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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 1999-06-16 to 1999-12-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A Calco (Italy)
- Age at study initiation: 8 weeks old
- Weight at study initiation: males: 225-250 g, Females: 200-225 g
- Fasting period before study: Not documented
- Housing: The rats were divided by sex and kept in Makrolon cages measuring 425 x 266 x 150mm, type 3D-Tecniplast Gazzada s.r.l., Buguggiate - Varese, each fitted with stainless steel cover-feed rack. During pre-mating, 2 males and 2 females were housed together. During the mating period, 1 male and female were housed together while during pregnancy and lactation, the females were housed individually. Dust free poplar/fir wood chips, heat processed for resin removal were used for bedding.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2°C
- Humidity (%): 55±15%
- Air changes: 15-20 air changes/hour
- Photoperiod: 12hrs dark / 12 hrs light

IN-LIFE DATES: From: June 16th 1999 To: July 25th 1999.

Administration / exposure

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

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil for solubility reasons
- Concentration in vehicle: 0, 6.25, 12.5 and 50 mg/ml
- Amount of vehicle: the volume administered was 10 ml/kg bw/day
Every day, appropriate amounts of the test substance were weighed , mixed in corn oil, magnetically stirred for few minutes and warmed up to 50-60°c for 15-20 minutes. Then the formulates were magnetically stirred again to obtain final suspensions at the final concentrations of 6.25, 12.5 and 50 mg/ml. Formulates were kept under magnetic stirring until the end of daily administration. All formulates were administered within 4 hours of the preparation.
Details on mating procedure:
- M/F ratio per cage: one male with one female
- Length of cohabitation: 7 evenings/week for a maximum of 14 evenings for 16 hours at a time
- Proof of pregnancy: sperm in vaginal smear] referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test substance were checked on two occasions with a two phase titration method according to ISO 2271.
Actual concentrations of 6.2, 13.2, 55.1 mg/ml for the first assay and 6.4, 13.4, 53.8 mg/ml for the second assay were found for the respective nominal concentrations of 6.25, 12.5 and 50 mg/ml.
Duration of treatment / exposure:
From 14 days prior to mating until a maximum dosing period of 28 days for males.
From 14 days prior to mating, during mating, pregnancy and up to day 3 of lactation for females. Females with positive smear who did not give birth were treated until killing (Day 25 of presumed pregnancy). Females without positive smear were treated up until 25 days after the end of the mating period.
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 62.5, 125 and 500 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale::
Dosage were selected on a basis of a maximum tolerated dose study in male and female rats (no further details available)
Positive control:
no

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: recorded daily

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed on a weekly basis during the pre-mating and mating periods. During the pregnancy period, the females were weighed on days 0, 7, 14 and 20 and during the lactation on days 0 (parturition day), and day 4.

FOOD CONSUMPTION :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Durign the pre-mating period of the male and female F0 generation, food was distributed in weighed amounts. The leftover amounts of the weighed food allocated to each cage were recorded once a week in order to calculate food consumption in g/rat/day. Food consumption was then recorded on days 7, 14 and 20 during pregnancy and on day 4 of lactation.

WATER CONSUMPTION: No data

Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Parameters examined in male parental generations:
In all males: testis weight, epididymis weight,
In the control and high dose-groups: histology with special emphasis on stages of spermatogenesis and histology on intersticial cell structure.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.



Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were killed the day after the end of treatment.
- Maternal animals: All surviving animals were killed on day 4 of lactation with their pups.

GROSS NECROPSY
Gross pathology examination was performed.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The ovaries and uteri (including horns, cerfix and vagina) testes, epididymides, accessory sex organs were weighed and then fixed in formalin (ovaries and uteri) or in Bouins fluid for histology

- The uteri of apparently non-pregnant females were stained using the method of Salewski and examined for the presence of implantation sites.

- Additional sections of testes were stained with PAS- hematoxylin to allow spermatogenesis to be classified into 14 stages, each stage based primarily upon the changes of the acrosome and head morphology of the younger generation of spermatids.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.

GROSS NECROPSY
- Gross necropsy consisted of external examinations

Statistics:
- The probablility of survival per group was determined using the product-limit procedure of Kaplan-Meyer. The Log-Rank test was applied in order to detect differences in survival among groups. A trend test was also applied.
- The following tests were used for the purpose of comparing treated and control groups:
To compare frequencies, the heterogeneity test (CHI square 2xN) and Fischers exact test were applied. The Trend test was also applied.
To compare the mating distribution during the time allowed to the F0 generation for mating, the Log-rank test was applied.
All the other data were compared with ANOVA and Dunnett's test (when necessary) for homogeneous variance and kruskal-Wallis non parametric ANOVA for not homogeneous variance.
Reproductive indices:
Mating index (F0): percent ratio of animals with positive smears plus females found to be pregnant but without positive smears to the animals mated.

Fertility index (F0): percent ratio of females having evident signs of pregnancy to females having positive vaginal smears plus females found to be pregnant but without positive vaginal smears.

Pregnancy index (F0): the precent ratio of females with live births to the pregnant females.

Pre-coital interval (F0): calculated on the dams which proved pregnant and was expressed for each group as the mean time lapse (in days) between the beginning of the mating period and the ascertainment that copulation had occurred.

Pregnancy period (F0): the duration of pregnancy was determined for all those dams that reached term of pregnancy as being the time that elapsed between the day vaginal smear proved positive and the day of parturition.

Post implantation losses: No. implantations-No. live pups / No. implantations x 100.
Offspring viability indices:
Birth Index: No. of live newborns at birth/No. of implantations x 100.

Viabililty index on Day 4: No. of live pups on day 4 after birth / No. of live births x 100.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
only high dose
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
only high dose
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
only high dose
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Description (incidence and severity):
Test substance intake: not applicable for gavage administration

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
only high dose

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
* At 500mg/kg/day:
- One female and one male died after 12 and 10 treatments respectively.
- All females treated with 500mg/kg/day showed dyspnea, loss of weight and soft stools after a few treatments. One female also displayed chromadacryorrhea and 5 females also showed slight to moderate dilation of the abdomen. Seven of the ten males at 500 mg/kg/day showed dyspnea and soft stools.
* At 125 and 62.5 mg/kg/day, there were no clinical signs or deaths.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- Pre-mating period:
* At 500mg/kg/day:
The body weight of the treated males and females decreased significantly in the first week of treatment. In the second week of treatment the body weight remained significantly lower than controls in the case of males and lower without reaching significance level in females. Consequently, body weight gain during the whole pre-mating period was lower in the case of females and significantly decreased in the case of males.
The mean daily food consumption of the treated males was significantly lower during the pre-mating period. The mean daily food consumption of the treated females was slightly lower than that of the control females, but without reaching significance.
* At 125 and 62.5 mg/kg/day, there were no effects on the body weight and food consumption during the pre-mating period.

- Pregnancy period:
* At 500mg/kg/day:
The mean body weight of the treated female was still lower than that of the control females during the pregnancy period, reaching significance level on days 14 and 20.
The mean daily food consumption of the 500mg/kg/day treated group was significantly lower only in the first week of pregnancy.
* At 125 and 62.5 mg/kg/day, there were no effects on the body weight and food consumption during the pre-mating period.

- Lactation period:
* At 500mg/kg/day:
The mean body weight of the treated females was still significantly lower than that of the control group on day 0 of lactation.
The mean daily food consumption of the treated female was slightly lower than that of the control group without reaching significance.
* At 125 and 62.5 mg/kg/day, there were no effects on the body weight and food consumption during the lactation period.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
No applicable.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Not examined.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Not examined.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
* At 500mg/kg/day:
The mating and fertility indices were slightly lower than those of the control group. From the 9 paired females 7 revealed to be sperm positive (77 %) after mating, 6 out of 9 (67 %) revealed to be pregnant, and 5 out of 6 (83 %) delivered live litters. One animal revealed to have fully resorbed. Mean pre-coital time was longer in this group (about 6.1 days) when compared to the controls and the lower dosage groups (1.5 to 2.1 days).No effects on parturition were found at any dose. The pregnancy length was similar in all experimental groups. The mean value per litter of live born was slightly lower in the 500 mg/kg/day treated group when compared to controls.
* At 125 and 62.5 mg/kg/day, there were no effects on reproductive performance.


ORGAN WEIGHTS (PARENTAL ANIMALS)
* At 500mg/kg/day:
For males, a lower absolute weight of epididymides was found at 500mg/kg/day which, in the absence of histopathological changes was considered to be of low toxicological significance.
* At 125 and 62.5 mg/kg/day, there were no effects on organ weights.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No effects mentioned.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histology of F0 testes, epididymides and ovaries did not show any compound-related changes. In particular, no changes were seen in the testicular stages performed in the PAS-hematoxylin stained sections.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
only high dose
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Description (incidence and severity):
not applicable in this type of test
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
* At 500mg/kg/day:
The birth index was statistically decreased compared to controls. The percentage of post implantation losses was increased to 19% per litter in comparison to about 6% per litter in the controls thus resulting in a lower rate of live borns of 83% in comparison to 94% in the controls.
Viability index on postnatal day 4 was in the range of those of the controls.
* At 125 and 62.5 mg/kg/day, there were no effects on the viability.

CLINICAL SIGNS (OFFSPRING)
No effects reported.

BODY WEIGHT (OFFSPRING)
No statistically significant differences were found between the body weight of the treated pups and the control pups during the 4 days of lactation.

SEXUAL MATURATION (OFFSPRING)
not applicable

ORGAN WEIGHTS (OFFSPRING)
Not examined

GROSS PATHOLOGY (OFFSPRING)
No effects reported.

HISTOPATHOLOGY (OFFSPRING)
Not examined.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: At the higher dose-level of 500 mg/kg bw (which is also the highest dose of the study), the live birth index was statistically decreased compared to controls (83% versus 94%).

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under these experimental conditions, , the NOAEL for the dams and their offspring was determined to be 125 mg/kg/day.
Executive summary:

The objective of this study was to evaluate the potential reproductive/developmental toxicity of dioctadecyldimethylammonium chloride (96.8% active) according to a GLP conform Reproduction/Developmental Toxicity Screening Test (OECD Guideline 421).

Groups of 10 rats (CRL:CD (SD) BR) per sex were treated with dosages of 0, 62.5, 125, and 500 mg/kg bw/day by gavage (administration volume 10 ml/kg bw/day) using corn oil as a vehicle for the control group. Males were treated daily from two weeks before mating, during mating and until a dosing period of a total of 28 days had been completed. Females were treated daily from two weeks before mating until the 4 th day of lactation. Subsequently these females weresacrificedwith their pups.

At daily doses of 500 mg/kg bw one male and one female died after 12 and 10 treatments respectively. Clinical observations revealed dyspnea, soft stools in all females and almost all males. Half of the females also showed slight to moderate dilation of the abdomen. Body weight loss of about 14 to 15 g was observed in both sexes during the first week of treatment. Further, statistically significantly lower mean daily food consumption was observed in the males during the premating period and in the dams during the first week of pregnancy. Statistically significantly lower mean dam body weights were observed after 14 and 20 days of gestation and at the day of birth after delivery.

No toxicologically relevant effects were observed at dosages of 125 and 62.5 mg/kg bw/day.

At sacrifice of the parental animals no significant differences were found in the organ weights of uterus, ovaries, testes and epididymides. Histopathology of testes, epididymides and of the ovaries of the animals of the 500 mg/kg dose groups did not show any compound related changes. No substance related changes were reported for the evaluation of testicular stages of spermatogenesis performed in the PAS-hematoxylin stained sections.

At the dosages of 62.5 and 125 mg/kg/day all of the 10 paired females revealed to be sperm positive after mating, all revealed to be pregnant and all delivered live litters. The numbers of corpora lutea had not been evaluated during this study.

At 500 mg/kg/day, from the 9 paired females 7 revealed to be sperm positive (77 %) after mating, 6 out of 9 (67 %) revealed to be pregnant, and 5 out of 6 (83 %) delivered live litters. One animal revealed to have fully resorbed. Mean pre-coital time was longer in this group (about 6.1 days) when compared to the controls and the lower dosage groups (1.5 to 2.1 days).

After birth, for the animals treated with 62.5 or 125 mg/kg/day there were no substance related biological differences in their pregnancy outcome in comparison to the control group. At 500 mg/kg/day, the percentage of post implantation losses was increased by 19% per litter in comparison to about 6% per litter in the controls and in the lower treatment groups, thus resulting in a statistically significantly lower rate of live borns of 83% in comparison to 94% in the controls and in the lower treatment groups. Viability index on postnatal day 4 was in the range of the controls and the lower treatment groups.

For all dose groups under investigation no statistically significant differences were found for the body weights of male and female pups at birth and on postnatal day 4. External abnormalities have not been reported.

Based on the findings of this study, it was concluded that the No-observed-adverse-effect level (NOAEL) regarding reproductive toxicity was 125 mg/kg bw/day.