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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:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study concerns the evaluation of results obtained in two individual studies (a OECD 421 study with its dose-range finding study) to compensate the potentially decreased statistical power in the main study that occured due to the decreased prengnancy rate in all groups including the control group.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
not applicable
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Test System:

Young healthy male and nulliparous, non pregnant female rats [strain: WISTAR rat Crl:WI(Han)] (Full-Barrier), were used in both (DRF and Main) studies. The animals were derived from a controlled full barrier maintained breeding system (SPF) (Source: Charles River, 97633 Sulzfeld, Germany).
According to Art. 9.2, No.7 of the German Act on Animal Welfare the animals were bred for experimental purposes.
At the start of treatment the age of the animals was 10-11 weeks. The range of the body weight was:
For DRF study-
Females: 152.80-229.20 g, (mean: 191.00g, ± 20%= 38.20 g)
Males: 248.00-372.00 g, (mean: 310.00 g, ± 20%= 62.00 g)

For main study-
Females: 150.52-225.78 g, (mean: 188.15g, ± 20%= 37.63 g)
Males: 218.48-327.72 g, (mean: 273.10 g, ± 20%= 54.62 g)

Housing and Feeding Conditions:

After an adequate acclimatisation period the animals were barrier maintained (full-barrier) in air conditioned rooms under the following conditions:
-Temperature: 22  3 °C
-Relative humidity: 55  10%
-Artificial light, sequence being 12 hours light, 12 hours dark
-Air change: 10 x / hour
-Free access to Altromin 1324 maintenance diet for rats and mice (Lot. No. 1130, 1455 and 1018)
-Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiol. controlled periodically)
-housed individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (Lot. No. 030511 and 040511)
Certificates of food, water and bedding are filed at BSL BIOSERVICE.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
In both DRF and main studies the test item was administered daily during 14 days pre mating and 14 days mating period in both male and in female; during gestation period and up to post natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 or 29 days has been completed.

The test item was administered by gavage using a gavaging canula. The maximum dose volume administered was 4 mL / kg body weight.

For each animal the individual dosing volume was calculated on the basis of the most recently measured body weight.
Details on mating procedure:
Animals were mated in the ratio of 1:1 (Male to Female). The subsequent morning and the next morning there after the vaginal smear of female were checked to confirm the pregnancy. Day of vaginal plug and/or sperm was considered as day 0 of gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The % nominal of formulation samples meant for the nominal concentration verification taken in study week 1 (first week of pre mating period), study week 3 (first week of mating), study week 5 (gestation) and study week 7 (gestation/lactation) was in the range of 86 -112 % in LD group, 103-114% in MD group and 105-110% in HD group.
The % nominal of formulation samples meant for the stability verification was 115 and 121% in LD group and 110 and 111% in HD group.
The % nominal of formulation samples meant for the homogeneity verification taken in study week 1 (first week of pre mating period) and study week 5 (gestation) was 112-120% in LD group and 108-119% in HD group.
Duration of treatment / exposure:
Males: 28 Days; Females: Approx. 54 Days
Frequency of treatment:
7 days a week
No. of animals per sex per dose:
For DRF study: 24 animals (3 females and 3 males /group)
For main study: 80 animals (10 females and 10 males /group)
Details on study design:
Preparation of the Animals:
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Animals were healthy and none of them had shown any pathological signs before the first administration. Before the first administration, all animals to be used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females. The animals were acclimatised for at least five days before the first dose administration.

Dosage:
In consultation with the sponsor the following doses were selected for the 3 dose groups (LD, MD and HD):

Dosage Group (mg/kg Body weight):

DRF Study
C 0
LD 250
MD 500
HD 750
Main Study
C 0
LD 250
MD 500
HD 750

The animals in the control group were handled in an identical manner to the test group subjects and received vehicle (corn oil) the same volume as used for the treatment groups.

Clinical Observation:
Animals were observed for clinical signs during the entire treatment period. General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. Twice daily (and once daily during weekends and holidays) all animals were observed for morbidity and mortality, Pertinent behavioral changes, signs of difficult or prolonged parturition and all signs of toxicity.

Body Weight and Food Consumption:
The body weight of all animals was recorded once before assignment to the experimental groups and on the first day of administration.
In males, the body weight was taken weekly during the entire study period. In females, the body weight was taken weekly during the pre-mating period, on gestation day 0, 7, 14, 20 and on post-natal day 0 (within 24 hours of parturition) and post-natal day 4 along with pups.
Food consumption was measured on the corresponding day of the body weight measurements after the beginning of the dose administration. Food consumption will not be measured during the mating period.

Litter observations:

The duration of gestation was recorded and was calculated from day 0 of pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.

Live pups were counted and sexed. Litters were weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by writing actual numbers on the back with the help of permanent marker. In addition to the observations on parent animals, any abnormal behavior of the offspring was recorded.
Positive control:
not included

Examinations

Parental animals: Observations and examinations:
Males were sacrificed on day 29 or 30 after the completion of mating period and females were sacrificed on respective post natal day 4 using Ketamine/Xylazine (2:1) at the dose of 1.4 mL/kg bw.
At the time of sacrifice during the study, the adult animals were subjected to a full, detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system.

In DRF and main studies, the ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicle with coagulating glands as a whole), and organs showing macroscopic lesions of adult animals were preserved for histopathological examination. Tissues were fixed in 10% neutral buffered formalin with the exception of testes and epididymides which were fixed in modified Davidson's solution for 24 hours and subsequently transferred to 10% neutral buffered formalin.

Organ Weight:
The testes and epididymides of all male adult animals and ovaries, uterus with cervix of all female adult animals were weighed.
Paired organs were weighed separately.

Histopathology:
Full histopathology was carried out on the preserved organs and tissues of all animals in the control and treatment groups. For testis, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional haematoxylin-PAS (Periodic Acid Schiff) stained slides.
All gross lesions were examined microscopically.

Oestrous cyclicity (parental animals):
Not Evaluated
Sperm parameters (parental animals):
Not Evaluated
Litter observations:
Dead pups and pups killed on day 4 post-partum were carefully examined for gross external abnormalities.
The number of implantation sites and corpora lutea was recorded in all pregnant females by gross observations.
Statistics:
For statistical analysis one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was carried out to reveal any differences between control- and test groups. Statistical analysis was performed with GraphPad Prism (version V) software (p<0.05 was considered as statistical significant).
Reproductive indices:
copulation index (%), fertility index (%), delivery index (%)
Offspring viability indices:
viability index (%)

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
hematuria at 500 and 750 mg/kg bw
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
at 500 and 750 mg/kg bw
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
at 500 and 750 mg/kg bw
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

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

Details on results (P0)

Day of Sacrifice:
In DRF study, all male animals were sacrificed on day 30. Lactating females along with pups were sacrificed on respective post natal day 4.
In main study, all male animals were sacrificed on day 29. Non pregnant females were sacrificed on the respective day 26 from the day of sperm positive vaginal smear as an evidence of mating or completion of mating period. Lactating females along with pups were sacrificed on respective post natal day 4.

Clinical Observation and Mortality:

There were few clinical signs observed in male and female control and treatment groups. The clinical signs recorded in males were, LD- nasal discharge (2/13 animals); MD- nasal discharge (1/13 animals), salivation (2/13 animals), moving the bedding (3/13 animals), piloerection (2/13 animals); HD- salivation (5/13 animals), moving the bedding (12/13 animals), piloerection (2/13 animals).

In females, the clinical findings were following, Control- alopecia (2/13 animals); LD- salivation (5/13 animals), piloerection (1/13 animals), reddish urine (1/13 animals); MD- alopecia (2/13 animals), salivation (6/13 animals), moving the bedding (6/10 animals), reddish bedding (due to red coloured urine) (3/13 animals), reddish urine (1/13 animals), nasal discharge (1/13 animals), piloerection (3/13 animals), reduced spontaneous activity (1/13 animals); HD- salivation (7/13 animals), moving the bedding (13/13 animals), reddish bedding (due to red coloured urine) (6/13 animals), reddish urine (2/13 animals), piloerection (13/13 animals), nasal discharge (2/13 animals), increased/decreased spontaneous activity (1/13 animals) and aggressive behaviour (1/13 animals).

The clinical signs salivation, moving the bedding, piloerection and reddish colour bedding were pronounced in female MD and HD groups and in male HD groups the clinical sign- moving the bedding was pronounced. These findings (except reddish colour bedding in females) could be attributed to the unpalatability instead of systemic toxicity of test item. Reddish urine or bedding (due to red coloured urine) was observed in 1/13 females of LD group, 4/13 females of MD and 8/13 females of HD group.
One female rat (animal no. 77, main study) of HD group was found dead on gestation day 23 with no previous record of clinical findings. The GD 23 being the normal parturition day for the average rats, the death due to complication in parturition is likely to be due to treatment.

Body Weight and Body Weight Change:

In males, statistical analysis of body weight data revealed significant decrease in HD group on day 28 and on the day of terminal sacrifice. Statistically significant difference was observed for body weight change in HD group between pre mating day 1-7. With the progress of the treatment days statistically no significant difference in body weight change was observed in treatment groups when compared to the corresponding control.

In females, there was no statistically significant difference observed for body weight measured during pre mating, gestation and lactation days. There was statistically significant difference observed for body weight change in HD group between GD 7-14. With the progress of the treatment days statistically no significant difference was observed in treatment groups when compared to the corresponding control.
However, in both males and females consistent decrease was observed for body weight and body weight change in MD and HD groups without having statistical significance. This change could be considered to have little toxicological relevance.
Food Consumption:

In males, statistically significant decrease was observed in HD group (between pre mating days 1-7). There was statistically no significant difference between pre mating day 7-14. In females, statistically significant decrease was observed in MD and HD groups (between pre mating days 1-7). There was statistically no significant difference between pre mating day 7-14. There was no statistically significant difference observed for food intake during gestation and lactation days in treatment groups when compared to the corresponding control.

However, there was consistent decrease in food intake in MD and HD groups without having statistical significance. This change corroborated the body weight change in MD and HD groups.

Gross Pathology:

At necropsy, the macroscopic findings observed in males were- nodule on epididymides (1/13 in MD), yellow spots on epididymides (2/13 in MD, 1/13 in HD), white crystalline mass in urinary bladder (1/13 in LD), enlarged stomach (1/13 in MD) and gas filled intestine (1/13 in MD). In females were- cyst on ovary (1/13 C, 3/13 LD, 1/13 HD), stomach with food/ bedding materials and mucosa with dark spots (1/13 in HD), bloody contents in urinary bladder (1/13 in HD) and misshapen liver (1/13 in HD). These findings were considered to be spontaneous and not related test item.

Organ Weight:

There was no treatment related changes observed for absolute and relative (to terminal body weight) organ weights in both males and females in treatment groups when compared to the corresponding control.

Histopathology:

In the main study, no clearly test item-related pathological findings were noted in the male reproductive organs. The occurrence of some spermatic granuloma(s) in MD and HD groups was not considered to be clearly test item-related.

No clearly test item-related pathological findings were noted in the female reproductive organs. When compared to the control group, a lower number of large ovarian corpora lutea was noted in MD and HD groups, but statistically no significant difference was observed for the number of corpora lutea counted at the time of necropsy. Intraluminal debris was more prominent in the vagina in HD group and cervical and/or vaginal epithelial mucification occurred in all treated groups. For these observations, in view of the limited number of pregnant females, a clear test item-relationship could not be established under the conditions of the main study.
In the DRF study, there was no indication of a test item-related effect on histomorphology of male or female reproductive organs. Especially, some inter-group differences noted in the main study could not be confirmed.
Taking the results of the main and DRF study together, it is therefore concluded that there is no convincing evidence of any test item-related effect on histomorphology of male or female reproductive organs.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No clear toxic effect observed at highest dose level tested.
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Hematuria and effect on body weight development was observed at 500 and 750 mg/kg bw

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

Litter Weight Data:
There was decrease in litter mean data, male litter weight (PND 4), female litter weight (PND 0 and 4) and total litter weight observed in LD, MD and HD groups when compared to the control. The Statistical analysis of litter weight data revealed significant decrease in total litter weight on PND 4 in MD and HD groups when compared to the control. The changes in litter weight data is considered to have toxicological relevance.

Precoital Interval and Duration of Gestation :
There was statistically no significant difference observed on precoital interval and duration of gestation in treatment groups when compared to corresponding control.

All females in control and treatment groups showed evidence of copulation during 14 days mating period. The pregnancy rate in Control and treatment groups were as follows, Control 53.85%, LD 92.31%, MD 69.23 % and HD 53.85%. The distribution of non pregnant females in Control, MD and HD groups was uniform and was not attributed to toxicity.

The pregnancy rate in dose range finding study (BSL Study 12565) was 100%.

Pre and Post Natal Data:
There was statistically no significant difference observed for corpora lutea, implantation sites, live pups and % pre and post implantation loss in treatment groups when compared to the corresponding controls. However, there was substantial increase in percent pre and post implantation loss in HD group when compared to control without reaching statistical significance. This finding could be considered to have toxicological relevance.
9.8.

Litter Data:
There was statistically no significant difference observed for number of pups born, number of male/female fetuses (PND 0 and 4), sex ratio, live pups (PND 0 and 4) and still birth in treatment groups when compared to control. However, there was consistent decrease in live pups (PND 0) in MD and HD groups, number of male/female pups in MD and HD groups; consistent increase in still births in MD and HD groups when compared to the control. This finding could be attributed to toxicity.

Reproductive Indices:
There was no statistical significant difference observed for copulation index (%), fertility index (%), delivery index (%) and viability index (%) when compared to the control. All pregnancies resulted in normal births except animal no. 77 (main study) in HD group. This animal was found dead on GD 23. The microscopic findings observed for this decedent are considered to be indicative of bad general condition and stress without representing the direct effects of toxicity, but the cause of death could be assumed to be due to some complication of parturition that went unnoticed. There was considerable decrease in mean viability index in HD group when compared to control group. This decrease is due to female no. 80, which had no surviving pups on PND 4.

Pup Survival Data:
One pup each from female no. DRF 13 (pup no. 7) and DRF 19 (pup no. 6) of control group and MD group was found missing (assumed to be cannibalized) on PND 1.The individual surviving fetus of animal number 80 (main study) of HD group was found dead on PND 1. The viability index of this animal on PND 4 was 0% and the mean viability index at HD group was 83.33%. Although this finding was observed in one individual animal of HD group, the treatment related effect could possibly be attributed.

Pup External Findings:
There were few external findings recorded in few pups of control and treatment groups, which are as follows- Control: reddish snout (Pup no. 6 and 12; animal 49); LD group: small size (pup no. 15; animal 56 of LD group), reddish snout (pup no. 3; animal 59); MD group: dark spot on snout (pup no. 6;animal 69); HD group: dark spot on snout (pup no. 9; animal 71), found dead (pup no. 1; animal 80). There was no dose response pattern observed for the type findings and were considered incidental.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Increased pre-and post implantation loss and reduced live pub numbers were obtained at 500 and 750 mg/kg bw.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The reproduction toxicity of DEGDBE was investigated according to OECD 421. The obtained parental NOAEL were 750 and 250 mg/kg bw for males and females respectively. The developmental NOAEL of 250 mg/kg bw was obtained based on the increased pre-and post implantation loss and reduced number of live pubs at 500 and 750 mg/kg bw.
Executive summary:

The reproduction toxicity of DEGDBE was investigated according to OECD 421. In order to compensate the potentially reduced statistical power due to the reduced number of pregnant animals in all groups in the main study, the results obtained in the dose-range finding study were combined for the robust evaluation.

The male wistar rats after being treated with diethyleneglycoldibutyether in corn oil for the period of 28 days at the dosage of 250, 500 and 750 mg/ kg body weight revealed treatment related changes for body weight development and food consumption at and above 500 mg/kg bw when compared to the corresponding controls. Due to the minor degree of the observed effect no toxicological meaning was attributed.

The female rats were dose administered for approximately 54 days at the same dosage. There was changes observed for body weight development and food consumption at and above 500 mg/kg bw; mortality of one female at 750 mg/kg body weight (due to assumed complication during parturition) and Reddish urine or bedding (LD 1/13, MD 4/13 and HD 8/13 females, observed only on dose application day 2) at and above 250 mg/kg bw. The reddish urine observed in LD group was considered minor due to its finding in an individual isolated female.

There was changes observed for the number of still births, % implantation loss, litter weight data, number of live male, female and total pups and viability index (%) at or above 250 mg/kg body weight. The changes observed at 250 mg/kg body weight were of minor degree and considered of no toxicological meaning.

Based on the result the NOAEL for adult males toxicity is believed to be at 750 mg/kg body weight and for females and fetal toxicity is believed to be at 250 mg/ kg body weight. .