Bis(2-ethylhexyl) cyclohexane-1,4-dicarboxylate

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2017-07-27 to 2018-08-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethylhexyl) cyclohexane-1,4-dicarboxylate

EC Number:

283-829-2

EC Name:

Bis(2-ethylhexyl) cyclohexane-1,4-dicarboxylate

Cas Number:

84731-70-4

Molecular formula:

C24H44O4

IUPAC Name:

1,4-bis(2-ethylhexyl) cyclohexane-1,4-dicarboxylate

Test material form:

liquid

Specific details on test material used for the study:

Batch No.: 20170214
Purity: >99%

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Rat is the preferred species for reproduction toxicity study and is accredited in the OECD Guideline.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Beijing Vital River Laboratory Animal Technology Co., Ltd.
- Age at study initiation: 35-41 days old (on arrival)
- Weight at study initiation: 112.80-138.55 for females and 122.29-162.59g for males (on arrival); 163.82-234.77g for males and 104.43-188.57g for females (on the final day of the acclimatization)
- Housing: plastic cages (L46.0×W31.5 ×H20.0cm) on cage racks (L170.0cm×W50.0cm×H160.0cm); two rats at most in per cage
- Diet: ad libitum. The sterilized SPF rodent growth and breeding feed were used in this test. All the nutrition components and contaminants were within the permitted limits described in the national standard.
- Water: ad libitum. Drinking water was purified with HT-R01000 purity system and routinely analyzed (at least annually), and all parameters were within the permitted limits described in the national standard.
- Acclimation period: eight days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.8-25.5 °C
- Humidity (%): 38-70%
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
As preparing the test item, calculate the theoretical weight and range of the test item according to the prepared volume and concentration shown below. Weigh the test item and place into a beaker. Add some solvent, mix and pour into a cylinder with lid. Wash the beaker with solvent and pour into the cylinder at least three times. Add solvent in the cylinder to the scale of prepared volume, mix and pour into a labeled bottle for use.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item has good solubility in corn oil, and the corn oil has no adverse effects by gavage as 4 ml/kg.bw to rats according to the experience of this lab, at the same time, it is accredited vehicle in the Guideline, so corn oil was selected as vehicle in this study.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: two weeks
- Proof of pregnancy: The presence of vaginal plugs was considered as the evidence of successful copulation. The day on which vaginal plugs was detected was considered as GD0.
- The mated females were caged individually for the birth and rearing of their pups.
- In the second week of the mating period, females without successful copulation were replaced with another male animal from the same group (a male that already has successfully mated with another female.)
- After the mating period, females without successful copulation were caged individually until sacrifice (more than 21 days after the last day of the mating period), at the same time, females with successful copulation but turning out to be non-pregnant were killed for necropsy.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical check for the concentrations of prepared test item was conducted for four times, respectively at the early stage and the last preparation, and twice in the course of the test.
Preparation of the sample:
Three samples were prepared by adding 0.5 mL of 10 mg/L standard solution to 99.5 mL of the dilution water (total volume: 100 mL) to obtain 0.05 mg/L of test solutions, respectively. They were transferred into each 250 mL separatory funnel. 30 mL of dichloromethane was added and shaked for 5 minutes. The dichloromethane layer was passed through sodium sulphate to remove moisture and collected in a round flask. This procedure was repeated one more time. Dichloromethane was evaporated using rotary evaporator at 30~40℃. Final volume of sample was adjusted to 1 mL using acetone and then analyzed. The recovery efficiency was calculated and this should be in the range 80-120%.
Calculation method of concentration:
Concentrations of the test item were quantitated by an external standard method. The peak area response for this test item in each calibration standard chromatogram was measured and a calibration curve constructed by linear regression of standard area response versus standard concentration. The measured concentration of the test item was calculated from the calibration curve using GC Chemstation . All value was expressed after correction of concentration factor (100-200 fold concentration) in this report.

Duration of treatment / exposure:

For F0-generation animals, the administration was continued during the premating period of at least 10 weeks, mating period of no longer than 2 weeks, gestation and lactation period (for females) until the weaning of their pups.
For F1-generation animals selected for mating, the administration was continued from PND22 to the weaning of their pups at the same doses with their mothers, including the premating period of at least 10 weeks, mating period of no longer than 2 weeks, gestation and lactation period (for females). The females in the process of parturition were not dosed.

Frequency of treatment:

once daily in the morning

Details on study schedule:

After all litters of F0-generation were weaned, 28 female pups and 28 male pups were selected at random selection from as many litters as possible in each group to rear F1-generation.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

40 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

28

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale:
In repeated dose 90-day oral toxicity study of the test substance in rats, no toxicologically significant effect was observed at the dose levels of 10, 100 and 1000mg/kg.d, and No Observed Adverse Effect Level (NOAEL) was considered to be 1000mg/kg.d.
Refer to the results in repeated dose 90-day oral toxicity study of DEHCH in rats supplied by sponsor, three dose levels were used in this study including 1000, 200 and 40mg/kg.d. A concurrent control group was included at the same time.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily in the morning hours; All animals were observed for morbidity and mortality twice daily at the beginning and the end of work (once daily on non working day).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once per week

BODY WEIGHT: Yes
- Time schedule for examinations: Once per week during the premating and mating period. Mated females were weighed on gestation day (GD) 0, 7, 14 and 21, and on day 0, 4, 7, 14 and 21 of lactation. All animals were weighed on their scheduled necropsy date.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
During the premating and gestation period, the ration food was added once weekly. The added food weight was 500±10 g per cage in the premating period and 200±10 g per cage in the gestation period. The food was weighed again one day (24 h± 1.5 h) later as surplus food weight. The results were expressed in g per animal per day.

OTHER
- Parturition observation: All pregnant females were observed for parturition from GD21 twice daily in the morning and afternoon respectively (once daily on non working day). Any difficulties and abnormalities occurring during parturition were recorded.

Oestrous cyclicity (parental animals):

For F0-generation and selected F1-generation females, vaginal smears to evaluate the estrus cycle length and normality were made for two weeks prior to mating. For each female, vaginal smear was made daily and was observed under the microscope to evaluate their estrus cycle stage.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
Testicular sperm count, Epididymal sperm morphology, Epididymal sperm motility and count

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups with 4 males and 4 females per litter, and if the number of one sex was not enough, another sex was added. The rest pups were euthanized with CO2.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring:
For pups in lactation period, a cage-side observation of the litters was conducted once daily in order to keep nest disturbance to a minimum, except for PND0, 4, 7, 14 and 21. On these days, pups were observed individually, and the number of live pups was recorded. On PND0, the number of each sex, stillbirth and grossly malformed pups was recorded and evaluated.
On PND0 and 4, litter weights were recorded. On PND4 after culling, and on PND7, 14 and 21, the pups were weighed individually.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Males were sacrificed after the end of the mating period, and dams were sacrificed after all litters were weaned.
- Maternal animals: Females without successful copulation and females with successful copulation but without delivery were sacrificed more than 21 days after the last day of the mating period.

GROSS NECROPSY
- The necropsy included carefully examinations of the external features of the carcass, external body orifices, the abdominal, thoracic, and cranial cavities and their contents of all animals, and any abnormal findings were recorded. For the parous females, the number of implantation sites in the uteri and the number of corpora lutea were recorded. For non-mated or non-pregnant females, the uteri were observed for the presence of implantation site.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Histopathology: Histopathology was examined on the following organs: Reproductive organs of the parental animals in the control and high-dose groups, and all males that failed to become sire and females that were non-mated or non-pregnant in the low- and mid- dose groups; all gross lesions found in the parental animals.
- Organ weights: For the parental animals of F0-generation and F1-generation, that included adrenals, brain, pituitary, kidneys, liver, spleen, thyroid, epididymides, uterus with cervix, ovaries, testes, prostate including seminal vesicles with coagulating glands; for the pups selected for necropsy, that included brain, spleen and thymus.
The organs were weighed as soon as possible after dissection to avoid drying. The pituitary and thyroid were weighed after the fixation in 10% neutral-buffered formalin. Organ-to-body weight ratios were calculated.

Postmortem examinations (offspring):

GROSS NECROPSY
One male and one female pup of each litter as possibly, and all pups with external abnormalities or clinical signs that had not recovered as necropsy were selected for necropsy. On the day of necropsy, the selected pups were euthanized by CO2 inhalation followed by exsanguinations from abdominal aorta and subjected to a full necropsy and general observation. The other live pups were humanely killed by CO2 euthanasia.
The stillborn pups or dead pups found on PND0~4, when not macerated, were observed for stomach contents (if containing milk) and external abnormality, and then were fixed in Bouin’s solution for examination of soft tissue. The dead pups found after PND5, when not macerated, were observed for external abnormality, and then were necropsied for any gross lesions and stomach contents (if containing milk).

HISTOPATHOLOGY / ORGAN WEIGTHS
- Histopathology: Histopathology was examined on the following organs: Reproductive organs of the parental animals in the control and high-dose groups, and all males that failed to become sire and females that were non-mated or non-pregnant in the low- and mid- dose groups; all gross lesions found in the parental animals.
- Organ weights: For F1-generation, that included adrenals, brain, pituitary, kidneys, liver, spleen, thyroid, epididymides, uterus with cervix, ovaries, testes, prostate including seminal vesicles with coagulating glands; for the pups selected for necropsy, that included brain, spleen and thymus.
The organs were weighed as soon as possible after dissection to avoid drying. The pituitary and thyroid were weighed after the fixation in 10% neutral-buffered formalin. Organ-to-body weight ratios were calculated.

Statistics:

The resulting data were analyzed using the methods mentioned below. P﹤0.05 was considered as a level of significance.
a) The data of clinical signs were analyzed by Chi-square test;
b) Data of the mean body weight and mean body weight gain, food consumption were statistic analyzed by decision tree; data of the food efficiency of the parental animals during the premating period will be statistic analyzed by decision tree;
c) The precoital interval and the gestation length were evaluated by Mann-Whitney U-test for grade analysis, and were statistic analyzed by decision tree;
e) Estrus cyclicity was evaluated by Chi-square test for number of acyclic animals and number of animals with prolonged estrus period, and by decision tree for mean cycle length and number of whole estrus cycle during the exam period;
f) Sperm parameters were evaluated by decision tree for sperm count and motility parameters, and by Chi-square test for sperm morphology;
g) Incidences of pathological findings were analyzed by the unilateral Fisher’s exact probability test.
All data analysis was done with test software in this facility or SPSS 17.0.
For the data that have no difference between the control group and dose group, it was not necessary to perform the statistical analysis.

Reproductive indices:

Female mating index = (number of mated females/number of females placed with males) ×100
Male mating index = (number of mated males/number of males placed with females) ×100
Male fertility index = (number of males siring a litter/number of males placed with females) ×100
Female fertility index = (number of pregnant females/number of females placed with males) ×100
Female fecundity index = (number of pregnant females/number of mated females) ×100
Gestation index = (number of females with live pups / number of pregnant females) ×100
Live birth index = (number of pups born alive / number of pups born) ×100
Viability index of lactation = (number of surviving pups on PND21 / number of pups after culling on PND4) ×100
Sex ratio = (number of live male pups on PND0/ number of live pups on PND0 ) ×100

Offspring viability indices:

Viability index of lactation = (number of surviving pups on PND21 / number of pups after culling on PND4) ×100
Sex ratio = (number of live male pups on PND0/ number of live pups on PND0 ) ×100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

For males, one animal in each dose group respectively was observed with dehairing on part of body during the premating and mating period, but that was not considered to be treatment-related clinical sign.
For females, dehairing, vaginal hemorrhage, soiled perineal region, scab around eye were observed on one or two animals in the different period, and two animals were observed with tumor, but the incidence of these signs had no significant increase in all dose groups compared with the control group, so that were not considered to be treatment-related clinical signs.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two females (No. 2015 and 2019) of the control group and one female (No. 2323) of the high-dose group were found dead on GD26, GD25 and GD23 respectively, at necropsy, embryos were found in their uterus, and no apparent cause of death was found. One male (No. 1300) of the high-dose group was found dead in the premating period, at necropsy, diaphragmatic hernia was found, that was considered as spontaneous death and without toxicological significance.
The mortality of male and female animals in the high-dose group had no significant increase compared with the control group, so that result was considered to be an incidental result without toxicological significance.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Premating period
For males and females, no significant difference was observed on the mean bodyweight and body weight gain in all dose groups compared with the control group (p>0.05).
Mating period
For males, no significant difference was observed in all dose groups compared with the control group (p>0.05).
Gestation period
No significant difference was observed in all dose groups compared with the control group (p>0.05).
Lactation period
No significant difference on the mean body weight of the parental females was observed in all dose groups compared with the control group (p>0.05). The body weight change from PND0 to PND4 had a statistically significant decrease in the high-dose group compared with the control group (p<0.05), but it recovered in the remaining lactation period. No significant difference on the body weight change was observed in the low- and mid- dose groups compared with the control group (p>0.05).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Premating period
For males, the mean food consumption in some test weeks had a statistically significant increase in one or more dose groups compared with the control group(p<0.05 or p<0.01), and in the 7th test week, the mean food consumption in the low-dose group had a statistically significant decrease(p<0.01), at the same time, the total food consumption in the premating period had a statistically significant increase in the high-dose group compared with the control group(p<0.01).
For females, the mean food consumption in some test weeks had a statistically significant increase in the mid- and/or high- dose groups compared with the control group(p<0.05 or p<0.01), and in the 5th test week, the mean food consumption in the low-dose group had a statistically significant decrease(p<0.05), at the same time, the total food consumption in the premating period had a statistically significant increase in the mid- and high- dose groups compared with the control group(p<0.05 or p<0.01).
Premating period
The mean food consumption of the pregnant females in the mid- and high- dose groups had a statistically significant increase in the last week compared with the control group (p<0.01).
Base on the results about the food consumption above, the animals had a tendency to eat more after the exposure of test item at the dose of 1000 and 200mg/kg.d, but at the same time, no adverse effect on the body weight in each dose group was observed, so these results were considered without toxicological significance.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Premating period
For males, the decrease of mean food efficiency was observed in the high-dose group in the second week (p<0.05), but the average food efficiency in the whole premating period had no statistically significant difference in all dose groups compared with the control group (p>0.05), these results were considered to be incidental without toxicological significance.
For females, the decrease of mean food efficiency was observed in the high-dose group in the second test week (p<0.05), in addition, the average food efficiency in the whole premating period had a statistically significant decrease in the mid-dose group, but that had no statistically significant change in the other two dose groups, these results were considered to be incidental without toxicological significance.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Scheduled necropsies
No treatment-related changes were found in males and females of high-dose group.
Based on the macroscopic findings, all gross lesions were examined microscopically. Testis atrophy, epididymis atrophy and prostate inflammatory cell infiltrate in histopathology were observed in the lesions of testis small, epididymis small and prostate small; kidney mineralization was observed in the lesions of kidney malformation; uterus amyloidosis was observed in the lesions of uterus nodule. All these abnormalities were facultative and were not treatment-related.
In males of all dose groups that failed to become sire, several cases of prostate inflammatory cell infiltrate and prostate abscess, one case of testis and epididymis atrophy were observed. In females that were non-mated or non-pregnant, ovary follicular cyst, decreased number/absent corpora lutea and uterus squamous metaplasia etc. were observed in some animals. No significant difference on the incidence of these abnormalities was observed in all dose groups compared with the control group (p>0.05), so that was considered not to be treatment-related.
Intercurrent dead animals
In F0 generation, the microscopic examination was conducted in four intercurrent dead animals, ovary hyperemia was observed in rat 2323.No abnormality was observed in the other animals.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

No differences were observed on all estrus cyclicity parameters in each dose group compared with the control group (p>0.05).

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Epididymal sperm motility and count
A statistically significantly increase on the total count of epididymal sperm was observed (p<0.05) in high-dose group, but that was not considered to be an adverse effect on the males. No statistically significant difference on the total count of epididymal sperm was observed in the other two dose groups compared with the control group (p>0.05).
Statistically significantly increases on the average path velocity (VAP), amplitude of lateral head displacement (ALH) in the high-dose group were observed (p<0.05), but these results were not considered to be the adverse effect. No statistically significant difference on the other sperm motility parameters was observed in all dose groups compared with the control group (p>0.05).
One male in the low-dose group (No.: 1104) was examined for epididymal sperm motility and count, but the report from TOX IVOS Sperm Analyzer showed that all sperms examined were static, it was considered that was not the actual state of the animal, but was caused by operation error, so the data of this animal were excluded from the statistical analysis.
Epididymal sperm morphology
No significant effect on the epididymal sperm morphology was observed in the examined males of all dose groups.
Testicular sperm count
No statistically significant difference on the testicular sperm count was observed in all dose groups compared with the control group (p>0.05).

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Fertility
In each group, twenty-eight males (27 males in the high-dose group) and 28 females were put together as one to one ratio for mating, and the mating period lasted for two weeks. 23, 24, 20 and 24 males were found to mate with female, and 18, 20, 15 and 21 males became sire in the control, low-, mid- and high- dose groups, respectively. The mating index and fertility index of males had no statistically significant difference in all dose groups compared with the control group (p>0.05).
25, 27, 24 and 27 females were found to mate with male, and 22, 22, 18 and 25 females were pregnant in the control, low-, mid- and high- dose groups, respectively. The mating index, fertility index and fecundity index of females had no statistically significant difference in all dose groups compared with the control group (p>0.05).
No difference was observed on precoital interval in females of all dose groups compared with the control group (p>0.05).
Pregnancy and Delivery Data
20, 22, 18 and 24 pregnant females survived delivery to obtain a litter with liveborn pups in the control, low-, mid- and high- dose groups, respectively. Two females in the control group and one female in the high-dose group were dead during the delivery. The gestation index had no statistically significant difference in all dose groups compared with the control group (p>0.05).
No statistically significant difference was observed on gestation length in all dose groups compared with the control group (p>0.05).

Details on results (P0)

Litter Data
All pregnant females with successful delivery obtained a litter with at least one liveborn pups, and 2, 0, 1 and 2 females delivered a litter with stillborn pup in the control, low-, mid- and high- dose groups, respectively. One female in the control group (No. 2016) and one female in the mid-dose group (No. 2216) had complete litter loss during the lactation period.
No statistically significant difference was observed on the mean number of corpora lutea and implantation sites in all dose groups compared with the control group (p>0.05). No difference was observed on the mean number of pups delivered and liveborn in all dose groups (p>0.05). No statistically significant difference was observed on the pre-implantation loss in all dose groups compared with the control group (p>0.05); the post-implantation loss in high-dose group had a significant decreased compared with the control group (p<0.01), but no toxicological significance; no statistically significant difference was observed on the post-implantation loss in low- and mid-dose groups compared with the control group (p>0.05).

Pup Data
- Pup status: There were 10, 0, 1 and 3stillborn pups in the control group and low-, mid-, high- dose groups, respectively. The live birth indexes in the low- and mid- dose groups were increased significantly compared with the control group (p<0.05 or p<0.01), but no significant adverse effect on the live birth index was observed in all dose groups. No pup in all groups was dead in the lactation period; and the viability index of lactation was 100% in each group.
- Mean number of pups: The mean number of live pups on PND0 had no statistically significant difference in all dose groups compared with the control group (p>0.05). The sex ratio of the live pups at birth had a statistically significant difference between the low-dose group and the control group (p<0.05), and more male pups were observed in the low-dose group, but no statistically significant difference on the sex ratio was observed in the mid- and high- dose groups (p>0.05), so that was considered to be an incidental result without toxicological significance. The mean number of live pups on the other time of the lactation period, including PND4, PND7, PND14 and PND21 had no statistically significant difference in all dose groups compared with the control group(p>0.05).
No statistically significant difference on the number of dead pups during the lactation period was observed in all dose groups (p>0.05).
- Pup clinical observations: During the lactation period, one litter including eight pups in the low-dose group were observed with sparse fur from PND13 to PND17, and recovered later. One pup in the litter of 2112 was observed with walking difficulty from PND19, and did not recover until being killed as scheduled. No pup with any abnormality and sign was observed in the other groups. The number of pups with signs in the low-dose group had statistically significant increase compared with the control group (p<0.01), but this was considered as an incidental result without toxicological significance.
- Pup body weights: The mean body weights of live pups in all dose groups had no statistically significant difference during the lactation period compared with the control group (p>0.05).
- Organ Weight of Pups
No statistically significant difference on absolute and relative organ weight was observed in all dose groups compared with the control group (p>0.05).
- Macroscopic Observations of Pups
Stillborn and dead pups: No abnormalities were observed in stillborn and dead pups.
Scheduled necropsies: No abnormality was observed in pups selected for necropsy of all dose groups.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

food consumption and compound intake

organ weights and organ / body weight ratios

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

For males, one or two animals were observed with dehairing and scar, abdominal distension or emaciation in the control, low- and mid- dose groups, but that were not considered to be treatment-related clinical signs.
For females, dehairing, and soiled perineal region were observed on one or two animals in the different period, and one animal in the low-dose group was observed with tumor, but the incidence of these signs had no significant increase in all dose groups, so that were not considered to be treatment-related clinical signs.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

One female (No. 2723) of the high-dose group was found dead on GD17, at necropsy, embryos were found in its uterus, and no apparent cause of death was found. Two males (No. 1408 and 1424) of the control group and one male (No. 1706) of the high-dose group were found dead in the premating period, at necropsy, thymus enlargement was found in rat 1408, and no obvious abnormality was found in rat 1424 and 1706.
The mortality of male and female animals in the high-dose group had no significant increase compared with the control group, so that result was considered to be an incidental result without toxicological significance.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Premating period
For males, no significant difference was observed on the mean bodyweight and body weight gain in all dose groups compared with the control group (p>0.05). For females, no significant difference was observed on the mean bodyweight in all dose groups compared with the control group (p>0.05). In the 5th, 7th, 10th and11th test weeks, the body weight gain in one or more dose groups had a statistically significant decrease or increase compared with the control group (p<0.05 or p<0.01), but the total body weight gain during the whole premating period in each dose group had no significant difference compared with the control group (p>0.05), so that was considered to be an incidental result without toxicological significance.
Mating period
For males, no significant difference was observed in all dose groups compared with the control group (p>0.05).
Gestation period
No significant difference was observed in all dose groups compared with the control group (p>0.05).
Lactation period
No significant difference on the mean body weight and body weight change of the parental females was observed in all dose groups compared with the control group (p>0.05).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Premating period
For males, the mean food consumption from the 6th test week to the 10th test week in the mid- and high- dose groups, and in the 8th test week in the low-dose group had a statistically significant increase compared with the control group(p<0.05 or p<0.01), at the same time, the total food consumption in the premating period had a statistically significant increase in the mid- and high- dose groups compared with the control group(p<0.01), and had no statistically significant difference in the low-dose (p>0.05).
For females, the mean food consumption in some test weeks had a statistically significant increase in the mid- and/or high- dose groups compared with the control group(p<0.05 or p<0.01), but no statistically significant increase on the total food consumption in the premating period was observed in these groups compared with the control group(p>0.05).
Gestation period
No statistically significant difference on the mean food consumption of the pregnant females was observed during the gestation period in all dose groups compared with the control group (p>0.05).
Base on the results about the food consumption above, the animals had a tendency to eat more after the exposure of test item at the dose of 1000 and 200mg/kg.d, but at the same time, no adverse effect on the body weight in each dose group was observed, so these results were considered without toxicological significance.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Premating period
For males, the decrease of mean food efficiency was observed in the high-dose group from the 8th to the 11th test week (p<0.05 or p<0.01), and in the mid-dose group in the 8th and 11th test week (p<0.05 or p<0.01) compared with the control group, at the same time, the average food efficiency in the whole premating period had a statistically significant decrease in the high-dose group compared with the control group(p<0.01), and had no statistically significant change in the other two dose groups. These decreases of the food efficiency were considered to be related with the increase of the food consumption.
For females, an increase or decrease of mean food efficiency was observed in one or more test weeks in all dose groups (p<0.05 or p<0.01), but the average food efficiency in the whole premating period had no statistically significant difference compared with the control group (p>0.05), these results were considered to be incidental without toxicological significance.

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

For the males of F1 generation, no statistically significant difference was observed on the day of achievement of preputial separation and the mean body weight on this day in all dose groups compared with the control group (p>0.05).
For the females of F1 generation, no statistically significant difference was observed on the day of achievement of vaginal opening and the mean body weight on this day in all dose groups compared with the control group (p>0.05).

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant difference compared with the control group was observed on organ weight:
Both of the absolute and relative pituitary weights in males of the low-dose group were statistically significant decreased compared with the control group (p<0.05 or p<0.01). The absolute pituitary weights in females of the mid- and high- dose groups were statistically significantly increased (p<0.05 or p<0.01), at the same time, the relative pituitary weight in females of the mid-dose group was statistically significantly increased (p<0.05).
The absolute liver weights in males and females of all dose groups were comparable with the control group, but the relative liver weight in males of the low-dose group was statistically significantly increased (p<0.05).
Both of the absolute and relative thyroid weights in males of the high-dose group and in females of the mid-dose group were statistically significantly increased (p<0.01).
Both of the absolute and relative spleen weights in males of the mid-dose group were statistically significantly increased (p<0.05).
The absolute brain weights in females of the mid- and high- dose groups were statistically significantly increased (p<0.05), but their relative brain weights were comparable with the control group, this was considered to be of no toxicological relevance.
The organ weights of the other organs in males and females of all dose groups were comparable with the control group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Scheduled necropsies
No treatment-related changes were found in males and females of all dose groups.
Intercurrent dead animals
One female (No. 2723) of the high-dose group was found dead on GD17, at necropsy, uterus enlargement with fetuses was observed; two males (No. 1408 and 1424) of the control group were found dead in the premating period, at necropsy, thymus enlargement was observed in rat 1408 and no obvious abnormality was observed in rat 1424; one male (No. 1706) of the high-dose group was found dead in the premating period, at necropsy, no obvious abnormality was observed because of autolyze.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Scheduled necropsies
No treatment-related changes were found in males and females of high-dose group.
Based on the macroscopic findings, all gross lesions were examined microscopically. No abnormality in histopathology was observed in the lesions of testis small and epididymis small.
In males of all dose groups that failed to become sire, prostate inflammatory cell infiltrate was observed in a few animals. In females that were non-mated or non-pregnant, ovary follicular cyst, decreased number/absent corpora lutea and uterus squamous metaplasia etc. were observed in some animals. No significant difference on the incidence of these abnormalities was observed in all dose groups compared with the control group (p>0.05), so that was considered not to be treatment-related.
Intercurrent dead animals
In F1 generation, the microscopic examination was conducted in four intercurrent dead animals, thymus hyperemia was observed in rat 1408, and ovary pigment was observed in rat 2723. No abnormality was observed in the other animals.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Estrus Cyclicity
No differences were observed on all estrus cyclicity parameters in each dose group compared with the control group (p>0.05).
Fertility
26, 28, 28 and 27males in the control, low-, mid- and high- dose groups and 28females in each group were put together as one to one ratio for mating, and the mating period lasted for two weeks. 22, 24, 23 and 23males in each group were found to mate with female, and 19, 20, 21 and 17 males became sire in the control, low-, mid- and high- dose groups, respectively. The mating index and fertility index of males had no statistically significant difference in all dose groups compared with the control group (p>0.05).
27, 25, 25 and 24 females were found to mate with male, and 24, 21, 22 and 20 females were pregnant in the low-, mid- and high- dose groups, respectively. The mating index, fertility index and fecundity index of females had no statistically significant difference in all dose groups compared with the control group (p>0.05). No difference was observed on precoital interval in all dose groups compared with the control group (p>0.05).
Pregnancy and Delivery Data
24, 21, 22 and 19 pregnant females survived delivery to obtain a litter with liveborn pups in the control, low-, mid- and high- dose groups, respectively. One female in the high-dose group was dead during the delivery. The gestation index had no statistically significant difference in all dose groups compared with the control group (p>0.05).
The mean gestation length in the mid-dose group had a significant extension compared with the control group (p<0.05), but that was within the range expected for SD rats, so this result was considered as an incidental result without toxicological significance. No statistically significant difference was observed on gestation length in the other two dose groups compared with the control group (p>0.05).

Sperm Analysis
- Epididymal sperm motility and count:
No statistically significant difference on the total count of epididymal sperm was observed in all dose groups compared with the control group (p>0.05).
Statistically significant decreases on the VAP, straight line velocity (VSL), curvilinear velocity (VCL), area of sperm motility in the low-dose group, and VSL, VCL, area of sperm motility in the mid-dose group were observed (p<0.01 or p<0.05). No statistically significant difference on all sperm motility parameters was observed in the high-dose group compared with the control group (p>0.05). No similar results were observed in F0 generation, at the same time, all the results of these parameters were absence of a dose relation among the dose groups, so it was considered the decreases on several sperm motility parameters in the low- and mid- dose groups were not an adverse effect of the treatment of the test item.
- Epididymal sperm morphology:
No significant effect on the epididymal sperm morphology was observed in the examined males of all dose groups.
- Testicular sperm count:
No statistically significant difference on the testicular sperm count was observed in all dose groups compared with the control group (p>0.05).

Details on results (F1)

Litter Data
All pregnant females with successful delivery obtained a litter with at least one liveborn pups, two females in the control group and one female in the mid-dose group delivered a litter with stillborn pup. One female in the control group (No. 2402) and one female in the high-dose group (No. 2721) had complete litter loss during the lactation period.
No statistically significant difference was observed on the mean number of corpora lutea and implantation sites in all dose groups compared with the control group (p>0.05). No difference was observed on the mean number of pups delivered and liveborn in all dose groups (p>0.05). The pre-implantation loss had a statistically significant decrease (p<0.05 or p<0.01) in the mid- and high- dose groups, but without toxicological significance. No statistically significant difference was observed in the low-dose group (p>0.05). No statistically significant effect on the post-implantation loss was observed in all dose groups (p>0.05).
Pup Data
- Pup status: There were 2, 0, 1 and 0 stillborn pups in the control group and low-, mid-, high- dose groups, respectively. No statistically significant effects on the live birth index was observed in all dose groups (p>0.05).1, 3, 2 and 0 pups respectively in the control, low-, mid- and high- dose groups were dead in the lactation period. No statistically significant effect on the viability index of lactation was observed in all dose groups (p>0.05).
- Mean number of pups: The mean number of live pups on PND0 had no statistically significant difference in all dose groups compared with the control group(p>0.05), at the same time, no statistically significant difference on the sex ratio at birth was observed in all dose groups(p>0.05). The mean number of live pups on the other time of the lactation period, including PND4, PND7, PND14 and PND21 had no statistically significant difference in all dose groups compared with the control group(p>0.05). No statistically significant difference on the number of dead pups during the lactation period was observed in all dose groups (p>0.05).
- Pup clinical observations: During the lactation period, one litter including eight pups in the control group were observed with hypothermia on PND2, and was missing later; one litter including six pups in the mid-dose group were observed with sparse fur from PND14 to PND18, and recovered later. No pup with any abnormality and sign was observed in the other groups. All signs observed were considered as an incidental result without toxicological significance.
- Pup body weights: The mean body weights of live pups in all dose groups had no statistically significant difference during the lactation period compared with the control group (p>0.05).
- Organ Weight of Pups: The absolute brain weights in female pups of all dose groups were statistically significantly increased (p<0.05 or p<0.01), but their relative brain weight was comparable with the control group, this was considered to be of no toxicological relevance.
- Macroscopic Observations of Pups
Stillborn and dead pups: No abnormalities were observed in stillborn and dead pups.
Scheduled necropsies: No abnormality was observed in pups selected for necropsy of all dose groups.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

food consumption and compound intake

organ weights and organ / body weight ratios

other: Reproductive and developmental toxicity

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

During the lactation period, one litter including eight pups in the control group were observed with hypothermia on PND2, and was missing later; one litter including six pups in the mid-dose group were observed with sparse fur from PND14 to PND18, and recovered later. No pup with any abnormality and sign was observed in the other groups. All signs observed were considered as an incidental result without toxicological significance.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weights of live pups in all dose groups had no statistically significant difference during the lactation period compared with the control group (p>0.05).

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The absolute brain weights in female pups of all dose groups were statistically significantly increased (p<0.05 or p<0.01), but their relative brain weight was comparable with the control group, this was considered to be of no toxicological relevance.

Details on results (F2)

Macroscopic Observations
- Stillborn and dead pups: No abnormalities were observed in stillborn and dead pups.
- Scheduled necropsies: No abnormality was observed in pups selected for necropsy of all dose groups.

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Result tables are provided in "Statistic data table" attached to the section of Attached background material below.

Individual data are provided in "Individual data appendix" attached to the section of Attached background material below.

Pathology detail results are provided in "Pathology Report" attached to the section of Attached background material below.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the two-generation reproduction toxicity study in rats, the No Observed Adverse Effect Level (NOAEL) for parental toxicity to males and females, for exposure to the test substance by gavage, is considered to be 1000 mg/kg.d. The NOAEL for reproductive toxic effects to parental males and females, for exposure to the test item by gavage, is considered to be 1000 mg/kg.d. The NOAEL for developmental toxic effects to pups, after parental exposure to the test substance by gavage, is considered to be 1000 mg/kg.d.

Executive summary:

A Two-generation reproduction toxicity study in rats was conducted on the test substance according to OECD Guideline 416.

In this study, twenty-eight rats/sex/group were exposed by gavage to 0, 40, 200 and 1000 mg/kg.d the test substance respectively. In F0 generation, males and females were exposed to the test item for at least ten weeks prior to mating, no longer than two weeks of the mating period; after that, males were killed for sperm analysis and necropsy, and females were exposed continuously during the gestation and lactation period until all pups were weaned. 28 rats/sex/group were selected from these pups as F1 generation. In F1 generation, males and females were exposed to the test item at the same doses with their parents from PND22, including the premating period of at least 10 weeks, mating period of at most 2 weeks, gestation and lactation period (for females), the males were killed for sperm analysis and necropsy after mating period, and the females were killed for necropsy after all pups were weaned. In two generations, one male and one female pup of each litter as possibly were selected for necropsy.

During the test, no treatment-related clinical signs and death of the parental animals in two generations was found at any dose level.

In two generations, no significant adverse effect on the body weight of parental males and females was observed during each period. At the same time, based on the results about the food consumption, the animals had a tendency to eat more after the exposure of test item at the dose of 1000 and 200mg/kg.d, but these results were considered without toxicological significance.

For the parental animals of two generations, no treatment-related effect was observed on all reproductive parameters, including the mating index, fertility index of males and females, pregnancy and delivery results and litter related results, etc. Additionally, estrus cyclicity and sperm parameters were not adversely affected by exposure to the test item in both generations.

For the pups of two generations, no treatment-related effect was observed on all developmental parameters, including the live birth index and the viability index of lactation, the mean number and body weight of live pups during lactation period. Additionally, no treatment-related clinical signs and abnormalities were observed in all pups. For the male and female pups of F0 generation selected for mating, the sexual maturation was not adversely affected in all dose groups.

At the necropsy of all parental animals and pups, no treatment-related gross change was observed; in microscopic examination, no toxicologically relevant change was observed in the reproductive organs of the examined parental animals. At the same time, no treatment-related effect was observed on the absolute and relative organ weights of all parental animals and pups selected for necropsy.

Under the conditions of the two-generation reproduction toxicity study in rats, the No Observed Adverse Effect Level (NOAEL) for parental toxicity to males and females, for exposure to the test item by gavage, is considered to be 1000 mg/kg.d. The NOAEL for reproductive toxic effects to parental males and females, for exposure to the test item by gavage, is considered to be 1000 mg/kg.d. The NOAEL for developmental toxic effects to pups, after parental exposure to the test item by gavage, is considered to be 1000 mg/kg.d.