compliance:
yes
Type of method:
in vivo

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Physical state: amber viscous liquid
- Storage conditions: ambient

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl:CD(SD).
- Age at study initiation: 22 - 41 days (6 - 8 days old on receipt).
- Weight at study initiation: 40.5 - 63.7 g.
- Housing: plastic maternity cages.
- Diet (e.g. ad libitum): ad libitum.
- Water (e.g. ad libitum): reverse osmosis-purified water ad libitum.
- Acclimation period: 14 - 16 days. During this time the pups were housed in plastic maternity cages (by litter with their own or a fostering dam).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3 - 21.7 °C.
- Humidity (%): 36.9 - 47.7 %.
- Air changes (per hr): approximately 10.
- Photoperiod (hrs dark / hrs light): 12 hour light (0600 to 1800 hours) / 12 hour dark controlled by calibrated light timers.

IN-LIFE DATES: From: 26 September 2006 To: 17 October 2006

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
VEHICLE
- Concentration in vehicle: the dosage volume for all groups was 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYSIS
-Three samples from the middle stratum of the control group formulation and three sets of samples from the top, middle and bottom strata of the test article formulations were collected prior to the initiation of dose administration. Additional samples (three sets) were collected for resuspension homogeneity from the same formulations following 10 days of room temperature storage.
-Two sets of samples for concentration verification were collected on each day of preparation and two sets were collected on the last day of use of each preparation. Two sets of homogeneity/stability samples and 6 sets of concentration samples were shipped under ambient conditions to the sponsor for homogeneity, stability and concentration analyses.
- Samples were analysed by high performance liquid chromatography (HPLC) using a reverse-phase technique.
-Homogeneity and stability of the test material in corn oil was established for all concentration groups. The nominal concentrations of all dosing suspensions were verified by the analytical data.

EXPERIMENTAL
SAMPLES
Samples were stored at room temperature and in the dark prior to analysis.

STANDARDS
Calibration standards used in the HPLC analysis were prepared from the same lot as that used in the study.

ANALYTICAL METHOD
-Six standards were analysed to generate a linear calibration curve. Samples were diluted with methylene chloride and analysed by reverse-phase HPLC employing a fluorescence detector.
-The limit of detection (LOD) and limit of quantitation (LOQ) were calculated to be 0.03 mg/mL and 0.07 mg/mL respectively. A reporting limit (RL) of 0.2 mg/mL was determined to be appropriate for vehicle results.
-Homogeneity was confirmed if the percent differences between the overall dose level mean and individual strata means were 10 % or less. Stability and concentration data were evaluated using percent difference between the nominal and measured concentrations. The acceptable tolerance was 15 %.

RESULTS
-All instrument control checks and quality control samples were within acceptable limits.
The dosing suspension homogeneity was confirmed. The percent difference of all strata means with their respective overall mean was below the 10 % tolerance.
-The percent difference between the theoretical and measured results was ≤ 15 %, confirming the stability of the test substance in corn oil.
-The nominal concentrations of all samples were verified (since the percent difference between the estimated and measured results is less than 15 %)
Duration of treatment / exposure:
20 consecutive days. Dosing occurred when the animals were 22 to 41 days of age; the start of dose administration was staggered based on age.
Frequency of treatment:
Animals were dosed once daily by gavage.
Duration of test:
All surviving animals were euthanised on postnatal day (PND) 42 following the 20 consecutive days of test material administration.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 50, 200 and 800 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
15 animals per dose group (all female).
Control animals:
yes, concurrent vehicle
Details on study design:
-For the control group, the appropriate amount of the vehicle was placed in a glass storage container and stirred throughout use.
-The test material formulations were prepared as follows: the appropriate amount of test material for each group was weighed into a tared, calibrated glass container. A stir bar and approximately 80 % of the vehicle were added to each container, and the mixtures were stirred until uniform. The appropriate volume of vehicle was added to bring each formulation to the calibration mark, and the preparations were stirred until uniform and during dose administration procedures. The test material formulations were prepared approximately weekly, divided into aliquots for daily dispensation and stored at room temperature.

OBSERVATIONS
Clinical Observations
-All animals were observed twice daily for appearance, behaviour, mortality and moribundity. A detailed physical examination was performed at the time of randomisation. The rats were also observed daily (prior to dosing).
-Clinical findings at the time of dose administration and approximately 1 hour following dose administration were recorded when observed. Individual body weights were recorded daily.
-Each female pup was observed daily for vaginal patency beginning on PND 23. Examination continued daily until vaginal patency was observed. Body weights were recorded on the day that vaginal patency was noted.

Observations on Estrous
- Beginning on the day that vaginal patency was observed, vaginal lavages were performed daily, through the day of euthanasia, and the slides were examined to determine the stage of estrus. The mean estrous cycle length was calculated and reported for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P] until the day of euthanasia).
-Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. In addition, the mean age at the onset of the first estrous cycle was calculated using the first day each animal was observed to be in estrus.

POSTMORTEM EXAMINATIONS
-A complete gross necropsy was performed only on animals that were found dead. All surviving animals were euthanised on PND 42 by carbon dioxide inhalation; blood was collected via the vena cava for evaluation of serum estradiol (E2), luteinising hormone (LH), thyroxine (T4) and thyroid stimulating hormone (TSH) levels.
-The uterus (wet and blotted), kidneys, liver, ovaries and oviducts, pituitary gland, spleen, adrenal glands, thymus gland and thyroid glands (with parathyroid glands) were weighed at the scheduled euthanasia. Luminal fluid weight was calculated by subtracting the blotted uterus weight from the wet uterus weight.
-The adrenal glands, axillary lymph nodes, ovaries, oviducts, uterus (horns and body), cervix, vagina, kidneys, liver, spleen, thymus gland, pituitary gland, thyroid glands (with parathyroid glands) and internal gross lesions were examined from all females, including those found dead; all of these tissues were retained with the exception of the adrenal glands, kidneys and liver.
Statistics:
STATISTICS 
Statistical tests were performed using appropriate computing devices or programs. Analyses were conducted using two-tailed tests for minimum significance levels of 1 and 5 %, comparing each test material-treated group to the control group.
Each mean was presented with the standard deviation (SD) and the number of animals (N) used to calculate the mean. In addition, percent difference from control was calculated for body weights, clinical pathology parameters and organ weights. Statistical analyses were not conducted if the number of animals was 2 or less.
Mean body weights, body weight changes, days of acquisition of vaginal patency, estrous cycle lengths, ages at the first occurrence of estrus, serum hormone levels, luminal fluid weights and absolute and relative organ weights were subjected to a parametric one-way analysis of variance (ANOVA) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test article-treated groups to the control group.

Results and discussion

Effect levels

Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: estrogenic effects.

Observed effects

CLINICAL SIGNS AND MORTALITY
Brown material on various body surfaces (primarily the anogenital and urogenital areas) was noted in the 800 mg/kg/day group, beginning as early as PND 24. Clear material around the mouth was noted in the 200 and 800 mg/kg/day group 1 hour following dose administration beginning as early as PND 29 and 27, respectively. This finding was attributed to the test material, but was not considered adverse as it did not persist to the daily examinations.
Salivation prior to dose administration was also noted in the 50, 200 and 800 mg/kg/day group beginning as early as PND 39, 28 and 28, respectively; however, this finding was not considered adverse because it generally did not persist to the daily examinations on the following day.

8 females in the 800 mg/kg/day group were found dead at 23, 25 or 26 days of age as a result of test material administration. 3 females in this group were found dead at approximately 1 - 2 hours following dose administration; the remaining 5 females were found dead on the morning following their last dose. In addition to the findings noted above, 5 of the 8 females in the 800 mg/kg/day group that were found dead had a hunched posture on the day prior to or the day of death; 1 of these females was observed rocking, lurching or swaying while ambulating. A hunched posture was also noted for 1 female in the 800 mg/kg/day group that survived to the scheduled necropsy. At the necropsy of the females that were found dead, 1 animal had dark red discoloration in the lungs and another had clear fluid contents in the uterus. No other internal findings were noted for animals in the 800 mg/kg/day group that died prematurely. All other animals survived to the scheduled euthanasia.


BODYWEIGHT
Test material-related lower mean bodyweight gains and bodyweight losses in the 800 mg/kg/day group were noted during the first 4 days of dose administration. The differences were statistically significant (p<0.01) compared to the control group. Throughout the remainder of the dosing period (following the deaths of the most sensitive animals) mean bodyweight gains in this group were sporadically lower than the control group; the difference was statistically significant (p<0.01) during PND 27-28.
However, when the entire treatment period (PND 22 - 42) was evaluated, mean bodyweight gain in the 800 mg/kg/day group was lower (not statistically significant) compared to the control group value. As a result of the decrements in mean bodyweight gains, mean bodyweights in the 800 mg/kg/day group were 9.7 - 16.1 % lower than the control group values during PND 24 - 42. The differences were statistically significant (p<0.05 or p<0.01) from PND 24 - 40.

Mean bodyweight gains in the 200 mg/kg/day group were similar to those in the control group during PND 22 - 23 through PND 27 - 28. Thereafter, mean bodyweight gain in this group was generally lower than that noted in the control group; the difference was statistically significant (p<0.01) during PND 39 - 40. Mean bodyweight gain in the 200 mg/kg/day group was lower (not statistically significant) than the control group value when the entire treatment period (PND 22 - 42) was evaluated. The decrements in mean bodyweight gain in this group were attributed to the test material and resulted in mean bodyweights that were 5.5 - 8.8 % lower (not statistically significant) than the control group values during PND 35 - 42.

Mean bodyweights and bodyyweight gains in the 10 and 50 mg/kg/day groups were generally similar to those in the control group throughout the study; none of the differences were statistically significant.


REPRODUCTIVE FUNCTION: VAGINAL PATENCY
The mean age of attainment of vaginal patency was earlier in the 50, 200 and 800 mg/kg/day groups; this change was attributed to the test material. The differences from the concurrent control group were statistically significant (p<0.01). Mean ages of attainment were 32.5, 33.3, 28.3, 28.2 and 28.9 days in the control, 10, 50, 200 and 800 mg/kg/day groups, respectively.
The values in the 50, 200 and 800 mg/kg/day groups were also lower than the mean minimum value in the laboratory historical control data for female pubertal studies (31.8 days of age). Mean body weights at the age of attainment (85.4, 83.4 and 73.9 g) in the 50, 200 and 800 mg/kg/day groups, respectively, were statistically significantly (p<0.01) lower than the concurrent control group value; once again the values were also below the minimum mean value in the laboratory historical control data (102.1 g).


REPRODUCTIVE FUNCTION: ESTROUS CYCLE
Consistent with an earlier age of attainment of vaginal patency, the ages of the first occurrence of estrus in the 50, 200 and 800 mg/kg/day groups (32.1, 31.2 and 28.9 days of age, respectively) were earlier than in the concurrent control group (34.4 days of age); the differences in the 200 and 800 mg/kg/day groups were statistically significant (p<0.05 or p<0.01). These values were also lower than the minimum mean value in the laboratory historical control data (34.1 days of age).
Estrous cycle lengths could only be determined for 5/15, 8/15, 13/15, 7/15 and 1 /7 females in the control, 10, 50, 200 and 800 mg/kg/day groups respectively, due to a high number of females with incomplete cycles. No differences in estrous cycle lengths were noted in the 10, 50 and 200 mg/kg/day groups with number of cycling animals available for evaluation.
Estrous cycle lengths in females of this age are highly variable. Abnormal estrous cycles (≥3 consecutive days estrus [E]) were noted in 12/15 and 6/7 females in the 200 and 800 mg/kg/day groups, respectively, as presented in Table 2.
The majority of females in the control, 10 and 50 mg/kg/day groups with abnormal estrous cycles had extended diestrus, whereas a shift toward extended estrus was noted in the 200 and 800 mg/kg/day groups. The persistent estrus noted at the 2 highest dosage levels was consistent with the earlier attainment (3.2 and 5.5 days earlier, respectively) of vaginal patency in these groups.


ORGAN WEIGHTS
Test material-related, generally statistically significant (p<0.05 or p<0.01) increases in mean absolute and relative (to final bodyweight) liver weights (800 mg/kg/day group) and decreases in mean absolute and relative spleen (800 mg/kg/day group), luminal fluid (absolute only), wet and blotted uterus and thymus gland (200 and 800 mg/kg/day groups) and ovary/oviduct weights (50, 200 and 800 mg/kg/day groups) were observed compared to the control group values. Table 3 presents these changes.
1 and 2 females in the 200 and 800 mg/kg/day groups had small ovaries at necropsy, and 1 of the 800 mg/kg/day group females had small oviducts, corresponding to the lower ovary/oviduct weights noted in these groups. The mean relative liver weight in the 200 mg/kg/day group was statistically significantly (p<0.05) higher than the control group value; however, the absolute value was comparable to that in the control group. Therefore, the increase in relative liver weight in this group was not considered test material-related.


HISTOPATHOLOGY
Selected histopathologic results are summarised in Table 4.
Microscopically, 14/15 rats from the 800 mg/kg/day group had absent corpora lutea. This effect was accompanied by moderate (9/15 rats) to severe (6/15 rats) granulosa cell necrosis and moderate (10/15 rats) to severe (5/15 rats) oocyte degeneration. In the 200 mg/kg/day group, there were 9 rats with absent corpora. Fourteen rats from this group had mild granulosa cell necrosis, and 1 rat had moderate granulosa cell necrosis.
The uterus of 7 rats from the 800 mg/kg/day group that were found dead had mild (4/14 rats) to severe (3/14 rats) atrophy, consisting of decreased endometrial and myometrial thickness as well as a reduced number of endometrial glands. The atrophic glandular epithelial cells had an inactive appearance with a reduced amount of cytoplasm. All the examined tissues from the control group were microscopically normal.
Mild follicular cell hypertrophy was noted in the thyroid gland of 3 rats from the 800 mg/kg/day group. The relationship of this finding to the test material was unclear. Also in the 800 mg/kg/day group, there was mild focal necrosis in ectopic thymic tissue that was present in the thyroid gland section of 1 rat that was found dead, and minimal focal lymphocytic infiltration was present in the thyroid gland of 1 rat from the 800 mg/kg/day group that was euthanised at the scheduled termination.
Remaining histologic changes were considered to be incidental findings, manifestations of spontaneous diseases or related to some aspect of experimental manipulation other than administration of the test material. There was no test material-related alteration in the incidence, severity or histologic character of those incidental and spontaneous tissue alterations.


OTHER EFFECTS
There were no test material-related effects in thyroid hormone levels (total T4 and TSH) at any dosage level. In addition, E2 and LH were unaffected by test material administration at all dosage levels. A high LH value was noted for 1 control group female, resulting in a higher-than-average mean for this group; this sample was re-analysed and the re-tested value was similar. No statistically significant differences from the control group were noted.

Any other information on results incl. tables

Table 1 Mean Ages of Onset of Vaginal Patency and First Estrus

Endpoint

(days)

Dose level (mg/kg/day)

0

10

50

200

800

Age vaginal patency was observed

32.5

33.3

28.3*

28.2*

28.9*

Age first estrus took place

34.4

35.6

32.1

31.2**

28.9**

*Values are statistically significant (p<0.01).

**Value is statistically significant (p<0.05 or p<0.01).

Table 2 Number of Females with Abnormal Estrous Cycles

Group

(mg/kg/day)

Number with

Extended D

Number with

Extended E

Number with Both Extended E and D

0

6/15

0/15

0/15

10

4/15

0/15

0/15

50

6/15

1/15

0/15

200

5/15

12/15

2/15

800

2/7

6/7

1/7

 

Table 3 Summary of Test Material-Related Effects on Absolute Organ Weights [g] with Percent Difference from the Control Group

Organ

0

mg/kg/day

10

mg/kg/day

50

mg/kg/day

200

mg/kg/day

800

mg/kg/day

Liver

8.2487

7.8837

(-4.4 %)

7.9892

(-3.1 %)

8.3159

(+0.8 %)

12.1045**

(+46.7 %)

Spleen

0.4681

0.4643

(-0.8 %)

0.4877

(+4.2 %)

0.4209

(-10.1 %)

0.3611**

(-22.9 %)

Wet Uterus

0.4201

0.3760

(-10.5 %)

0.413

(-4.5 %)

0.2177**

(-48.2 %)

0.2143**

(-49.0 %)

Blotted Uterus

0.3371

0.3277

(-2.8 %)

0.3083

(-8.5 %)

0.2022**

(-40.0 %)

0.1992**

(-40.9 %)

Luminal Fluid

0..0829

0.0482

(41.9 %)

0.0930

(+12.2 %)

0.0155

(-81.3 %)

0.0151

(-81.8 %)

Thymus

0.5948

0.5867

(-1.4 %)

0.5768

(-3.0 %)

0.4598**

(-22.7 %)

0.3105**

(-47.8 %)

Ovaries/Oviducts

0.0936

0.0855

(-8.7 %)

0.0787*

(-1539 5)

0.0548**

(-41.5 %)

0.0393**

(-58.0 %)

* = p<0.05

** = p<0.01     

Table 4 Incidence of Selected Histopathologic Findings

 

0 mg/kg/day

200 mg/kg/day

800 mg/kg/day

Ovaries

 

 

 

No. tissues examined

15

15

15

Corpora lutea absent, bilateral

0

9

14

Necrosis, granulose cell, bilateral

0

15

15

    Mild

0

14

0

    Moderate

0

1

9

    Severe

0

0

6

Degeneration, oocyte, bilateral

0

15

15

    Mild

0

14

0

    Moderate

0

1

10

    Severe

0

0

5

Uterus

 

 

 

No. tissues examined

15

15

14

Atrophy

0

0

7

    Mild

0

0

4

    Severe

0

0

3

†Missing 1 uterus and cervix at trimming. This was only noted for females that were found dead.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, oral administration of the test material to juvenile female rats resulted in estrogenic effects for females at 50, 200 and 800 mg/kg/day. The NOAEL for estrogenic effects was therefore 10 mg/kg bw/day.
Executive summary:

The ability of the test material to induce effects on pubertal development in the intact juvenile female rat was investigated in accordance with the standardised guideline EPA OPPTS 890.1450.

The test material, in the vehicle corn oil, was administered orally by gavage once daily for 20 consecutive days to 4 groups each of 15 Crl:CD(SD) immature female rats. Dosage levels were 10, 50, 200 and 800 mg/kg/day.

Administration of the test material resulted in estrogenic effects for females at 50, 200 and 800 mg/kg/day, evidenced by earlier attainment of vaginal patency (with corresponding lower mean body weight on the day of attainment) and at 200 and 800 mg/kg/day by earlier age at the first occurrence of estrus. In addition, estrous cycle disturbances were noted in the 200 and 800 mg/kg/day groups with 12/15 and 6/7 females, respectively, exhibiting persistent estrus (≥3 consecutive days of estrus).

No test material-related effects on mean serum E2, LH, T4 or TSH levels were observed at any dosage level. Mean absolute and relative (to final body weight) spleen weight in the 800 mg/kg/day group and mean absolute and relative wet and blotted uterus weights (and thus, luminal fluid weight) and thymus gland weights in the 200 and 800 mg/kg/day groups were lower than the control group values.

 Lower mean absolute and relative ovary/oviduct weights were observed in the 50, 200 and 800 mg/kg/day groups. Mean absolute and relative liver weights in the 800 mg/kg/day groups were higher than the control group values. Microscopic findings corresponding to the lower mean ovary and uterus weights were observed in the 800 mg/kg/day group. The 800 mg/kg/day dosage level resulted in complete absence of corpora lutea in 14 of 15 rats. All ovaries in this group had moderate to severe granulosa cell necrosis and moderate to severe oocyte degeneration. Mild or severe atrophy of the uterus was present in 7 rats from the 800 mg/kg/day group that were found dead. In the 200 mg/kg/day group, similar but milder morphologic changes in ovaries were present.

 

In conclusion, based on the results of this study, oral administration of the test material to juvenile female rats resulted in estrogenic effects for females at 50, 200 and 800 mg/kg/day.