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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Title:
Unnamed
Year:
1999

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD TG 421
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Dicyclohexylamine
EC Number:
202-980-7
EC Name:
Dicyclohexylamine
Cas Number:
101-83-7
IUPAC Name:
N-cyclohexylcyclohexanamine
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
dicylohexylamine, purity: 99.89 %

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:Charles River Japan
- Age at study initiation: 8 weeks
- Weight at study initiation: 306 – 335 g for males and 201 – 224 g for females
- Fasting period before study: N/A
- Housing: , the animals were kept separate in bracket-type wire mesh cages (W 410 × D 350 × H 170 mm: Lead Engineering Co., Ltd.) divided in two. A total of two animals, one male and one female, were housed together in the aforementioned cages during the mating period and the dam and pups were housed together in plastic econ cages (W 340 × D 400 × H 185 mm: Clea Japan) with a floor covering (white flakes: Charles River Japan) for the period from 17 days of gestation through four days of lactation.
- Diet (e.g. ad libitum): ad libitum solid food NMF (irradiation-sterilized and autoclave-sterilized)
- Water (e.g. ad libitum): ad libitum tap water (Gotenba Municipal Waterworks water: automated water supply equipment used).
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3°C
- Humidity (%): 50 ± 20%,
- Air changes (per hr): of 10 - 15 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours (7:00 a.m. to 7:00 p.m.).

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water):concentrations of the corn oil solution were stable after being stored for eight days refrigerated (approximately 4°C) away from the light followed by being stored at room temperature away from the light for 24 hours
- Concentration in vehicle:0, 4, 8 and 16 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg
- Lot/batch no. (if required): chemical grade, Nacalai Tesque, lot number: V7R2020
- Purity: Chemical grade
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In formulating the test solution, the necessary amount of the test substance was measured for each concentration, it was dissolved in corn oil (chemical grade, Nacalai Tesque, lot number: V7R2020), and the specified amount was formulated. Test solution formulation was performed at a frequency of once per week as a general rule because it was confirmed at the Bozo Research Center that the test substance 4 and 40 mg/mL concentrations of the corn oil solution were stable after being stored for eight days refrigerated (approximately 4°C) away from the light followed by being stored at room temperature away from the light for 24 hours (Attached Data 2). After preparation, the daily amounts of the test solution were dispensed into brown glass bottles and stored refrigerated (approximately 4°C) away from light.
The percentage of the indicated value was always within the range of 97 – 101% and the concentrations were appropriate when each concentration of the test solution was confirmed at the Bozo Research Center using two administrations, one from before the start of administration to males and females and one in the final week of administration to males.
Details on mating procedure:
After having administered the test substance for the 14-day pre-mating period, males and females of the same group were housed together overnight in one-on-one pairs. The cohabitation period lasted until copulation was confirmed, up to 14 days at most. The formation of a copulatory plug or the confirmation of sperm in the vaginal smear was regarded as confirmation of copulation and this day was considered ‘Day 0’ of gestation.
Duration of treatment / exposure:
Exposure period: males: 49 days; females from 14 days before mating to day 3 of lactation
Premating exposure period (males): 14 days
Premating exposure period (females): 14 days
Duration of test: males: 50 days, females: day 4 post partum
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
80 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 (twelve)
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for dosages
The dosages were determined using the results of the preliminary repeated dose 2-week oral administration study (Bozo Research Center, test number: U-1417, dosage: 0, 20, 40, 80, and 160 mg/kg) as a reference. Two of the five males and four of the five females in the 160 mg/kg dosage group died. There were no cases of death in the 80 mg/kg dosage group, but salivation was observed in both males and females in the clinical sign observations. No obvious changes in weight or food consumption were observed in the males of that group, but inhibited weight growth and a decrease in food consumption were observed in the females. However, no effects from administration of the test substance were observed on the female estrous cycle, necropsy findings, or organ weight. No obvious effects from the administration of the test substance were observed in the 20 or 40 mg/kg dosage groups other than the salivation that was observed in the 40 mg/kg dosage group. Consequently, the high dosage in this study was set at 80 mg/kg, at which effects on female body weight and food consumption was observed but at which there were no cases of death and the intermediate and low dosages were set at 40 and 20 mg/kg by dividing by a common ratio of two.

For the assignment of animals to each group, the animals selected by the criteria shown in ‘Section 2. Test animals’ were stratified by weight on the day of grouping (administration start day) and this was conducted through a combination of a block placement method and a random sampling method (the necessary groups were configured with the block placement method and the test groups and the individual numbers in the groups were allotted randomly) such that each group’s average weight was as equal as possible.

62 males and 62 females were purchased at seven weeks old, and after one week of quarantine and habituation, 48 males with no abnormal clinical indications and that showed good weight gain were selected, and after obtaining vaginal smears for all of the females for five days from the day after transport and observing the estrous cycles, 48 females that indicated normal estrous cycles and that had no abnormal clinical indications and showed good weight gain, just like with the males, were selected and administration was started at eight weeks old. The weight range at the start of administration was 306 – 335 g for males and 201 – 224 g for females. The surplus males after the groups were formed and the females with abnormal estrous cycles were euthanized under deep ether anesthesia after the group formation was completed and the other females were put down in the same manner after the mating period was ended.

Examinations

Maternal examinations:
Females (P)
(1) Clinical sign observation
Clinical sign observation was conducted on the outer body surface, nutrition status, and behavior three times a day, pre-administration, immediately post-administration, and two hours after administration. However, the observation on Saturdays and holidays occurred only pre-administration and immediately post-administration.

(2) Body weight measurement
Weight was measured between 8:30 a.m. – 12:30 p.m. during the pre-mating period before administration at one day, then at four days, eight days, 11 days, and 15 days, followed by at zero days, seven days, 14 days, and 21 days of gestation during the gestation period, followed by at zero days and four days of lactation during the lactation period.

(3) Food consumption measurement
For food consumption, the remaining food was measured between 8:30 a.m. – 12:30 p.m. on the same days that weight was measured during the pre-mating period, then at one day, seven days, 14 days, and 21 days of gestation during the gestation period, followed by at one day and four days of lactation during the lactation period, and daily food consumption was calculated from the difference from the previous day’s food consumption.

(4) Vaginal smear examination
Vaginal smears were obtained from all females during the pre-mating administration period until copulation was established and were examined microscopically. The vaginal smear images during the pre-mating administration period were classified into proestrus, estrus, postestrus, and anestrous, and the frequency of estrus stage images and the number of days from one estrus stage until the next estrus stage (estrous cycle) were examined. The presence of sperm in the vaginal smear was checked during the mating period.

(5) Delivery and nursing observations
The dams delivered naturally and the presence of any delivery abnormalities was observed. Confirmation of delivery completion was conducted every day from 21 days through 23 days and if delivery had ended by 10:30 a.m., that day was considered ‘Day 0’ of lactation. If delivery occurred after 10:30 a.m., the following day was considered ‘Day 0’ of lactation. For dams for which a completed delivery had been confirmed, they were left to care for their pups until Day 4 of lactation and their lactation condition was observed using nest building, retrieving, and lactation as the indicators.

(6) Necropsy
After the weight of all cases was measured at four days of lactation, the subjects were exsanguinated through the abdominal aorta under ether anesthesia, necropsied, the internal organs and tissue were observed macroscopically, and the number of corpora lutea and the number of implantation sites were checked. In addition, the organs and tissue in which abnormalities were observed in the necropsy were fixed in a 10% formalin solution in a phosphate buffer and preserved. The two cases (No. 4102, 4110) from the 80 mg/kg dosage group that died during gestation and the six cases from the same group (No. 4101, 4106, 4108, 4109, 4111, 4112) in which all of the pups were stillborn or all of the pups died during the lactation period were necropsied in the same manner when they were discovered.

(7) Histopathological testing
The ovaries of all cases were embedded in paraffin, segments were prepared for the control group and the high dosage group, hematoxylin-eosin staining was performed, and they were examined microscopically.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data
- Other:
Fetal examinations:
1) Pup observation
At zero days of lactation, we counted the number of live born and the number of stillborn, and observed the existence of external malformations. Pups with external malformations were fixed in a 10% formalin solution in a phosphate buffer and preserved. The gender of the live born pups was determined and their weight was measured, then they were cared for by the dam. The stillborn pups were also fixed in a 10% formalin solution in a phosphate buffer and preserved.
(2) Nursling observation and measurement
Nurslings were observed once a day each day to determine whether they were alive or dead. Weight was measured at zero days (lactation Day 0) and at four days after birth. At four days after birth, all pups were exsanguinated under ether anesthesia, necropsies were performed, and the presence of internal organ abnormalities was checked.

Statistics:
Statistical analysis was performed using the following methods and the two-sided significance level was 5 and 1%. For the delivery rate, birth rate, and viability rate, the mean value and the rate were obtained for each dam and compared. In addition, the data of one female from the 20 mg/kg dosage group for which incorrect administration was thought to have occurred was excluded from the copulation rate and the fertility rate data and was excluded from the count.
1) Multiple comparison tests
A variance uniformity assay was performed for each group using the Bartlett method. As a result, when the variance was uniform, a one-way analysis of variance was performed and if a significant difference between groups was observed, a paired comparison test was performed for the control group and each administration group using the Dunnett method. If the variance was not uniform, a Kruskal-Wallis rank test was performed, and if it was significant, a Dunnett’s test was performed for the difference in the average of the ranks for the control group and each administration group.
Weight, food consumption, frequency of estrus images, estrous cycle, number of days of cohabitation, gestation period, organ weight, number of corpora lutea, number of implantation sites, number of live born pups, sex ratio, implantation rate, delivery rate, birth rate, viability rate

2) x2 test
Copulation rate, fecundation rate, delivery rate

3) Mann-Whitney U test
This was performed for the frequency of occurrence and intensity of degree for findings in the histopathological testing.

Indices:
Gender ratio = Males/ (males + females)
Viability rate (%) = (number of surviving pups at Day 4 of lactation/number of live born pups at Day 0 of
lactation) × 100
Delivery rate (%) = (Total number of pups delivered/number of implantation sites) × 100
Birth rate (%) = (number of surviving pups at Day 0 of lactation/total number of pups delivered) × 100
Implantation rate (%) = (number of implantation scars/number of corpora lutea) × 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
80 mg/kg: salivation, unkempt fur, prone position, oligopnea, hypothermia, and poor pup retrieval
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
80 mg/kg: 2 females GD 21/22
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Weight gain was inhibited in the 80 mg/kg dosage group and a significant difference was observed between that group and the control group in the measurement values at four days, eight days, and 11 days, as well as at 21 days of gestation and at four days of lactation
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption in the 80 mg/kg dosage group was lower than in the control group and significant differences were observed between the two groups throughout the study
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
80 mg/kg- poor pup retrieval
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
80 mg/kg: A diaphragmatic hernia and dark red spots in the glandular stomach were observed in one case
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
control and 80 mg/kg: only the ovaries examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
see attachment
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
see attachment
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
see attachment
Early or late resorptions:
no effects observed
Description (incidence and severity):
see attachment
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
80mg/kg live birth index = 58.5% +- 38.9
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): see attachment
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
In females (P), one case died at 21 days of gestation and one died at 22 days of gestation in the 80 mg/kg dosage group. In addition, unkempt fur was recorded in the case that died at 22 days of gestation and a prone position, oligopnea, hypothermia, and poor pup retrieval were recorded in one case after delivery had been completed at 22 days of gestation. Unkempt fur and poor retrieval were observed as clinical sign changes during the lactation period in that group. Furthermore, weight gain was inhibited and low values were indicated for weight gain and food consumption in the pre-mating administration period, the gestation period, and the lactation period. However, no effects were observed in the necropsy findings or the histopathological test findings of the ovaries in that group due to administration of the test substance. No effects on clinical signs, weight, food consumption, or necropsy findings were observed in the groups with dosages of 40 mg/kg or lower.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
dead fetuses
mortality

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
80 mg/kg- m/f significantly lower at PND0
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
80mg/kg live birth index = 58.5% +- 38.9
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
no treatment effect on Delivery Index, body weight significantly reduced in 80mg/kg vs controls.
Changes in postnatal survival:
effects observed, treatment-related
Description (incidence and severity):
80 mg/kg viability index = 20.8% vs 99% for controls
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Many dams with poor pup retrieval were observed during lactation in the 80 mg/kg dosage group and only four dams had surviving pups up to four days of lactation. However, lactation abnormalities were not observed in the groups with dosages of 40 mg/kg or lower. At the same time, no effects were observed on the number of dams with live born pups, the gestation rate, the number of corpora lutea, the number of implantation sites, or the implantation rate.
A significantly lower number of viable pups and a significantly lower birth rate were observed. However, no effect was observed on the number of live born pups, the number of stillborn pups, or on the gestation rate due to administration of this test substance in the groups with dosages of 40 mg/kg or lower. In addition, no effect was observed on the delivery rate in any of the groups due to administration of the test substance.
No effect was observed on the sex ratio of pups due to administration of the test substance and external malformations caused by administration of the test substance were not observed. There were many pup deaths in the 80 mg/kg dosage group during the lactation period and the viability rate in that group was remarkably low. However, no effect was observed on the viability rate of groups with dosages of 40 mg/kg or lower due to administration of the test substance. Pup weight was low for both males and females of the 80 mg/kg dosage group at zero days and four days after birth, but no effect on weight was observed in the groups with dosages of 40 mg/kg or lower due to administration of the test substance. No abnormalities thought to be caused by administration of the test substance were observed in the necropsies of the pups at four days after birth.


Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in litter size and weights
changes in postnatal survival

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
80 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

With respect to developmental toxicity:
findings which differ from controls:
low - mid - high dose versus control
---gestation index
100% - 100% - 90% versus 100%
1/10 surviving high dose dam without pups  
---live birth index 
99.5% - 99.0% - 58.5%(p<0.01) versus 94.9% 
---viability index 
99.4% - 97.8% - 20.8%(p<0.01) versus  99.0% 
---body weight of pups on day 0
male: 7.0g, 6.8g, 6.1g(p<0.01) versus 7.3g
female: 6.7g, 6.5g, 5.5g(p<0.01) versus 7.0g 
---body weight on day 4
male: 11.3g, 11.0g, 9.0g versus 11.8g
female: 10.6g, 10.4g, 8.6g versus 11.1g

Applicant's summary and conclusion

Conclusions:
Many dams with poor pup retrieval were observed in the 80 mg/kg dosage group during lactation and effects were observed on pup survival and development due to administration of the test substance. Consequently, the reproductive development no observed effect level was determined to be 40 mg/kg/day for females, and 40 mg/kg/day for pups.
Executive summary:

A preliminary reproduction toxicity screening test of dicyclohexylamine was performed at dosages of 0 (control group), 20, 40, and 80 mg/kg with 12 males and 12 females Sprague-Dawley SPF rats [Crj; CD (SD)] per group in which it was administered to males for a 14-day pre-mating period and through the mating period until the day before necropsy and to females for a 14-day pre-mating period, through the mating period, the gestation period, and delivery until Day 3 of lactation.

 

In males (P), inhibited weight growth was observed at 80 mg/kg. However, no effects on clinical signs, food consumption, necropsy findings, testes or epididymis weight or the histopathological findings of these organs were observed in that group due to administration of the test substance. At the same time, no effects on clinical signs, weight, food consumption, necropsy findings, testes or epididymis weight were observed in the groups with dosages of 40 mg/kg or lower.

In females (P), one case died at 21 days of gestation and one died at 22 days of gestation in the 80 mg/kg dosage group. In addition, unkempt fur was recorded in the case that died at 22 days of gestation and a prone position, oligopnea, hypothermia, and poor pup retrieval were recorded in one case after delivery had been completed at 22 days of gestation. Unkempt fur and poor retrieval were observed as clinical sign changes during the lactation period in that group. Furthermore, weight gain was inhibited and low values were indicated for weight gain and food consumption in the pre-mating administration period, the gestation period, and the lactation period. However, no effects were observed in the necropsy findings or the histopathological test findings of the ovaries in that group due to administration of the test substance. No effects on clinical signs, weight, food consumption, or necropsy findings were observed in the groups with dosages of 40 mg/kg or lower.

 

In the 80 mg/kg dosage group, two females died, inhibited weight gain was observed in males and females, and decreased food consumption was observed in females. Consequently, we determined the general toxicity no observed effect level for males and females to be 40 mg/kg/day.

 

Looking at the parent animal (P) reproductive development toxicity, no effects on the female estrous cycle were observed due to administration of this test substance, nor were any effects observed for male and female copulation or fertility, or for the gestation period. In addition, delivery abnormalities were not observed in the control group or in any of the test substance administration groups. Many dams with poor pup retrieval were observed during lactation in the 80 mg/kg dosage group and only four dams had surviving pups up to four days of lactation. However, lactation abnormalities were not observed in the groups with dosages of 40 mg/kg or lower. At the same time, no effects were observed on the number of dams with live born pups, the gestation rate, the number of corpora lutea, the number of implantation sites, or the implantation rate.

A significantly lower number of viable pups and a significantly lower birth rate wereobserved. However, no effect was observed on the number of live born pups, the number of stillborn pups, or on the gestation rate due to administration of this test substance in the groups with dosages of 40 mg/kg or lower. In addition, no effect was observed on the delivery rate in any of the groups due to administration of the test substance.

No effect was observed on the sex ratio of pups due to administration of the test substance and external malformations caused by administration of the test substance were not observed. There were many pup deaths in the 80 mg/kg dosage group during the lactation period and the viability rate in that group was remarkably low. However, no effect was observed on the viability rate of groups with dosages of 40 mg/kg or lower due to administration of the test substance. Pup weight was low for both males and females of the 80 mg/kg dosage group at zero days and four days after birth, but no effect on weight was observed in the groups with dosages of 40 mg/kg or lower due to administration of the test substance. No abnormalities thought to be caused by administration of the test substance were observed in the necropsies of the pups at four days after birth.

 

No effects were observed on the estrous cycle, copulation, or fertility, or in the gestation period or delivery in any of the administration groups due to administration of the test substance. However, many dams with poor pup retrieval were observed in the 80 mg/kg dosage group during lactation and effects were observed on pup survival and development due to administration of the test substance. Consequently, the reproductive development no observed effect level was determined to be 80 mg/kg/day for males, 40 mg/kg/day for females, and 40 mg/kg/day for pups.