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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 based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
From 05 Jan 2012 to 31 Jul 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 421, GLP-compliant)
Cross-reference
Reason / purpose:
reference to same study

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Number of Animals: 44 males (11 per group) and 44 females (11 per group)
- Age (at delivery): 10 weeks
- Body Weight Range (at Start of Treatment): 301 to 344 g (males) and 191 to 228 g (females)
- Identification: Cage card and individual animal number (ear tattoo).
- Randomization: Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Accommodation: 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; batch/lot no. 02105111001) with paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK), batch/lot no. 55/T-6060). 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 2914C (batch no. 73/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. Results of representative analyses for contaminants were included in the study report.
- Water: Community tap-water from Itingen was available ad libitum in water bottles. Results of bacteriological assay, chemical and contaminant analyses of representative samples were included in the study report.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study. At least 5 days.

ENVIRONMENTAL CONDITIONS
Standard laboratory conditions, continuously monitored.
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 to 70
- Air changes (per hr): . Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12 (with at least eight hours music during the light period)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
DOSE FORMULATIONS
The dose formulations were not corrected for purity and were prepared as supplied by the Sponsor. The dose formulations were prepared weekly as indicated by the results of stability analyses in the non-GLP dose range-finding study. The test item was weighed into a glass beaker on a tared Mettler balance and the vehicle was added. The mixtures were stirred using a magnetic stirrer and stored at room temperature (20 °C +/- 5 °C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATIONS
The dose formulations were stored at room temperature (20 +/- 5 °C) in glass beakers. They were stable for at least 8 days, based upon the results of stability analyses performed during the non-GLP dose range-finding study.

TREATMENT
- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of studies.
- Frequency of Administration: Once daily
- Daily Target Dose Level: 0 mg/kg/day (control group), 100 mg/kg/day (group 2), 300 mg/kg/day (group 3), 1000 mg/kg/day (group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Wistar rats, Harlan Laboratories study D33665 (non-GLP).
- Dose Volume: 10 mL/kg body weight
- Dose Concentrations: 0 mg/mL/day (control group), 10 mg/mL/day (group 2), 30 mg/mL/day (group 3), 100 mg/mL/day (group 4)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
METHOD
After experimental start, a sample from the control group as well as three samples (top, middle and bottom) of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples were also taken to confirm stability (4 hours and 8 days at room temperature). During week 3 of treatment, samples were also taken to confirm homogeneity and concentration. The aliquots for analysis of dose formulations were stored at -20 +/- 5°C until analysis. The test item was used as the analytical standard.

RESULTS
The linearity of the analytical systems used for sample analyses was demonstrated with a good relationship between absorbance measured and working standard concentrations. All calibration points met the acceptance limit of +/-20% variation from the calibration curve derived by linear regression analysis. The regression coefficients calculated were found to be better than 0.99.
Blank samples showed no significant absorbance and, therefore, it was confirmed that only bidistilled water was applied within the control experiment.
The application formulations investigated during the study were found to comprise test material in the range of 93.8% to 108.3% and, thus, the required content limit of +/-20% with reference to the nominal content was met. The homogeneous distribution of the test item in the preparations was approved because single results found did not deviate more than 3% (acceptance criterion: <15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept four hours or eight days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item and bidistilled water as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.
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.

If a female did not mate during the 14-day pairing period, a second pairing of this female with a male in the same group, which had already mated successfully, was considered. If mating was not recorded during this additional pairing period of a maximum of 14 days, the female was sacrificed and, if indicated, the reproductive organs examined histopathologically in order to ascertain the reason for the infertility.

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.
Duration of treatment / exposure:
Minimum 4 weeks (males) and approximately 7 weeks (females)
Frequency of treatment:
Once daily
Duration of test:
MALES
- Acclimatization: 5 days minimum
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Treatment Ends: On day before sacrifice
- Necropsy: After a minimum of 28 days treatment

FEMALES
- Acclimatization: 5 days minimum
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Gestation: Approximately 21 days
- Treatment Ends: On day 3 post partum
- Necropsy: On day 4 post partum
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
11
Control animals:
yes, concurrent vehicle
Details on study design:
The purpose of this study was to generate preliminary information concerning the effects of the test material on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition and to assess major effects on pup development.

Examinations

Maternal 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: Pre-pairing period days 1 - 8 and 8 - 14, gestation period days 0 - 7, 7 - 14 and 14 - 21 post coitum and lactation period days 1 - 4 post partum. No food consumption was recorded during the pairing period.

BODY WEIGHTS: Recorded daily from treatment start to day of necropsy.

TERMINATION AND NECROPSY
Dams were sacrificed on day 4 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.
All animals sacrificed or found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. At the scheduled sacrifice, all animals were killed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.
All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.
For the parent animals, special attention was directed at the organs of the reproductive system. 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.

TISSUE PRESERVATION: The ovaries from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution.

HISTOTECHNIQUE
All organ and tissue samples to be examined by the principal investigator for histopathology were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Special stains were used at the discretion of the principal investigator for histopathology.

HISTOPATHOLGOLGY: Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator for histopathology. The same applied to all occurring gross lesions and to all animals, which died spontaneously or had to be terminated in extremis. Special emphasis was made on the histopathology of interstitial cell structure. Histological examination of ovaries was carried out on any female that did not give birth.
Ovaries and uterine content:
The ovaries and uterus were examined after termination on day 5 post partum. Examinations included the number of corpora lutea and the number of implantation sites.
Fetal examinations:
Not performed
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 (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 (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 was applied if the variables could be dichotomized without loss of information.
Indices:
From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, post-implantation losses, mean litter size, pup sex ratios and viability indices, dead/live pups at first litter check, pups sex ratio

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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

Details on maternal toxic effects:
1. IN-LIFE DATA
VIABILITY / MORTALITY: All animals survived the scheduled study period.

CLINICAL SIGNS OR OBSERVATIONS
There were no findings of toxicological relevance at any dose level.
In females, yellow discoloration of the feces was noted from day 5 of the pre-pairing period and continued throughout the subsequent pairing, gestation and lactation periods. The severity of this finding was generally dose-dependent, but was considered to be a typical passive effect that results from oral administration of a dyestuff and was of no toxicological relevance. Slight hair loss was noted in a single female during the gestation period, but in view of the late onset and low incidence, was considered to be without toxicological implications.

FOOD CONSUMPTION OF FEMALES
Pre-pairing, Gestation and Lactation Periods
The mean daily food consumption values of the test item-treated females compared favorably with the control females throughout the pre-pairing, gestation and lactation periods.

BODY WEIGHTS OF FEMALES
Pre-Pairing, Pairing, Gestation and Lactation Periods
No test item-related effects on body weights and body weight gain of females were observed at any dose level.
In females treated with 1000 mg/kg/day, a significant increase (p<0.05) in mean body weight was noted on day 4 of the gestation period. This isolated finding was considered to be incidental. The body weights recorded during all other periods were similar to those of the control females.
At 300 mg/kg/day, all body weights were similar throughout the various phases.
At 100 mg/kg/day, significantly elevated mean body weights (p<0.05) were noted on days 0 - 5 of the gestation period only. In the absence of a dose-response relationship, these differences were considered to be incidental. The body weights recorded during all other periods were similar to the controls.

REPRODUCTION AND BREEDING DATA
 No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level.
 At the dose level of 1000 mg/kg bw/day, higher incidence of post-implantation loss and higher postnatal loss as well as lower litter size at first litter check and on day 4 post partum were noted.
 The mean post implantation loss of the dams at 1000 mg/kg/day was close to the upper limit of the historical control data and was below the mean post implantation loss of the dams treated with 300 mg/kg/day; therefore this was considered to be unrelated to the treatment with the test item.

2. TERMINAL FINDINGS
MACROSCOPICAL FINDINGS
Discoloration of the ovaries found in one female at 1000 mg/kg/day and was considered to be within the range of normal background alterations.

HISTOPATHOLOGY FINDINGS
All microscopic findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age. There was no morphological evidence of toxicological properties in ovaries of female animals examined in this study.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Remarks:
for general toxicity (P)
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: effect type not specified
Dose descriptor:
NOAEL
Remarks:
for reproduction (P)
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: effect type not specified
Dose descriptor:
NOAEL
Remarks:
for development (F1)
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: effect type not specified

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:not examined

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

1. REPRODUCTION, BREEDING AND PUP DATA

SUMMARY OF PERFORMANCE

 

P Animals Breeding for F1 Litters 

Group

(mg/kg/day)

1
(0)

2
(100)

3
(300)

4
(1000)

Female numbers

45 - 55

56 - 66

67 - 77

78 - 88

Number of females paired

11

11

11

11

Number of females mated

11

11

11

11

Number of females which were not pregnant

1

0

0

2

Number of females which reared their pups until day 4 post partum

10

11

11

9

 

MATING PERFORMANCE AND FERTILITY

  The mean and median precoital times for all groups were similar.

  The respective parameters of the mating performance were unaffected as all dose levels and similar to those of the control values.

  Mating of female no. 84 was not detected. Three females were not pregnant: no. 55 (control group) and nos. 81 ad 83 (1000 mg/kg/day).

 

DURATION OF GESTATION

  No effects on duration of gestation were observed at any dose level.

 

CORPORA LUTEA COUNT

  No effects on corpora lutea count were observed at any dose level.

 

IMPLANTATION RATE AND POST-IMPLANTATION LOSS

  The implantation rate was unaffected in all groups.

 

Although the mean post-implantation loss was slightly higher at 300 mg/kg/day and 1000 mg/kg/ day when compared with the control females, the differences were not dose-related and therefore considered to be unrelated to the test item treatment. The total post implantation loss of the dams treated with 300 mg/kg/day was significantly elevated (p<0.01) when compared with the controls, but not dose-dependent and therefore considered to be unrelated to the treatment with the test item. The mean post implantation loss in females at 100 mg/kg/day was nearly identical to the controls.

 

LITTER SIZE AT FIRST LITTER CHECK

  The mean litter sizes of the control group and those at 1000 mg/kg/day were similar. The marginally lower mean litter size noted at 300 mg/kg/day coincided with the slightly higher post-implantation loss.

  The overall mean numbers of living pups per dam at first litter check compared favorably in the control and test item-treated groups; three pups of litter no. 79 (1000 mg/kg/day), two pups of litter no. 70 (300 mg/kg/day) and four pups of litter no. 49 (control group) were found dead.

  The respective birth indices (number of pups borne alive as a percentage of implantations) were similar in test item-treated and control animals.

 

2. IN-LIFE DATA OF PARENTAL MALES

 

VIABILITY / MORTALITY

  All animals survived the scheduled study period.

 

CLINICAL SIGNS OR OBSERVATIONS

  There were no findings of toxicological relevance at any dose level.

  In males, yellow discoloration of the feces was noted from treatment day 5 of the pre-pairing period and continued throughout the pairing period. The severity of this finding was generally dose-dependent, but was considered to be a typical passive effect that results from oral administration of a dyestuff and was of no toxicological relevance.

 

FOOD CONSUMPTION OF MALES

  Pre-pairing Period

  In males, although the mean daily food consumption values at 300 mg/kg/day and 1000 mg/kg/ day were significantly lower (p<0.05) than that of the control males during days 8 - 14, these differences were not dose-related and were considered to be a result of an incidentally elevated control value rather than to the treatment with the test item.

 

BODY WEIGHTS OF MALES

  Pre-Pairing and Pairing Periods

  There were no differences of toxicological relevance between the mean body weights of test item-treated or control males. The mean body weight gain of all groups compared favorably.

 

3. TERMINAL FINDINGS OF PARENTAL MALES

ORGAN WEIGHTS

 No test item-related changes in organ weights were noted at any dose level.

At 300 mg/kg/day, the mean absolute epididymide weight (left side only) were significantly reduced (p<0.05) when compared with the controls. This finding was considered to be incidental as the mean relative organ weight was normal and unilateral organ weight differences are not associated with toxicological relevance.

At 100 mg/kg/day, the mean absolute epididymide weights (bilateral) were significantly reduced (p<0.05) when compared with the controls. These differences were considered to be related to the lower mean terminal body weights; the mean relative organ weights were unaffected.

No further changes of organ weights were noted at any dose level.

 

MACROSCOPICAL FINDINGS

A small number of typical background changes were noted in test item-treated and control males. Neither type nor incidence were considered to be commensurate with findings of toxicological relevance in males.

 

HISTOPATHOLOGY FINDINGS

All microscopic findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age. There was no morphological evidence of toxicological properties in the testes, epididymides, prostate and seminal vesicles of male animals examined in this study.

 

Detailed examination of the testes (including sperm staging) was performed on control males (nos. 1 - 11) and males at 1000 mg/kg/day. The stages were checked for completeness of cell populations, completeness of stages and degenerative changes. No differences on the completeness of stages or cell populations of the testes were recorded between the control males (nos. 1 - 11) and the males at 1000 mg/kg/day (nos. 34 - 44). In male no. 6 of the control group, unilateral Sertoli cell vacuolation (minimal in degree) was noted. Male no. 41 (treated with 1000 mg/kg/day) showed unilateral tubular degeneration/atrophy that was considered to be minimal in degree.

 

There were no findings or microscopical changes to the reproductive organs that were considered to be related to the infertility noted in male no. 4

Applicant's summary and conclusion

Conclusions:
This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item to rats (OECD421, GLP compiant). The test material was administered in water as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. The test material was administered to male rats for 14 days prior to pairing and for 28 days in total, 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.

Test item-related effects were restricted to secondary, passive effects only (discolored feces) and there were no findings of toxicological relevance. Body weight and food consumption of parent animals were unaffected, and no differences in organ weights were noted. There were no macroscopical or microscopical findings related to the treatment with the test item.

No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level. Minor differences in the mean post-implantation loss, postnatal loss, and litter size at first litter check and day 4 post partum were either unrelated to dose or within the limits of the historical control data and therefore considered to be incidental.

No test item-related findings were noted at first litter check and during lactation in pups at any dose level. The sex ratios of the pups in test item-treated groups were normal and pup weights recorded until day 4 post partum compared favorably in all groups.

Because fecal discoloration (i.e. a secondary passive finding) was noted in treated rats at all dose levels, a NOEL (No Observed Effect Level) could not be established. The NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females, as well as for reproduction data and F1 offspring was considered to be 1000 mg/kg bw/day, the highest dose level tested.
Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of the test material on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition. 

Four groups of 11 males and 11 females were treated by gavage with test item 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.

 The following dose levels were used:

 Group 1: 0 mg/kg body weight/day (control group)

Group 2: 100 mg/kg body weight/day

Group 3: 300 mg/kg body weight/day

Group 4: 1000 mg/kg body weight/day

 A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (bidistilled water).

 

The following results were obtained:

 

MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

 All animals survived the scheduled study period.

 No clinical signs of toxicological relevance were noted at any dose level. In both sexes, yellow discoloration of the feces was noted from treatment day 5 of the pre-pairing period and continued throughout treatment. The severity of this finding was generally dose-dependent, but was considered to be a typical secondary passive effect that results from oral administration of a dyestuff and was of no toxicological relevance.

 

FOOD CONSUMPTION OF PARENTAL ANIMALS

 Minor intergroup differences in the mean daily food consumption of males were not considered to be related to the treatment with the test item. The daily food consumption values of the test item-treated females were unaffected.

 

BODY WEIGHTS OF PARENTAL ANIMALS

 There were no effects upon body weights of either males or females.

 

REPRODUCTION AND BREEDING DATA

 No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level.

 At the dose level of 1000 mg/kg bw/day, higher incidence of post-implantation loss and higher postnatal loss as well as lower litter size at first litter check and on day 4 post partum were noted.

 The mean post implantation loss of the dams at 1000 mg/kg/day was close to the upper limit of the historical control data and was below the mean post implantation loss of the dams treated with 300 mg/kg/day; therefore this was considered to be unrelated to the treatment with the test item.

 

ORGAN WEIGHS OF PARENTAL ANIMALS

 No test item-related effects on organ weights were noted at any dose level.

 

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATIONS OF PARENTAL ANIMALS

 No test item-related macroscopical findings were noted at any dose level.

 Under the conditions of this study, the test item produced no morphological evidence of toxicological properties in testes, epididymides, prostate and seminal vesicles of male animals and ovaries of female animals.

 

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

 No test item-related findings were noted in pups at any dose level.

 The sex ratio of the test item-treated pups did not indicate any effects of toxicological relevance.

 

PUP WEIGHTS TO DAY 4 POST PARTUM

 No effects on pup body weights were noted at any dose level.

 

MACROSCOPICAL FINDINGS OF PUPS

 No test item-related findings were found in pups at any dose level.