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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 Aug 2018 - 31 Oct 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 Aug 2018 - 31 Oct 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test.
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI (Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Males approximately 11 weeks, Females Approximately 13 weeks
- Weight at study initiation: Males: 319.2 to 382.6 g; Females: 210.5 to 269.5 g
- Fasting period before study: no
- Housing: During pre-mating period animals were group housed (up to 5 animals of the same sex and same dosing group together in plastic cages meeting European directive 2010/63/EU requirements. During mating phase males and females were cohabitated on a one-to-one basis. During post-mating phase males were group housed (5 males/cage). During gestation, females were individually housed. During lactation phase pups were housed with the dam. Cellulose bedding (Serlab, Montataire, France) or dust-free sawdust (SDS/Dietex, Argenteuil, France) made from spruce tree wood. Animals received a small amount (handful) of shredded paper (SDS/Dietex) and the isolated animals had free access to a wooden gnaw block (Aspen Bricks, Dietex France).
- Diet: Rat powdered complete diet ad libitum (Diet reference A04C-10) sterilised by irradiation. Each batch of diet is supplied with a certificate of analysis which is verified and authorized for release by a veterinarian.
- Water: Softened and filtered (0.2 µm) mains drinking water was available ad libitum (via an automatic watering system or bottles).
- Acclimation period: 7 days between animal arrival and start of pre-test estrous cycle smears (females) or start of treatment (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): > 35%
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 06 August 2018 To 31 October 2018
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
TEST ITEM PREPARATION
The test item was prepared as a solution in the vehicle at concentrations of 20, 60 and 200 mg/mL according to Standard Operating Procedures of the Test Facility. No correction factor was used for dose calculations.
Test item was prepared at least weekly.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Quadruple accurately weighed (4 x 1g) samples were taken from each formulation (top, middle, bottom), including the vehicle used on the first day of treatment. Homogeinicity was also measured. The samples were stored at room temperature (+15 to +25 °C) or refrigerated (+2 to +8 °C) within the established stability periods. One set of samples (2 x 1g) was analyzed, the second set was not required and was discarded after the end of the stability period.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 of gestation (G0)
- After successful mating each pregnant female was caged individually
Duration of treatment / exposure:
For males: 14 days before mating, throughout the mating period and up to and including the day before scheduled necropsy (i.e. 28 days).
For females: 14 days before mating, throughout the mating period. During gestation (the first day of gestation was designated as G0) and 13 days after parturition up to and including the day before necropsy (the day of birth is designated as L0), and at least 25 days post-coitum for not pregnant females and females with total litter resorption and at least 22 days of gestation for females sacrificed unable to deliver.
Frequency of treatment:
Once daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: the dose levels are based on an OECD TG 407 with the test substance, information on its key metabolite Ethylene Glycol, developmental toxicity effect dose at ca 900 mg/kg bw and Dodecanedioc acid for which absence of effects was seen at 1000 mg/kg bw.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights of the males were recorded weekly from the first day of exposure (prior to the first exposure) up to necropsy.
Individual body weights for females were recorded twice pretest and then weekly during pre-mating and mating periods (only pre-mating data are reported), on Days 0, 3, 6, 9, 12, 15, 18 and 20 of gestation, and on Days 1, 4, 7 and 14 of lactation.

FOOD CONSUMPTION: Yes
- Food consumption of males was recorded weekly during the pre-mating period.
- Food consumption of females was recorded for the following periods: weekly during the pre-mating period, gestation: Days 0 to 3, 3 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 20, lactation: Days 1 to 4, 4 to 7 and 7 to 14.

TERMINAL EXAMINATIONS: Yes
All adult females were weighed before necropsy (except killed moribund) and killed by carbon dioxide inhalation followed by exsanguination and necropsied according to the following schedule:
- Females: on Day 14 of lactation, on Days 26, 27 or 28 post-coitum for mated females that failed to produce a viable litter (not pregnant or total litter resorption), on Days 22 or 23 post-coitum for females that were unable to deliver.


SACRIFICE
All adult females were weighed before necropsy (except killed moribund) and killed by carbon dioxide inhalation followed by exsanguination and necropsied according to the following schedule: on Day 14 of lactation, on Days 26, 27 or 28 post-coitum for mated females that failed to produce a viable litter (not pregnant or total litter resorption), on Days 22 or 23 post-coitum for females that were unable to deliver.

GROSS NECROPSY
- All animals (including any killed moribund) were submitted to full necropsy procedures including examination of external surface, all orifices, cranial cavity, the carcass, thoracic and abdominal cavities and organs and their contents. Special attention was paid to the organs of the reproductive system.
- The following organs were observed macroscopically: Cervix, Clitoral gland, Kidneys, Liver, Mammary gland, Ovaries with oviduct, Pituitary gland, Thyroid glands, Uterus, Vagina

ORGAN WEIGHTS
- Paired organs were weighed together. Organs were weighed after dissection of fat and other contiguous tissues. The following organs were weighed: Kidneys, Liver, Ovaries with oviduct, Parathyroid glands (thyroid and parathyroid glands were weighed together)

HISTOPATHOLOGY
Histopathological examinations were performed as follows:
- for all organs/tissues from all adult animals killed moribund during the study
- for all macroscopic lesions from all dose group animals
- for all organs/tissues from all adult males and females of groups 1 (control) and 4 (high dose) (for females, only those with live pups)
- for the reproductive organs and thyroid of all males that failed to sire, females which failed to deliver and females with total litter loss.
- the kidneys (identified histologically as a target organ for males in the 1000 mg/kg/day group) were examined from all animals of groups 2 and 3 and the remaining animals not initially examined in groups 1 and 4. For the confirmation of the presence of crystals (oxalates) polarized light was used.
- The following organs were observed macroscopically: Cervix, Clitoral gland, Kidneys, Liver, Mammary gland, Ovaries with oviduct, Pituitary gland, Thyroid glands, Uterus, Vagina
Ovaries and uterine content:
Vaginal smears were taken daily and used to determine the cycle stage beginning 14 days prior to treatment (not reported), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal smears continued for those females with no evidence of copulation until termination of the mating period.
Fetal examinations:
For each F1 litter, the following data were recorded:
– number of pups born (live and dead);
– external abnormalities of the pups;
– number, weight and sex of pups alive on PND 1, 4, 7 and 13.
– anogenital distance (AGD) was measured on PND 1. The AGD was normalized to the cube root of body weight.
– all males in each litter were examined for the number of areola/nipples on PND 13.
On Day 4 post-partum, the size of each litter was adjusted (culling) to 8 pups, by eliminating extra pups by random selection to yield 4 males and 4 females per litter where possible.
Blood samples were collected from pups culled on PND 4 and two surplus pups where possible on PND 13, pooled and used for determination of serum T4 levels.
No pups were eliminated when litter size was below the culling target (8 pups/litter). When there was only one pup available above the culling target, only one pup was eliminated and used for blood collection.
Statistics:
Statistical analyses were performed by the Provantis data acquisition system, where appropriate, as follows:
The best transformation for the data (none, log or rank) was determined depending upon: the kurtosis of the data, the probability of the Bartlett's test for homogeneity of the variances and, an assessment of whether the size of the groups were approximately equal or not.
Non- or log-transformed data were analysed by parametric methods.
Rank transformed data were analysed using non-parametric methods.
Data were then analysed to test for a dose-related trend to detect the lowest dose at which there was a significant effect, based on the Williams test for parametric data or the Shirley's test for non-parametric data.
Homogeneity of means was assessed by analysis of variances (ANOVA) for parametric data or Kruskal-Wallis test for non-parametric data.
If no trend was found and means were not homogeneous, the data were analysed by parametric or non-parametric Dunnett's test to look for significant differences from the control group.
Selected incidence data were analysed using the Provantis data acquisition system and/or a SAS software package. A chi2 test was used for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 was significant.
Estrous cycle, pre-coital interval, anogenital distance and sperm analysis data were analysed using a SAS software package. Levene’s test was used to test the equality of variance across groups and Shapiro-Wilk's test was used to assess the normality of the data distribution in each group. Data with homogenous variances and normal distribution in all groups were analysed using ANOVA followed by Dunnett’s test. Data showing non-homogenous variances or a non-normal distribution in at least one group were analysed using Kruskal-Wallis test followed by the Wilcoxon's rank sum test.
Indices:
Irregularity index: Mean standard deviation of length of the oestrous cycle/ √ (length of the oestrous cycle)
Days in estrus: (number of estrus days/ number of smears) x100
Pre-coital interval (in days): sum of days until successful insemination/ number of inseminated females
Copulation index (%): (number of inseminated females/ number of paired animals) x100
Fertility index (%): (number of pregnant females/ number of inseminated females) x100
Pre-birth loss (%): (number of implantations - number of offspring born/ number of implantations) x100
Live birth index (in %): (number of pups born alive/ number of pups born) x 100
Viability index (in %): (number of pups alive on PND 4 (before culling)/ number of pups alive at birth) x 100
Lactation index (%): (number of pups alive on PND 13/ number of pups alive on PND 4 (after culling)) x 100
Sex ratio (proportion of male) (in %): (number of males/ number of pups) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Compound-related clinical signs were restricted to higher incidences (animals and/or occasions) of hypersalivation, abnormal foraging and pedalling for females in all treated groups generally in a dose-related manner during all phases of the study, including gestation and lactation, compared with sporadic cases in the control group.
Other isolated clinical signs noted such as sporadic bleeding, chromodacryorrhea, localized hairloss and teeth anomalies were considered incidental.
- An overview of the clinical signs can be found in tabular form in the attached study report on page 128 to 137 (pre-mating), page 139 to 163 (gestation), page 165 to 192 (lactation), page 194 to 199 (excluded).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two females in the 1000 mg/kg bw/day group were unable to deliver pups and were sacrificed in a moribund condition on gestation day 22 or 23. One female had a thin appearance with marked piloerection, irregular breathing, decreased activity and soiled urogenital region prior to sacrifice. In the absence of an indication of prolonged pregnancy or peri-natal pup death for the remaining 8 females in the group (gestation length of 22 or 23 days), these cases were considered to be incidental and not compound-related. Their presence in the high dose group only was considered fortuitous.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, overall mean body weight gain was slightly lower for the females (25%) during the pre-mating period (Days 1 to 15) compared with the control. In addition, at 100 mg/kg bw/day, overall mean body weight gain was lower for the females (25%) during the pre-mating period (Days 1 to 15) compared with the control. However, in the absence of any effect on absolute mean body weight through to termination, the differences were considered to be of no toxicological significance. In addition, the body weight gain reduction in femals was not dose-dependent. Although there were some intergroup differences, there was no consistent finding indicative of a compound-related effect on mean body weight or mean body weight gain in the lower dose groups.
- An overview of mean body weight can be found in tabular form in the attached study report on page 43 and 44 (pre-mating), page 51 to 53 (gestation), page 60 (lactation).
- Historical control data on body weight gain (g) during gestation and lactation can be found in tabular form in the attached study report on page 609 and 611, respectively.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no compound-related effect on mean food consumption in any group.
- An overview of mean food consumption can be found in tabular form in the attached study report on page 70 (pre-mating), page 74 and 75 (gestation), page 79 (lactation).
- Historical control data on maternal food consumption (g/day) during gestation and lactation can be found in tabular form in the attached study report on page 610 and 612, respectively.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
An overview of absolute and relative organ weights can be found in tabular form in the attached study report on page 395.
Gross pathological findings:
no effects observed
Description (incidence and severity):
An overview of macroscopic observations can be found in tabular form in the attached study report on page 401 to 404.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
An overview of microscopic observations can be found in tabular form in the attached study report on 410 to 414 (females).
Number of abortions:
no effects observed
Description (incidence and severity):
There were 9/9, 9/9, 8/9 and 8/10 pregnant females in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively, that completed delivery. All females that delivered had liveborn pups.
- An overview of Summary of Mating Performance and Fertility can be found in tabular form in the attached study report on page 86.
- Historical control data can be found in tabular form in the attached study report on page 613.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
There was no compound-related effect on the percentage pre-birth loss in any group. The percentage pre-birth loss was higher in the 300 mg/kg/day group (2.8 times higher) compared with the control but in the absence of a similar finding in the 1000 mg/kg/day group this was considered to be incidental.
Although there were some intergroup differences, the mean number of pups delivered in each of the control and treated groups was within the historical control range so there was no compound-related effect.
- An overview of pre- and post implantation loss can be found in tabular form in the attached study report on page 94.
- Historical control data can be found in tabular form in the attached study report on page 613.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
There was one pregnant female in the 300 mg/kg bw/day group with a total litter resorption. This isolated finding in the intermediate dose group was considered incidental and due to an atypical low number of implantation sites (n=3) compared with the group mean of 12.8.
- An overview of resorption can be found in tabular form in the attached study report on page 86.
Dead fetuses:
no effects observed
Description (incidence and severity):
There was no compound-related effect on the live birth index for the treated groups and were consequently comparable with those in the concurrent control and/or the historical control data.
- An overview of the live birth index, viability index, and lactation index can be found in tabular form in the attached study report on page 96.
- Historical control data can be found in tabular form in the attached study report on page 613.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
The mean duration of gestation was comparable (approximately 22 days) in all groups.
- An overview of the individual gestation length data can be found in tabular form in the attached study report on page 94.
- Historical control data can be found in tabular form in the attached study report on page 613.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There were 9/9, 9/9, 8/9 and 8/10 pregnant females in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively, that completed delivery.
- An overview of Summary of Mating Performance and Fertility can be found in tabular form in the attached study report on page 86.
- Historical control data can be found in tabular form in the attached study report on page 613.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Mean pup body weight was comparable for both sexes in all groups from birth through to termination on PND 13.
- An overview of the mean pup weight can be found in tabular form in the attached study report on page 98 to 100.
- Historical control data can be found in tabular form in the attached study report on page 614.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There was no compound-related effect on pup viability in any group from birth through to termination. The live birth index, viability index (pre-cull on PND 4) and lactation index for the treated groups were consequently comparable with those in the concurrent control and/or the historical control data.
- An overview of the live birth index, viability index, and lactation index can be found in tabular form in the attached study report on page 96.
- Historical control data can be found in tabular form in the attached study report on page 613.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
An overview of the sex ratio can be found in tabular form in the attached study report on page 96.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
An overview of the litter size can be found in tabular form in the attached study report on page 96.
An overview of the litter weight can be found in tabular form in the attached study report on page 96.
Historical control data can be found in tabular form in the attached study report on page 613 and 614.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
The live birth index, viability index (pre-cull on PND 4) and lactation index for the treated groups were consequently comparable with those in the concurrent control and/or the historical control data.
- An overview of the live birth index, viability index, and lactation index can be found in tabular form in the attached study report on page 96.
- Historical control data can be found in tabular form in the attached study report on page 613.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There was no compound-related effect on gross pathology findings in pups.
- An overview of the Individual Pup Macroscopic Observations can be found in tabular form in the attached study report on page 582 to 607.
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone analysis:
- No biologically relevant differences in Total T4 levels were noted among the different groups of PND 13 pups.
- An overview of the Mean Thyroid Hormone levels can be found in tabular form in the attached study report on page 360.
Nipple retention
- There was no compound-related effect on male areola/nipple retention in any group. No nipples were observed for any male in any group on PND13.
- An overview of the Individual Male Pup Areola/Nipple Number can be found in tabular form in the attached study report on page 304 to 307.
AGD:
- Anogenital distance (normalized for body weight) for male and female pups was considered not to be affected by treatment. A statistically significantly longer mean anogenital distance for female pups at 300 mg/kg/day (9%) was attributed to relatively high intra-group differences and in the absence of any dose-related trend these variations were considered not to be related to treatment.
- An overview of the Mean Pup Anogenital Distance can be found in tabular form in the attached study report on page 105.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Analysis of dose preparations: All analyzed samples for formulations prepared at nominal concentrations of 20, 60 and 200 mg/mL of Zenolide in vehicle (corn oil), taken from each preparation, including the vehicle, on the first day of treatment were in agreement with acceptance criteria (± 15 %). The deviations from the nominal concentrations ranged from -3.8 % to 1.1 %. Furthermore, the test item prepared as a suspension in the vehicle was homogenous as the RSD on the six aliquots (Top, Middle, Bottom) was ≤ 1.5 %. No test item was present in the vehicle sample.

Conclusions:
Under the conditions of the test (OECD 421, GLP), the NOAEL for maternal and developmental toxicity was determined to be 1000 mg/kg bw/day.
Executive summary:

A repeated dose study with screening for reproductive/developmental effects was performed according to OECD/EC guidelines and GLP principles. The test substance, was administered by daily oral gavage to female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of 10 rats/sex received the same dose volume (5 mL/kg/day) of the vehicle (corn oil). Females were treated during 2 weeks prior to mating, during mating, during gestation and up to the day before necropsy (i.e. on day 13 of lactation for females that delivered. Formulation analysis showed that formulations were prepared accurately and homogeneously. Two females in the 1000 mg/kg bw/day group were unable to deliver pups and were sacrificed in a moribund condition on gestation day 22 or 23. Their presence in the high dose group only was considered fortuitous and this mortality is not considered test item-related. There were higher incidences (animals and/or occasions) of hypersalivation, abnormal foraging and pedaling in all treated groups generally in a dose-related manner during all phases of the study, including gestation and lactation, compared with sporadic cases in the control group, but those clinical signs were not considered compound-related. At 1000 mg/kg bw/day, overall mean body weight gain was slightly lower for the females during the pre-mating period (Days 1 to 15) compared with the control. However, in the absence of any effect on absolute mean body weight through to termination, the differences were considered to be of no toxicological significance. There was no compound-related effect on mean food consumption. No biologically relevant differences in total T4 levels were noted among tthe different groups of PND 13 pups. There were no compound-related effects on estrous cycles, mating performance or fertility. There were 9/9, 9/9, 8/9 and 8/10 pregnant females in the control, 100, 300 and 1000 mg/kg/day groups, respectively, that completed delivery. The mean duration of gestation was comparable (approximately 22 days) in all groups. All females that delivered had liveborn pups. Two pregnant females in the 1000 mg/kg bw/day group were unable to complete delivery and were sacrificed for ethical reasons. There were no test item-related effects on the number of implantation, pre-birth loss, litter size, pup viability or pup body weight. There was no compound-related effect on male areola/nipple retention in any group. No nipples were observed for any male in any group on PND13. There was no compound-related effect on anogenital distance (normalized for body weight) for the male or female pups. Therefore, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity was 1000 mg/kg bw/day. The developmental No Observed Adverse Effect Level (NOAEL) was set in 1000 mg/kg bw/day for both sexes.

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 Aug 2018 - 31 Oct 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test.
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI (Han)
Details on species / strain selection:
Rat is one of the rodent species acceptable to the regulatory authorities. Historical control data for the strain are available at the Test Facility.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Males approximately 11 weeks, Females Approximately 13 weeks
- Weight at study initiation: Males: 319.2 to 382.6 g; Females: 210.5 to 269.5 g
- Fasting period before study: no
- Housing: During pre-mating period animals were group housed (up to 5 animals of the same sex and same dosing group together in plastic cages meeting European directive 2010/63/EU requirements. During mating phase males and females were cohabitated on a one-to-one basis. During post-mating phase males were group housed (5 males/cage). During gestation, females were individually housed. During lactation phase pups were housed with the dam. Cellulose bedding (Serlab, Montataire, France) or dust-free sawdust (SDS/Dietex, Argenteuil, France) made from spruce tree wood. Animals received a small amount (handful) of shredded paper (SDS/Dietex) and the isolated animals had free access to a wooden gnaw block (Aspen Bricks, Dietex France).
- Diet: Rat powdered complete diet ad libitum (Diet reference A04C-10) sterilised by irradiation. Each batch of diet is supplied with a certificate of analysis which is verified and authorized for release by a veterinarian.
- Water: Softened and filtered (0.2 µm) mains drinking water was available ad libitum (via an automatic watering system or bottles).
- Acclimation period: 7 days between animal arrival and start of pre-test estrous cycle smears (females) or start of treatment (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): > 35
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 06 August 2018 To 31 October 2018
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
TEST ITEM PREPARATION
The test item was prepared as a solution in the vehicle at concentrations of 20, 60 and 200 mg/mL according to Standard Operating Procedures of the Test Facility. No correction factor was used for dose calculations.
Test item was prepared at least weekly.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Quadruple accurately weighed (4 x 1g) samples were taken from each formulation (top, middle, bottom), including the vehicle used on the first day of treatment. Homogeinicity was also measured. The samples were stored at room temperature (+15 to +25 °C) or refrigerated (+2 to +8 °C) within the established stability periods. One set of samples (2 x 1g) was analyzed, the second set was not required and was discarded after the end of the stability period.
Duration of treatment / exposure:
For males: 14 days before mating, throughout the mating period and up to and including the day before scheduled necropsy (i.e. 28 days).
For females: 14 days before mating, throughout the mating period. During gestation (the first day of gestation was designated as G0) and 13 days after parturition up to and including the day before necropsy (the day of birth is designated as L0), and at least 25 days post-coitum for not pregnant females and females with total litter resorption and at least 22 days of gestation for females sacrificed unable to deliver.
Frequency of treatment:
Once daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
MATING PROCEDURE
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 of gestation (G0)
- After successful mating each pregnant female was caged individually
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights of the males were recorded weekly from the first day of exposure (prior to the first exposure) up to necropsy.
Individual body weights for females were recorded twice pretest and then weekly during pre-mating and mating periods (only pre-mating data are reported), on Days 0, 3, 6, 9, 12, 15, 18 and 20 of gestation, and on Days 1, 4, 7 and 14 of lactation.

FOOD CONSUMPTION: Yes
- Food consumption of males was recorded weekly during the pre-mating period.
- Food consumption of females was recorded for the following periods: weekly during the pre-mating period, gestation: Days 0 to 3, 3 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 20, lactation: Days 1 to 4, 4 to 7 and 7 to 14.

CLINICAL CHEMISTRY: Yes, hormone analysis

Other examinations
- Thyroid hormone analysis. Blood samples were collected from males and females on the day of necropsy under Isoflorane. Animals were not fasted before sampling. Thyroxine (T4) was analysed in male rats
- Estrous cyclicity: Vaginal smears were taken daily and used to determine the cycle stage beginning 14 days prior to treatment (not reported), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal smears continued for those females with no evidence of copulation until termination of the mating period.
- Sperm parameters: Sperm counts and motility were assessed using automated equipment (Hamilton Thorne Research IVOS). The left cauda epididymis was sampled and used for the assessment of sperm motility. Sperm counts were performed using the left testis following removal of the tunica albuginea. Sperm morphology was evaluated (at least 200 sperm per sample, where possible). The numbers of sperm with each type of abnormality was recorded.
Sacrifice and pathology:
SACRIFICE
All adult males and females were weighed before necropsy (except killed moribund) and killed by carbon dioxide inhalation followed by exsanguination and necropsied according to the following schedule:
- Males: after completion of the mating period on study Day 30 (28 days of dose administration)
- Females: on Day 14 of lactation, on Days 26, 27 or 28 post-coitum for mated females that failed to produce a viable litter (not pregnant or total litter resorption), on Days 22 or 23 post-coitum for females that were unable to deliver.

GROSS NECROPSY
- All animals (including any killed moribund) were submitted to full necropsy procedures including examination of external surface, all orifices, cranial cavity, the carcass, thoracic and abdominal cavities and organs and their contents. Special attention was paid to the organs of the reproductive system.
- The following organs were observed macroscopically: Cervix, Clitoral gland, Coagulating gland, Epididymides, Kidneys, Liver, Mammary gland (females and males, inguinal area), Ovaries with oviduct, Pituitary gland, Preputial gland, Prostate, Seminal vesicles, Testes, Thyroid glands, Uterus, Vagina

ORGAN WEIGHTS
- Paired organs were weighed together. Organs were weighed after dissection of fat and other contiguous tissues. The following organs were weighed: Epididymides (right), Kidneys, Liver, Ovaries with oviduct, Parathyroid glands (thyroid and parathyroid glands were weighed together), Prostate, Seminal vesicles with coagulating gland, Testes (left)

HISTOPATHOLOGY
Histopathological examinations were performed as follows:
- for all organs/tissues from all adult animals killed moribund during the study
- for all macroscopic lesions from all dose group animals
- for all organs/tissues from all adult males and females of groups 1 (control) and 4 (high dose) (for females, only those with live pups)
- for the reproductive organs and thyroid of all males that failed to sire, females which failed to deliver and females with total litter loss.
- the kidneys (identified histologically as a target organ for males in the 1000 mg/kg/day group) were examined from all animals of groups 2 and 3 and the remaining animals not initially examined in groups 1 and 4. For the confirmation of the presence of crystals (oxalates) polarized light was used.
- The following organs were observed microscopically: Cervix, Clitoral gland, Coagulating gland, Epididymides, Kidneys, Liver, Mammary gland (females and males, inguinal area), Ovaries with oviduct, Pituitary gland, Preputial gland, Prostate, Seminal vesicles, Testes, Thyroid glands, Uterus, Vagina
Statistics:
Statistical analyses were performed by the Provantis data acquisition system, where appropriate, as follows:
The best transformation for the data (none, log or rank) was determined depending upon: the kurtosis of the data, the probability of the Bartlett's test for homogeneity of the variances and, an assessment of whether the size of the groups were approximately equal or not.
Non- or log-transformed data were analysed by parametric methods.
Rank transformed data were analysed using non-parametric methods.
Data were then analysed to test for a dose-related trend to detect the lowest dose at which there was a significant effect, based on the Williams test for parametric data or the Shirley's test for non-parametric data.
Homogeneity of means was assessed by analysis of variances (ANOVA) for parametric data or Kruskal-Wallis test for non-parametric data.
If no trend was found and means were not homogeneous, the data were analysed by parametric or non-parametric Dunnett's test to look for significant differences from the control group.
Selected incidence data were analysed using the Provantis data acquisition system and/or a SAS software package. A chi2 test was used for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 was significant.
Estrous cycle, pre-coital interval, anogenital distance and sperm analysis data were analysed using a SAS software package. Levene’s test was used to test the equality of variance across groups and Shapiro-Wilk's test was used to assess the normality of the data distribution in each group. Data with homogenous variances and normal distribution in all groups were analysed using ANOVA followed by Dunnett’s test. Data showing non-homogenous variances or a non-normal distribution in at least one group were analysed using Kruskal-Wallis test followed by the Wilcoxon's rank sum test.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Compound-related clinical signs were restricted to higher incidences (animals and/or occasions) of hypersalivation, abnormal foraging and pedalling for both males and females in all treated groups generally in a dose-related manner during all phases of the study, including gestation and lactation, compared with sporadic cases in the control group.
Other isolated clinical signs noted such as sporadic bleeding, chromodacryorrhea, localized hairloss and teeth anomalies were considered incidental.
- An overview of the clinical signs can be found in tabular form in the attached study report on page 108 to 126 ( Males), page 127 to 137 (females: pre-mating), page 138 to 163 (females: gestation), page 164 to 192 (females: lactation), page 193 to 199 (females: excluded).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two females in the 1000 mg/kg bw/day group were unable to deliver pups and were sacrificed in a moribund condition on gestation day 22 or 23. One female had a thin appearance with marked piloerection, irregular breathing, decreased activity and soiled urogenital region prior to sacrifice. In the absence of an indication of prolonged pregnancy or peri-natal pup death for the remaining 8 females in the group (gestation length of 22 or 23 days), these cases were considered to be incidental and not compound-related. Their presence in the high dose group only was considered fortuitous.
There was no unscheduled death amongst the males in any group.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, overall mean body weight gain was lower (36%) for the males during the dosing period (Days 1 to 29) and for the females (25%) during the pre-mating period (Days 1 to 15) compared with the control. In addition, at 100 mg/kg bw/day, overall mean body weight gain was lower for the females (25%) during the pre-mating period (Days 1 to 15) compared with the control. However, in the absence of any effect on absolute mean body weight through to termination for either sex, the differences were considered to be of no toxicological significance. In addition, the body weight gain reduction in femals was not dose-dependent. Although there were some intergroup differences, there was no consistent finding indicative of a compound-related effect on mean body weight or mean body weight gain for either sex in the lower dose groups.
- An overview of mean body weight can be found in tabular form in the attached study report on page 35 and 36 (males), page 43 and 44 (females: pre-mating), page 51 to 53 (females: gestation), page 60 (females: lactation).
- An overview of mean body weight change can be found in tabular form in the attached study report on page 40 and 41 (males) and 48 and 49 (females: pre-mating), page 57 and 58 (females: gestation), page 64 (females: lactation).
- Historical control data on body weight gain (g) during gestation and lactation can be found in tabular form in the attached study report on page 609 and 611, respectively.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no compound-related effect on mean food consumption for either sex in any group.
- An overview of mean food consumption can be found in tabular form in the attached study report on page 66 (males), page 70 (females: pre-mating), page 74 and 75 (females: gestation), page 79 (females: lactation).
- Historical control data on maternal food consumption (g/day) during gestation and lactation can be found in tabular form in the attached study report on page 610 and 612, respectively.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
When compared with controls, treatment-related increased absolute and relative mean kidney weights were noted in males at 1000 mg/kg bw/day (55 and 60%, respectively) and with minor increasing mean weights at 300 or 100 mg/kg bw/day (only statistically significant for absolute weight). These values showed statistical significance in treated animals from the high dose group and were histologically correlated with minimal or moderate interstitial inflammation, cortical tubular degeneration/regeneration with tubular dilatation accompanied by multifocal intratubular crystals.
Other occasional organ weight differences were considered to be incidental and unrelated to treatment.
- An overview of absolute and relative organ weights can be found in tabular form in the attached study report on page 392 to 394 (males) and 395 (females).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There was no evidence at any dose level of macroscopic-related changes in the genital tract for either sex. Irregular surface (bilateral) of the kidney was noted in 7/10 males given 1000 mg/kg bw/day, accompanied by bilateral enlarged appearance in 2/10 males. Any other macroscopic observations were considered to be incidental and unrelated to test substance administration.
- An overview of macroscopic observations can be found in tabular form in the attached study report on page 397 to 399 (males) and 401 to 404 (females).
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The main treatment-related findings noted in the kidneys of males at 1000 mg/kg bw/day consisted in minimal to slight and in some cases moderate multifocal cortical tubular degeneration/regeneration characterized by tubular basophilia, karyomegaly, nuclear crowding with intratubular cell debris observed in the corticomedullary junction following acute tubule epithelial injury, accompanied by interstitial inflammation and tubular dilatation. Microscopic examination with polarised light showed presence of intratubular translucent crystals of Calcium oxalate. These crystals are expected to be the result of one metabolite of Zenolide, which is Oxalic acid (third metabolite) based on work on Ethylene glycol (ATSDR review 2010). The formation of these crystals is rat species dependent and Wistar rats (used in the present study and mainly males) are more sensitive compared with other rat strains (Cruzan et al., 2004 and Corley et al., 2008). In addition, no treatment-related findings in the kidneys were noted for either sex at 100 or 300 mg/kg bw/day. The microscopic tubular effects and the crystals in males at 1000 mg/kg/day were associated with one of Zenolide’s metabolites, which is Ethylene glycol.
There were no treatment-related findings in the genital tract for either sex at 1000 mg/kg/day. All animals examined showed normal tissular morphology.
Any other microscopic observations were considered to be incidental and unrelated to the test substance administration.
- An overview of microscopic observations can be found in tabular form in the attached study report on page 406 to 408 (males) and 410 to 414 (females).
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone analysis:
- No biologically relevant differences in Total T4 levels were noted among the different groups of F0-males.
- An overview of mean Total T4 can be found in tabular form in the attached study report on page 360.
Estrous Cycle:
- There was no compound-related effect on mean length and regularity of the estrous cycles in any group. Most females had regular cycles of approximately 4 days. Two females in the 100 mg/kg bw/day group were acyclic or had an acyclic period during the pre-mating period. These isolated cases in the low dose group only were incidental and there was no compound-related effect.
- An overview of mean estrous cycle data can be found in tabular form in the attached study report on page 84.
Sperm parameters:
- There was no compound-related effect on mean sperm counts (testis and cauda epididymis) or motility, including the percentage of progressively motile sperm, in any group. Similarly, the percentages of sperm with normal and abnormal morphology were comparable in all groups.
- An overview of mean sperm parameters can be found in tabular form in the attached study report on page 88 (sperm morphology), page 90 (Sperm Motility), Page 91 and 92 (Sperm counts).
- Historical control data can be found in tabular form in the attached study report on page 615 to 617.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Analysis of dose preparations: All analyzed samples for formulations prepared at nominal concentrations of 20, 60 and 200 mg/mL of Zenolide in vehicle (corn oil), taken from each preparation, including the vehicle, on the first day of treatment were in agreement with acceptance criteria (± 15 %). The deviations from the nominal concentrations ranged from -3.8 % to 1.1 %. Furthermore, the test item prepared as a suspension in the vehicle was homogenous as the RSD on the six aliquots (Top, Middle, Bottom) was ≤ 1.5 %. No test item was present in the vehicle sample.

Conclusions:
Under the conditions of the test (OECD 421, GLP), the NOAEL was determined to be 300 and 1000 mg/kg bw/day for males and females, respectively.
Executive summary:

A repeated dose study with screening for reproductive/developmental effects was performed according to OECD/EC guidelines and GLP principles. The test substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of 10 rats/sex received the same dose volume (5 mL/kg/day) of the vehicle (Corn oil). Males were treated for 28 days, i.e. 2 weeks prior to mating, during mating and up to termination. Females were treated during 2 weeks prior to mating, during mating, during gestation and up to the day before necropsy (i.e. on day 13 of lactation for females that delivered. Formulation analysis showed that formulations were prepared accurately and homogeneously. Two females in the 1000 mg/kg bw/day group were unable to deliver pups and were sacrificed in a moribund condition on gestation day 22 or 23. Their presence in the high dose group only was considered fortuitous and this mortality is not considered test item-related. There were higher incidences (animals and/or occasions) of hypersalivation, abnormal foraging and pedaling for both males and females in all treated groups generally in a dose-related manner during all phases of the study, including gestation and lactation, compared with sporadic cases in the control group, but those clinical signs were not considered compound-related. At 1000 mg/kg bw/day, overall mean body weight gain was slightly lower for the males during the dosing period (Days 1 to 29) and for the females during the pre-mating period (Days 1 to 15) compared with the control. However, in the absence of any effect on absolute mean body weight through to termination for either sex, the differences were considered to be of no toxicological significance. There was no compound-related effect on mean food consumption for either sex in any group. No biologically relevant differences in total T4 levels were noted among the different groups of F0-males. There were no compound-related effects on estrous cycles, mean sperm counts (testis and cauda epididymis), sperm motility and sperm morphology. Pathology findings at 1000 mg/kg bw/day included irregular surface of the kidneys (bilateral), on a few occasions accompanied by bilateral enlarged appearance, and increased absolute and relative mean kidney weights that correlated histologically with minimal to moderate interstitial inflammation, cortical tubular degeneration/regeneration with tubular dilatation accompanied by multifocal intratubular crystals for males. These kidney effects are similar to those found for Ethylene glycol. Microscopic examination with polarized light showed presence of intratubular translucent crystals of Calcium oxalate. These crystals are expected to be the result of one metabolite of the test substance, which is Oxalic acid (third metabolite) based on work on Ethylene glycol. The formation of these crystals is rat species dependent and Wistar rats (used in the present study and mainly males) are more sensitive compared with other rat strainsand is considered for the determination of the No Observed Adverse Effect Level. Only minor increases in mean kidney weights were noted at 300 or 100 mg/kg bw/day but with no crystal formation. Therefore, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was 300 mg/kg bw/day for males and 1000 mg/kg bw/day for females due to the presence and absence of compound-related kidney changes, respectively.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test.
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

1
Chemical structure
Reference substance name:
1,4-dioxacyclohexadecane-5,16-dione
EC Number:
259-423-6
EC Name:
1,4-dioxacyclohexadecane-5,16-dione
Cas Number:
54982-83-1
Molecular formula:
C14H24O4
IUPAC Name:
1,4-dioxacyclohexadecane-5,16-dione
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI (Han)
Details on species / strain selection:
Rat is one of the rodent species acceptable to the regulatory authorities. Historical control data for the strain are available at the Test Facility.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Males approximately 11 weeks, Females Approximately 13 weeks
- Weight at study initiation: Males: 319.2 to 382.6 g; Females: 210.5 to 269.5 g
- Fasting period before study: no
- Housing: During pre-mating period animals were group housed (up to 5 animals of the same sex and same dosing group together in plastic cages meeting European directive 2010/63/EU requirements. During mating phase males and females were cohabitated on a one-to-one basis. During post-mating phase males were group housed (5 males/cage). During gestation, females were individually housed. During lactation phase pups were housed with the dam. Cellulose bedding (Serlab, Montataire, France) or dust-free sawdust (SDS/Dietex, Argenteuil, France) made from spruce tree wood. Animals received a small amount (handful) of shredded paper (SDS/Dietex) and the isolated animals had free access to a wooden gnaw block (Aspen Bricks, Dietex France).
- Diet: Rat powdered complete diet ad libitum (Diet reference A04C-10) sterilised by irradiation. Each batch of diet is supplied with a certificate of analysis which is verified and authorized for release by a veterinarian.
- Water: Softened and filtered (0.2 µm) mains drinking water was available ad libitum (via an automatic watering system or bottles).
- Acclimation period: 7 days between animal arrival and start of pre-test estrous cycle smears (females) or start of treatment (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): > 35
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 06 August 2018 To 31 October 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
TEST ITEM PREPARATION
The test item was prepared as a solution in the vehicle at concentrations of 20, 60 and 200 mg/mL according to Standard Operating Procedures of the Test Facility. No correction factor was used for dose calculations.
Test item was prepared at least weekly.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 of gestation (G0)
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Quadruple accurately weighed (4 x 1g) samples were taken from each formulation (top, middle, bottom), including the vehicle used on the first day of treatment. Homogeinicity was also measured. The samples were stored at room temperature (+15 to +25 °C) or refrigerated (+2 to +8 °C) within the established stability periods. One set of samples (2 x 1g) was analyzed, the second set was not required and was discarded after the end of the stability period.
Duration of treatment / exposure:
For males: 14 days before mating, throughout the mating period and up to and including the day before scheduled necropsy (i.e. 28 days).
For females: 14 days before mating, throughout the mating period. During gestation (the first day of gestation was designated as G0) and 13 days after parturition up to and including the day before necropsy (the day of birth is designated as L0), and at least 25 days post-coitum for not pregnant females and females with total litter resorption and at least 22 days of gestation for females sacrificed unable to deliver.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: the dose levels are based on an OECD TG 407 with the test substance, information on its key metabolite Ethylene Glycol, developmental toxicity effect dose at ca 900 mg/kg bw and Dodecanedioc acid for which absence of effects was seen at 1000 mg/kg bw.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights of the males were recorded weekly from the first day of exposure (prior to the first exposure) up to necropsy.
Individual body weights for females were recorded twice pretest and then weekly during pre-mating and mating periods (only pre-mating data are reported), on Days 0, 3, 6, 9, 12, 15, 18 and 20 of gestation, and on Days 1, 4, 7 and 14 of lactation.

FOOD CONSUMPTION: Yes
- Food consumption of males was recorded weekly during the pre-mating period.
- Food consumption of females was recorded for the following periods: weekly during the pre-mating period, gestation: Days 0 to 3, 3 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 20, lactation: Days 1 to 4, 4 to 7 and 7 to 14.

CLINICAL CHEMISTRY: Yes, hormone analysis

TERMINAL EXAMINATIONS: Yes
All adult males and females were weighed before necropsy (except killed moribund) and killed by carbon dioxide inhalation followed by exsanguination and necropsied according to the following schedule:
- Males: after completion of the mating period on study Day 30 (28 days of dose administration)
- Females: on Day 14 of lactation, on Days 26, 27 or 28 post-coitum for mated females that failed to produce a viable litter (not pregnant or total litter resorption), on Days 22 or 23 post-coitum for females that were unable to deliver.
- Pups: at culling (PND 4) or at terminal sacrifice (PND 13)

Other examinations
- Thyroid hormone analysis. Blood samples were collected from males and females on the day of necropsy under Isoflorane. Animals were not fasted before sampling. Thyroxine (T4) was analysed in male rats
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily and used to determine the cycle stage beginning 14 days prior to treatment (not reported), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal smears continued for those females with no evidence of copulation until termination of the mating period.
Sperm parameters (parental animals):
Sperm counts and motility were assessed using automated equipment (Hamilton Thorne Research IVOS). The left cauda epididymis was sampled and used for the assessment of sperm motility. Sperm counts were performed using the left testis following removal of the tunica albuginea. Sperm morphology was evaluated (at least 200 sperm per sample, where possible). The numbers of sperm with each type of abnormality was recorded.
Litter observations:
For each F1 litter, the following data were recorded:
– number of pups born (live and dead);
– external abnormalities of the pups;
– number, weight and sex of pups alive on PND 1, 4, 7 and 13.
– anogenital distance (AGD) was measured on PND 1. The AGD was normalized to the cube root of body weight.
– all males in each litter were examined for the number of areola/nipples on PND 13.
On Day 4 post-partum, the size of each litter was adjusted (culling) to 8 pups, by eliminating extra pups by random selection to yield 4 males and 4 females per litter where possible.
Blood samples were collected from pups culled on PND 4 and two surplus pups where possible on PND 13, pooled and used for determination of serum T4 levels.
Postmortem examinations (parental animals):
SACRIFICE
All adult males and females were weighed before necropsy (except killed moribund) and killed by carbon dioxide inhalation followed by exsanguination and necropsied according to the following schedule:
- Males: after completion of the mating period on study Day 30 (28 days of dose administration)
- Females: on Day 14 of lactation, on Days 26, 27 or 28 post-coitum for mated females that failed to produce a viable litter (not pregnant or total litter resorption), on Days 22 or 23 post-coitum for females that were unable to deliver.

GROSS NECROPSY
- All animals (including any killed moribund) were submitted to full necropsy procedures including examination of external surface, all orifices, cranial cavity, the carcass, thoracic and abdominal cavities and organs and their contents. Special attention was paid to the organs of the reproductive system.
- The following organs were observed macroscopically: Cervix, Clitoral gland, Coagulating gland, Epididymides, Kidneys, Liver, Mammary gland (females and males, inguinal area), Ovaries with oviduct, Pituitary gland, Preputial gland, Prostate, Seminal vesicles, Testes, Thyroid glands, Uterus, Vagina

ORGAN WEIGHTS
- Paired organs were weighed together. Organs were weighed after dissection of fat and other contiguous tissues. The following organs were weighed: Epididymides (right), Kidneys, Liver, Ovaries with oviduct, Parathyroid glands (thyroid and parathyroid glands were weighed together), Prostate, Seminal vesicles with coagulating gland, Testes (left)

HISTOPATHOLOGY
Histopathological examinations were performed as follows:
- for all organs/tissues from all adult animals killed moribund during the study
- for all macroscopic lesions from all dose group animals
- for all organs/tissues from all adult males and females of groups 1 (control) and 4 (high dose) (for females, only those with live pups)
- for the reproductive organs and thyroid of all males that failed to sire, females which failed to deliver and females with total litter loss.
- the kidneys (identified histologically as a target organ for males in the 1000 mg/kg/day group) were examined from all animals of groups 2 and 3 and the remaining animals not initially examined in groups 1 and 4. For the confirmation of the presence of crystals (oxalates) polarized light was used.
- The following organs were observed microscopically: Cervix, Clitoral gland, Coagulating gland, Epididymides, Kidneys, Liver, Mammary gland (females and males, inguinal area), Ovaries with oviduct, Pituitary gland, Preputial gland, Prostate, Seminal vesicles, Testes, Thyroid glands, Uterus, Vagina
Postmortem examinations (offspring):
Pups (extra pups on PND4, any moribund pups and surviving pups on PND13) were killed by intraperitoneal injection of sodium pentobarbitone.
Pups (including any found dead or killed moribund) were necropsied. Any external abnormalities observed were recorded but not preserved. For any pups found dead or killed moribund, the stomach were examined for the presence of milk and defects or cause of death were evaluated, if possible.
Each pup was sexed and examined for external defects with special attention being paid to the external reproductive organs.
For pups on PND 13, the thyroid glands of one male and one female of each litter were fixed and preserved (if possible pups sampled for hormone analysis).
Statistics:
Statistical analyses were performed by the Provantis data acquisition system, where appropriate, as follows:
The best transformation for the data (none, log or rank) was determined depending upon: the kurtosis of the data, the probability of the Bartlett's test for homogeneity of the variances and, an assessment of whether the size of the groups were approximately equal or not.
Non- or log-transformed data were analysed by parametric methods.
Rank transformed data were analysed using non-parametric methods.
Data were then analysed to test for a dose-related trend to detect the lowest dose at which there was a significant effect, based on the Williams test for parametric data or the Shirley's test for non-parametric data.
Homogeneity of means was assessed by analysis of variances (ANOVA) for parametric data or Kruskal-Wallis test for non-parametric data.
If no trend was found and means were not homogeneous, the data were analysed by parametric or non-parametric Dunnett's test to look for significant differences from the control group.
Selected incidence data were analysed using the Provantis data acquisition system and/or a SAS software package. A chi2 test was used for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 was significant.
Estrous cycle, pre-coital interval, anogenital distance and sperm analysis data were analysed using a SAS software package. Levene’s test was used to test the equality of variance across groups and Shapiro-Wilk's test was used to assess the normality of the data distribution in each group. Data with homogenous variances and normal distribution in all groups were analysed using ANOVA followed by Dunnett’s test. Data showing non-homogenous variances or a non-normal distribution in at least one group were analysed using Kruskal-Wallis test followed by the Wilcoxon's rank sum test.
Reproductive indices:
Irregularity index: Mean standard deviation of length of the oestrous cycle/ √ (length of the oestrous cycle)
Days in estrus: (number of estrus days/ number of smears) x100
Pre-coital interval (in days): sum of days until successful insemination/ number of inseminated females
Copulation index (%): (number of inseminated females/ number of paired animals) x100
Fertility index (%): (number of pregnant females/ number of inseminated females) x100
Offspring viability indices:
Pre-birth loss (%): (number of implantations - number of offspring born/ number of implantations) x100
Live birth index (in %): (number of pups born alive/ number of pups born) x 100
Viability index (in %): (number of pups alive on PND 4 (before culling)/ number of pups alive at birth) x 100
Lactation index (%): (number of pups alive on PND 13/ number of pups alive on PND 4 (after culling)) x 100
Sex ratio (proportion of male) (in %): (number of males/ number of pups) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Compound-related clinical signs were restricted to higher incidences (animals and/or occasions) of hypersalivation, abnormal foraging and pedalling for both males and females in all treated groups generally in a dose-related manner during all phases of the study, including gestation and lactation, compared with sporadic cases in the control group.
Other isolated clinical signs noted such as sporadic bleeding, chromodacryorrhea, localized hairloss and teeth anomalies were considered incidental.
- An overview of the clinical signs can be found in tabular form in the attached study report on page 109 to 126 (Males), page 128 to 137 (females: pre-mating), page 139 to 163 (females: gestation), page 165 to 192 (females: lactation), page 194 to 199 (females: excluded)
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two females in the 1000 mg/kg bw/day group were unable to deliver pups and were sacrificed in a moribund condition on gestation day 22 or 23. One female had a thin appearance with marked piloerection, irregular breathing, decreased activity and soiled urogenital region prior to sacrifice. In the absence of an indication of prolonged pregnancy or peri-natal pup death for the remaining 8 females in the group (gestation length of 22 or 23 days), these cases were considered to be incidental and not compound-related. Their presence in the high dose group only was considered fortuitous.
There was no unscheduled death amongst the males in any group.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, overall mean body weight gain was lower (36%) for the males during the dosing period (Days 1 to 29) and for the females (25%) during the pre-mating period (Days 1 to 15) compared with the control. In addition, at 100 mg/kg bw/day, overall mean body weight gain was lower for the females (25%) during the pre-mating period (Days 1 to 15) compared with the control. However, in the absence of any effect on absolute mean body weight through to termination for either sex, the differences were considered to be of no toxicological significance. In addition, the body weight gain reduction in femals was not dose-dependent. Although there were some intergroup differences, there was no consistent finding indicative of a compound-related effect on mean body weight or mean body weight gain for either sex in the lower dose groups.
- An overview of mean body weight can be found in tabular form in the attached study report on page 35 and 36 (males), page 43 and 44 (females: pre-mating), page 51 to 53 (females: gestation), page 60 (females: lactation).
- An overview of mean body weight change can be found in tabular form in the attached study report on page 40 and 41 (males) and 48 and 49 (females: pre-mating), page 57 and 58 (females: gestation), page 64 (females: lactation).
- Historical control data on body weight gain (g) during gestation and lactation can be found in tabular form in the attached study report on page 609 and 611, respectively.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no compound-related effect on mean food consumption for either sex in any group.
- An overview of mean food consumption can be found in tabular form in the attached study report on page 66 (males), page 70 (females: pre-mating), page 74 and 75 (females: gestation), page 79 (females: lactation).
- Historical control data on maternal food consumption (g/day) during gestation and lactation can be found in tabular form in the attached study report on page 610 and 612, respectively.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The main treatment-related findings noted in the kidneys of males at 1000 mg/kg bw/day consisted in minimal to slight and in some cases moderate multifocal cortical tubular degeneration/regeneration characterized by tubular basophilia, karyomegaly, nuclear crowding with intratubular cell debris observed in the corticomedullary junction following acute tubule epithelial injury, accompanied by interstitial inflammation and tubular dilatation. Microscopic examination with polarised light showed presence of intratubular translucent crystals of Calcium oxalate. These crystals are expected to be the result of one metabolite of Zenolide, which is Oxalic acid (third metabolite) based on work on Ethylene glycol (ATSDR review 2010). The formation of these crystals is rat species dependent and Wistar rats (used in the present study and mainly males) are more sensitive compared with other rat strains (Cruzan et al., 2004 and Corley et al., 2008). In addition, no treatment-related findings in the kidneys were noted for either sex at 100 or 300 mg/kg bw/day. The microscopic tubular effects and the crystals in males at 1000 mg/kg/day were associated with one of Zenolide’s metabolites, which is Ethylene glycol.
There were no treatment-related findings in the genital tract for either sex at 1000 mg/kg/day. All animals examined showed normal tissular morphology.
Any other microscopic observations were considered to be incidental and unrelated to the test substance administration.
- An overview of microscopic observations can be found in tabular form in the attached study report on page 406 to 408 (males) and 410 to 414 (females).
Other effects:
no effects observed
Description (incidence and severity):
Fertility:
- There was no compound-related effect on fertility in any group. There was one mated female that did not become pregnant in each of the control, 100 and 300 mg/kg bw/day groups. The fertility index in the treated groups was therefore the same as, or higher than, that in the control group (90% or 100%).
- An overview of Mating Performance and Fertility data can be found in tabular form in the attached study report on page 86.
- Historical control data can be found in tabular form in the attached study report on page 613.
Thyroid hormone analysis:
- No biologically relevant differences in Total T4 levels were noted among the different groups of F0-males.
- An overview of mean Total T4 can be found in tabular form in the attached study report on page 360.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There was no compound-related effect on mean length and regularity of the estrous cycles in any group. Most females had regular cycles of approximately 4 days. Two females in the 100 mg/kg bw/day group were acyclic or had an acyclic period during the pre-mating period. These isolated cases in the low dose group only were incidental and there was no compound-related effect.
- An overview of mean estrous cycle data can be found in tabular form in the attached study report on page 84.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There was no compound-related effect on mean sperm counts (testis and cauda epididymis) or motility, including the percentage of progressively motile sperm, in any group. Similarly, the percentages of sperm with normal and abnormal morphology were comparable in all groups.
- An overview of mean sperm parameters can be found in tabular form in the attached study report on page 88 (sperm morphology), page 90 (Sperm Motility), Page 91 and 92 (Sperm counts).
- Historical control data can be found in tabular form in the attached study report on page 615 to 617.
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating performance and fertility
- There was no compound-related effect on mating performance in any group.
- There were 10 mated pairs of animals in each of the control, 100, 300 and 1000 mg/kg bw/day groups. The mating index was therefore 100% in all groups. The majority of mated females showed evidence of insemination within the first 4 days of pairing (approximate duration of a normal estrous cycle). There were some intergroup differences in the mean pre-coital interval but this was due to the disproportionate influence of one or two females in the control, 100 and 300 mg/kg bw/day groups that did not mate until the end of the second week of mating. In the absence of a similar case in the 1000 mg/kg bw/day group, these isolated findings were considered incidental and there was no compound-related effect.
- An overview of Summary of Mating Performance and Fertility can be found in tabular form in the attached study report on page 86.
- Historical control data can be found in tabular form in the attached study report on page 613.

Pre-birth loss
- There was no compound-related effect on the percentage pre-birth loss in any group. The percentage pre-birth loss was 2.8 times higher in the 300 mg/kg/day group compared with the control but in the absence of a similar finding in the 1000 mg/kg/day group this was considered to be incidental. There was also one pregnant female in the 300 mg/kg/day group with a total litter resorption. This isolated finding in the intermediate dose group was considered incidental and due to an atypical low number of implantation sites (n=3) compared with the group mean of 12.8. Although there were some intergroup differences, the mean number of pups delivered in each of the control and treated groups was within the historical control range so there was no compound-related effect.
- An overview of Summary of Delivery and Litter Data can be found in tabular form in the attached study report on page 94 and 95.
- Historical control data can be found in tabular form in the attached study report on page 613.

Details on results (P0)

Parturition:
- There were 9/9, 9/9, 8/9 and 8/10 pregnant females in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively, that completed delivery. All females that delivered had liveborn pups. Two pregnant females in the 1000 mg/kg bw/day group were unable to complete delivery and were sacrificed for ethical reasons.
- An overview of Summary of Mating Performance and Fertility can be found in tabular form in the attached study report on page 86.
- Historical control data can be found in tabular form in the attached study report on page 613.
Gestation length
- The mean duration of gestation was comparable (approximately 22 days) in all groups.
- An overview of the individual gestation length data can be found in tabular form in the attached study report on page 94
- Historical control data can be found in tabular form in the attached study report on page 613.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs, principally including incomplete hair growth, were noted sporadically across the groups, including the control, and are part of the background of clinical changes amongst rat litters.
- An overview of the Individual Clinical observations for pups can be found in tabular form in the attached study report on page 164 to 192.
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was no compound-related effect on pup viability in any group from birth through to termination. The live birth index, viability index (pre-cull on PND 4) and lactation index for the treated groups were consequently comparable with those in the concurrent control and/or the historical control data.
- An overview of the live birth index, viability index, and lactation index can be found in tabular form in the attached study report on page 96.
- Historical control data can be found in tabular form in the attached study report on page 613.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean pup body weight was comparable for both sexes in all groups from birth through to termination on PND 13.
- An overview of the mean pup weight can be found in tabular form in the attached study report on page 98 to 100.
- Historical control data can be found in tabular form in the attached study report on page 614.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There was no compound-related effect on gross pathology findings in pups.
- An overview of the Individual Pup Macroscopic Observations can be found in tabular form in the attached study report on page 582 to 607.
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone analysis:
- No biologically relevant differences in Total T4 levels were noted among the different groups of PND 13 pups.
- An overview of the Mean Thyroid Hormone levels can be found in tabular form in the attached study report on page 360.
Nipple retention
- There was no compound-related effect on male areola/nipple retention in any group. No nipples were observed for any male in any group on PND13.
- An overview of the Individual Male Pup Areola/Nipple Number can be found in tabular form in the attached study report on page 304 to 307.
AGD:
- Anogenital distance (normalized for body weight) for male and female pups was considered not to be affected by treatment. A statistically significantly longer mean anogenital distance for female pups at 300 mg/kg/day (9%) was attributed to relatively high intra-group differences and in the absence of any dose-related trend these variations were considered not to be related to treatment.
- An overview of the Mean Pup Anogenital Distance can be found in tabular form in the attached study report on page 105.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Analysis of dose preparations: All analyzed samples for formulations prepared at nominal concentrations of 20, 60 and 200 mg/mL of Zenolide in vehicle (corn oil), taken from each preparation, including the vehicle, on the first day of treatment were in agreement with acceptance criteria (± 15 %). The deviations from the nominal concentrations ranged from -3.8 % to 1.1 %. Furthermore, the test item prepared as a suspension in the vehicle was homogenous as the RSD on the six aliquots (Top, Middle, Bottom) was ≤ 1.5 %. No test item was present in the vehicle sample.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test (OECD 421, GLP), the NOAEL for parental toxicity was determined to be 300 and 1000 mg/kg bw/day for males and females, respectively. The NOAEL for reproductive toxicity was determined to be 1000 mg/kg bw/day.
Executive summary:

A repeated dose study with screening for reproductive/developmental effects was performed according to OECD/EC guidelines and GLP principles. Zenolide, was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of 10 rats/sex received the same dose volume (5 mL/kg/day) of the vehicle (Corn oil). Males were treated for 28 days, i.e. 2 weeks prior to mating, during mating and up to termination. Females were treated during 2 weeks prior to mating, during mating, during gestation and up to the day before necropsy (i.e. on day 13 of lactation for females that delivered. Formulation analysis showed that formulations were prepared accurately and homogeneously. Two females in the 1000 mg/kg bw/day group were unable to deliver pups and were sacrificed in a moribund condition on gestation day 22 or 23. Their presence in the high dose group only was considered fortuitous and this mortality is not considered test item-related. There were higher incidences (animals and/or occasions) of hypersalivation, abnormal foraging and pedaling for both males and females in all treated groups generally in a dose-related manner during all phases of the study, including gestation and lactation, compared with sporadic cases in the control group, but those clinical signs were not considered compound-related. At 1000 mg/kg bw/day, overall mean body weight gain was slightly lower for the males during the dosing period (Days 1 to 29) and for the females during the pre-mating period (Days 1 to 15) compared with the control. However, in the absence of any effect on absolute mean body weight through to termination for either sex, the differences were considered to be of no toxicological significance. There was no compound-related effect on mean food consumption for either sex in any group. No biologically relevant differences in total T4 levels were noted among the different groups of F0-males nor among the different groups of PND 13 pups. There were no compound-related effects on estrous cycles, mating performance or fertility, including mean sperm counts (testis and cauda epididymis), sperm motility and sperm morphology. There were 9/9, 9/9, 8/9 and 8/10 pregnant females in the control, 100, 300 and 1000 mg/kg/day groups, respectively, that completed delivery. The mean duration of gestation was comparable (approximately 22 days) in all groups. All females that delivered had liveborn pups. Two pregnant females in the 1000 mg/kg bw/day group were unable to complete delivery and were sacrificed for ethical reasons. There were no test item-related effects on the number of implantation. Pathology findings at 1000 mg/kg bw/day included irregular surface of the kidneys (bilateral), on a few occasions accompanied by bilateral enlarged appearance, and increased absolute and relative mean kidney weights that correlated histologically with minimal to moderate interstitial inflammation, cortical tubular degeneration/regeneration with tubular dilatation accompanied by multifocal intratubular crystals for males. These kidney effects are similar to those found for Ethylene glycol. Microscopic examination with polarized light showed presence of intratubular translucent crystals of Calcium oxalate. These crystals are expected to be the result of one metabolite of Zenolide, which is Oxalic acid (third metabolite) based on work on Ethylene glycol.The formation of these crystals is rat species dependent and Wistar rats (used in the present study and mainly males) are more sensitive compared with other rat strains and is considered for the determination of the No Observed Adverse Effect Level. Only minor increases in mean kidney weights were noted at 300 or 100 mg/kg bw/day but with no crystal formation. Therefore, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was 300 mg/kg bw/day for males and 1000 mg/kg bw/day for females due to the presence and absence of compound-related kidney changes, respectively. The reproduction No Observed Adverse Effect Level (NOAEL) was set in 1000 mg/kg bw/day for both sexes.