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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
September 06 to November 05, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline test with GLP
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Test animals

Species:
rat
Strain:
other: RccHanTM: WIST(SPF)
Sex:
male/female
Details on test animals and environmental conditions:
Animals: Rat, RccHanTM: WIST(SPF)
Rationale: Recognized by international guidelines as a recommended test system.
Breeder: Harlan Laboratories, B.V. Kreuzelweg 53 5961 NM Horst / Netherlands
Number of Animals: 44 males: 11 per group 44 females: 11 per group

Age (at Start of Treatment): 11 weeks
Body Weight Range
(at Start of Treatment): Males: 312 to 351 g Females: 208 to 244 g
Identification: Cage card and individual animal number (ear tattoo).
Pups: On day 1 post partum, pups were individually tattooed with Indian ink.
Randomization: Performed after at least three days of acclimatization using a computer-generated random algorithm. Body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
Husbandry
Room Numbers, Füllinsdorf: E0441A (acclimatization) and E0441 (after acclimatization) Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). Values outside of these ranges occasionally occurred, usually following room cleaning, which was considered not to have any influence on the study. These data were not reported but were retained in the raw data. There was 12-hour fluorescent light / 12-hour dark cycle with music during the light period.
Accommodation: In groups of three to five animals in Makrolon type-4 cages with wire mesh tops up to the day of ran-domization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg/Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V., Netherlands), batch/ lot nos. 100099. During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.

Diet: Pelleted standard Harlan Teklad 2018C (batch no. 43/12) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. .
Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Four groups of 11 males and 11 females were treated by gavage with Neo Heliopan OS once daily. Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to the day 3 post partum.

Vehicle and Control Item
Identification: Corn oil
Source: Carl Roth GmbH
Batch Number: 292189296
Expiry Date (Retest Date): 02-Aug-2017
Storage Conditions: Room temperature (20 ± 5 °C)
Details on mating procedure:
During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if:

- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.

The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 1 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. To confirm the stability (8 days) samples of about 1 g of each concentration were taken from the middle of each aliquot used on day 7 of the treatment. During the last week of the treatment, samples were taken from the middle to confirm concentration.
The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to B. Bürkle (Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.

The samples were analyzed by GC coupled to an FID detector following an analytical procedure provided by the Sponsor and adapted at Harlan Laboratories. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.

In conclusion, the results indicate the accurate use of the test item and corn oil as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.
Duration of treatment / exposure:
Males: 28 days Females: Approximately 7 weeks
Frequency of treatment:
Once daily
Details on study schedule:
Two females did not mate during the 14-day pairing period and a second pairing of these females with a male in the same group, which had already mated successfully, was carried out. Although mating was not observed also during the second pairing period, both females had body weight gain indicating pregnancy. Therefore the second mating period was interrupted.
All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 25, 80, 250 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
11
Control animals:
yes, concurrent vehicle
Details on study design:
Method: Oral, by gavage
Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type studies.
Frequency of Administration: Once daily
Target Dose Levels: Group 1: 0 mg/kg/day (control group)
Group 2: 25 mg/kg/day
Group 3: 80 mg/kg/day
Group 4: 250 mg/kg/day
Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories Study D54872, using dose levels of 0, 100, 300 and 1000 mg/kg/day, resulting in mortality and adverse toxic effects at the dose level
of 1000 mg/kg bw/day and adverse toxic effects at the dose level of 300 mg/kg bw/day.
Dose Volume: 4 mL/kg body weight
Dose Concentrations: Group 1: 0.00 mg/mL
Group 2: 6.25 mg/mL
Group 3: 20.00 mg/mL
Group 4: 62.50 mg/mL
Duration of Acclimatization Period: Minimum 5 days
Duration of Treatment Period: Males: 28 days Females: Approximately 7 weeks
Positive control:
no data

Examinations

Parental animals: Observations and examinations:
Viability / Mortality: Twice daily
Clinical Signs: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.
Food Consumption: Males: Pre-Pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; after pairing period weekly.
Females: Pre-Pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; gestation days 0 - 7, 7 - 14 and 14 - 21 and days 1 - 4 of the lactation.
No food consumption was recorded during the pairing period.
Body Weights: Recorded daily from treatment start to day of necropsy.
Oestrous cyclicity (parental animals):
not applicable
Sperm parameters (parental animals):
no data
Litter observations:
Pup Data: The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.
Postmortem examinations (parental animals):
Males were sacrificed after treatment for 28 days, when no longer needed for the assessment of reproductive effects.
If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.
At the scheduled sacrifice, all animals were weighed and sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.
All parent animals and pups, except those excessively cannibalized, were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred to establish, if possible, the cause of death. For the parent animals, special attention was directed at the organs of the reproductive system. For the parent animals, special attention was directed at the organs of the reproductive system.
Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.
The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.
Organ Weights and Preservation
Organs were trimmed from any adherent tissue, and their wet weight taken. If not indicated otherwise in the table, organs were preserved in neutral phosphate buffered 4% formaldehyde solution.
Histotechnique
All organ and tissue samples to be examined by the principal investigator were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin.
Histopathology
Slides of all organs and tissues listed collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator. The same applied to all occurring gross lesions and to all animals, which died spontaneously or had to be terminated in extremis.

Qualitative assessment of the male reproductive organs was performed. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made.
Postmortem examinations (offspring):
Pups and dams were sacrificed on day 4 post partum. All parent animals and pups, except those excessively cannibalized, were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred to establish, if possible, the cause of death.
Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:

• Means and standard deviations of various data were calculated.

• The Dunnett-test [see References (2)] (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.

• The Steel-test [see References (3)] (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.

• Fisher's exact-test [see References (4)] was applied if the variables could be dichotomized without loss of information.
Reproductive indices:
fertility indices, mean precoital time, post-implantation loss,
Offspring viability indices:
mean litter size, pup sex ratios and viability indices.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At the dose level of 250 mg/kg bw/day, one female (no. 78) was found dead on day 23 of the gestation period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At the dose levels of 250 and 80 mg/kg bw/day, reduced food consumption was noted during lactation.At the dose level of 250 mg/kg bw/day, statistically significant reduction in body weight gain was noted on day 4 of the lactation period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At the dose levels of 250 and 80 mg/kg bw/day, reduced food consumption was noted during lactation.At the dose level of 250 mg/kg bw/day, statistically significant reduction in body weight gain was noted on day 4 of the lactation period.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Details on results (P0)

1 Viability / Mortality
At the dose level of 250 mg/kg bw/day, one female (no. 78) was found dead on day 23 of the gestation period. During necropsy, fetuses were found in uterus of this female. A slight body weight loss was noted in this female on day 22 of the gestation period but no other signs and no macroscopical or microscopical findings indicating bad condition of this female were noted.
Death of the female was most probably a result of difficult parturition and considered to be test-item related.
All remaining animals of P generation survived the scheduled study period.
2 Clinical Signs or Observations
No test item-related findings were noted in males or females at any dose level.
In one female (67) at the mid-dose level, hunched posture and ruffled fur were noted from day 2 of the lactation period onwards. Further, slight swelling in axillary region was observed in one female (no. 79) at the high-dose level from day 9 of the gestation period onwards. Because of isolated occurrence, these findings were considered not to be related to the treatment with the test item.
No further findings were noted in males or females at any dose level.
3 Food Consumption of Males
The overall differences in mean food consumption at the dose levels of 25, 80 and 250 mg/kg bw/day were respectively: +0.9%, -0.9% and +1.7% during the pre-pairing period (percentages refer to the respective values in the control group).
4 Food Consumption of Females
Pre-Pairing, Gestation and Lactation Periods

At the dose levels of 250 and 80 mg/kg bw/day, reduced food consumption was noted during lactation. Mean food consumption was 16.4 and 19.8 g/animal/day at the dose levels of 250 and 80 mg/kg bw/day, respectively and 22.2 g/animal/day in the control group. Although the differences were not statistically significant, they were dose dependent and therefore considered to probably be related to the treatment with the test item.

No effects on food consumption were noted at the dose level of 80 mg/kg bw/day.

The overall differences in mean food consumption at the dose levels of 25, 80 and 250 mg/ kg bw/day were respectively: -5.6%, -0.6% and -4.4% during the pre-pairing period, -2.4%, +6.2% and +4.8% during the gestation period and +1.4%, -10.8% and -26.1% during the lactation period (percentages refer to the respective values in the control group).
5 Body Weights of Males
Pre-Pairing and Pairing Periods

At the dose level of 250 mg/kg bw/day, slight but statistically significant reduction in body weight gain was noted on day 13 of the pre-pairing period. Body weight gain was also slightly lower on further days at the end of this period but without statistical significance. No significant differences in absolute body weights were noted at this dose level.
At the dose levels of 25 and 80 mg/kg bw/day, no test item-related effects on absolute body weights or body weight gain were noted.
The overall differences in mean body weight gain at the dose levels of 0, 25, 80 and 250 mg/kg bw/day were respectively: +15%, +15%, +13% and +13% during the pre-pairing period and +11%, +9%, +9% and +10% during the pairing period (percentages refer to the body weight gain within the period).
At the low- and mid-dose levels, statistically significantly lower body weight gains were noted on individual days during the pairing period. Because the differences were minor and did not follow dose dependency, they were considered not to be related to the treatment with the test item.
6 Body Weights of Females
Pre-Pairing, Pairing, Gestation and Lactation Periods
At the dose level of 250 mg/kg bw/day, statistically significant reduction in body weight gain was noted on day 4 of the lactation period. No significant differences in absolute body weights were noted at this dose level at any time.
At the dose levels of 25 and 80 mg/kg bw/day, no effects on absolute body weights or body weight gain were noted.
The overall differences in mean body weight gain at the dose levels of 0, 25, 80 and 250 mg/kg bw/day were respectively: +8%, +7%, +9% and +6% during the pre-pairing period, +51%, +55%, +56% and +48% during the gestation period and +5%, +2%, +2% and ±0% during the lactation period (percentages refer to the body weight gain within the period).
Mating Performance and Fertility:
No effects on mating performance, fertility index or conception rate were noted at any dose level.
No effects on gestation index were noted at the dose level of 25 mg/kg bw/day; it was 100% at this dose level.
Duration of Gestation :
At the dose levels of 250 and 80 mg/kg bw/day, prolonged gestation period was noted. Mean duration of gestation was 22.6 and 22.0 days at the dose levels of 250 and 80 mg/kg bw/day, respectively, compared to 21.5 days in the control group.
Mean prolongation of the gestation at the high- and mid-dose levels was not statistically significant. However, it was dose dependent and the values were beyond the biological background (historical control data included values of gestation length from 21.2 to 21.8 days).
Therefore this finding was considered to be test item-related.
At the dose level of 25 mg/kg bw/day, mean gestation of duration was the same like the control value; 21.5 days and therefore not affected by the treatment.
Corpora Lutea Count :
No effects on corpora lutea count were observed at any dose level.
Implantation Rate and Post-Implantation Loss :
Number of implantations was not affected by the treatment with the test item at any dose level. At the dose level of 25 mg/kg bw/day, no effects on post-implantation loss were noted.






Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Effect level:
80 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the unclear death of one female at 250 mg/kg.
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Based on slight changes in body weight gain
Dose descriptor:
NOAEL
Remarks:
(fertility)
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Observed effect not relevant to impairment of the fertility.

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Higher post-implantation loss resulted in lower litter size at the dose levels of 250 and 80 mg/kg bw/day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At the dose level of 250 mg/kg bw/day, reduced body weights of pups were noted.
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

Litter Size at First Litter Check

Higher post-implantation loss resulted in lower litter size at the dose levels of 250 and 80 mg/kg bw/day.
Mean number of living pups per dam was 5.3 and 9.2 at the high- and mid-dose levels, respectively, compared to 12.0 in the control group. Further, birth index (number of pups borne alive as a percentage of implantations) was also reduced and it was 42.9% and 66.2% at the high- and mid-dose levels, respectively, compared to 88.2% in the control group. Reduction in the mean number of pups per dam was statistically significant at the high-dose level whereas reduction in birth index was statistically significant at both dose levels. These effects were considered to be test item-related.

No effects on litter size were noted at the dose level of 25 mg/kg bw/day. Mean number of pups was 13.1 per dam and birth index was 94.2% at this dose level.

Postnatal Loss Days 0 - 4 Post Partum

Postnatal loss was considered not to be affected by the treatment with the test item.

During lactation, a total number of 18, 3, 10 and 13 pups (which corresponded to mean number per dam of 1.8, 0.3, 1.0 and 1.4) were lost at the dose levels of 0, 25, 80 and 250 mg/kg bw/day, respectively.
Litter Data - F1 Pups
1 External Examination at First Litter Check and during Lactation

No test item-related observations were noted in pups during the first litter check or during lactation at any dose level.

Following findings were recorded: no milk in the stomach and/or pups cold to touch in one litter at each low- and mid-dose levels and in two litters at the high-dose level; these findings were predominantly observed in the litters affected by post-natal loss. One pup at the high-dose level had a wound. Several pups which were dead at first litter check were partially cannibalized. All these findings were considered to remain within the normal biological background.
2 Sex Ratios

Pups sex ratio was not affected by exposure to the test item at any dose level.
3 Body Weights to Day 4 Post Partum
At the dose level of 250 mg/kg bw/day, reduced body weights of pups were noted. Mean body weights of pups were 5.0 g compared to 6.0 g in the control group on day 1 of the lactation period; this difference was statistically significant. Body weights of pups at the high dose level remained lower than the respective control value also on day 4 of the lactation period. Mean body weights were 7.6 g compared to 9.2 in the control group; this difference was however not longer statistically significant. Body weight gain of pups at the high-dose level was similar to the body weight gain in the control group.
No test item related effects on body weights or body weight gain in pups were noted at the dose levels of 25 and 80 mg/kg bw/day.
In general, mean body weights of pups on day 1 post partum were: 6.0 g, 5.9 g, 6.3 g and 5.0 g, at the dose levels of 0, 25, 80 and 250 mg/kg/day respectively, body weight gain of pups during the first four days of the lactation period was +44.4%, +42.7%, +48.0% and +44.6%, respectively.

At the mid-dose level, statistically significantly higher body weight gain was noted. In the absence of increased body weight gain at the high dose level, this was considered not to be related to the treatment with the test item.
4 Macroscopical Findings
No test item related findings were found in pups at any dose level.
Incidentally, tan discoloration of kidneys was noted in one pup at the low-level. No further findings were noted at any dose level.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the NOEL of general systemic toxicity is considered to be 80 mg/kg bw/day in females and 250 mg/kg bw/day in males. The NOAEL for fertility is 250 mg/kg bw/day. Observed effects are considered to be related to developmental toxicity and are described in the relevant section.
Executive summary:

This study was performed to generate preliminary information concerning the effects of test article on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition. The test item was administered in corn oil as vehicle at dosages of 25, 80, and 250 mg/kg body weight/day, and controls received the vehicle only. Test article was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

At the high dose level, one female was found dead on day 23 of the gestation period which was considered to be a result of birth complications. No further adverse effects were noted in males or females at any dose level. At the dose levels of 250 and 80 mg/kg bw/day a reduction in gestation index as well as an increase in incidence of post-implantation loss resulting in a lower litter size were noted. These effects were statistically significant and dose dependent and therefore considered to be test item-related and adverse. Based on the individual data, increased post-implantation loss occurred predominantly in females with prolonged gestation. A prolonged gestation was noted at the high- and mid-dose level, which were considered to be test item-related. No compensation for lower body weights occurred during lactation. Reduction of pup absolute body weights was considered to be adverse.

Based on the results of this study, where in males only slight changes in body weight gain were noted at the high-dose level, the NOAEL (No Observed Adverse Effect Level) for general toxicity in males was considered to be 250 mg/kg bw/day. Because of the death of one female at the dose level of 250 mg/kg bw/day, NOAEL (No Observed Adverse Effect Level) for general toxicity in females was considered to be 80 mg/kg bw/day. The observed effects of prolonged gestation, reduced gestation index and increased post-implantation loss resulting in lower litter size at the dose levels of 250 and 80 mg/kg bw/day are considered to be related to developmental toxicity. As a consequence, the NOAEL for fertility was determined to be 250 mg/kg bw/d.