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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 22 November 1993 to 01 August 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Physical state: Colourless liquid
- Analytical purity: 30.2% aqueous solution
- Lot/batch No.: PN-93-12
- Storage condition of test material: In the dark at ambient temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, England
- Age at study initiation: (P) 4 weeks; (F1) 25 d
- Weight at study initiation: (P) ca. 90 g
- Housing: Initially 2 per cage in popypropylene cages with stainless steel grid bottoms, mesh tops and food hoppers (42 x 27 x 20 cm). 3 d prior to mating the males were transferred to individual cages. For mating the females were transferred to the cage of the appropriate co-group male. Mated females were transferred to individual solid bottomed cages of the same dimensions, where they remained with the litters until termination. F1 animals selected as parents for the next generation were housed 2 per grid bottomed cage, sexes separated, and then followed the same caging regime as P animals.
- Diet: Rat and Mouse Breeder Diet No. 3 (Expanded) SQC, ad libitum
- Water: Domestic mains water, ad libitum
- Acclimation period: 13 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2
- Humidity (%): 50 ± 15
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared at intervals of up to 1 week. For the high dose concentration, the test substance was weighed into a labelled dose container and the requisite quantity was added and mixed by inversion as required. For the lower dose level, formulation was by serial dilution of the next highest concentration. A quitable aliquot of each formulation was dispensed to the animal room for dosing on that day. Solutions were used within 8 days of preparation.

VEHICLE
- Concentration in vehicle: 0, 1, 3 and 8 mg/mL (for 30.2% aqueous solution of the test substance)
- Amount of vehicle (if gavage): 10 mL/kg
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Until mating or 7 nights elapsed
- Proof of pregnancy: Vaginal plug or sperm in vaginal smear, referred to as Day 0 of pregnancy
- After 7 d of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged (how): Individual solid bottomed cages (42 x 27 x 20 cm)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On 6 occasions during the study, triplicate samples of dosing solutions were taken and analysed for concentration and homogeneity. The method of analysis was reported the provision of IRI Project 370340.
Duration of treatment / exposure:
Parental: 10 weeks prior to mating to weaning of F1 generation
F1: From Day 25 after birth to weaning of F2 generation.
Frequency of treatment:
Daily
Details on study schedule:
F1 parental animals not mated until 11 weeks after weaning
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 10, 30 and 90 mg/kg bw/day (30.2% aqueous solution)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 3, 9 and 27 mg/kg bw/day (pure substance)
Basis:
actual ingested
No. of animals per sex per dose:
28/sex for parental generation and 24/sex for F1 generation
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on evaluation of existing data.
- Rationale for animal assignment (if not random): Computer generated randomly sequenced number, but ensuring that siblings were not placed in the same treatment group.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily, plus at the beginning of each day and as late as possible for viability.

BODY WEIGHT: Yes
- Time schedule for examinations: One week prior to commencement of treatment, then on each day of dosing.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was recorded weekly for each animal, commencing 1 week prior to treatment. Food consumption monitoring was suspended during the mating period and then, for males, recommenced as before. For females, consumption was measured over Days 0-7, 7-14 and 14-20 of gestation, and Days 0-7 and 7-14 of lactation.

Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED
The following parameters were examined in F1/F2 offspring: Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical abnormalities

GROSS EXAMINATION OF DEAD PUPS: Yes where appropriate, for external and internal abnormalities.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, termination after weaning of the litters
- Maternal animals: All surviving animals, termination after weaning of the litters

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cranial, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were weighed: Ovaries, uterus, cervix and vagina, testes (weighed individually), epididymides (weighed individually), seminal vesicle, coagulating gland, prostate gland, pituitary gland. The female reproductive tract was examined for signs of pregnancy and the number of visible implantation sites was recorded.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 7 weeks of age without necropsy.

GROSS NECROPSY
Offspring found dead or killed before Day 14 of lactation were sexed, examined for the externally visible abnormalities and for the presence of milk in the stomach. Offspring dying on or after Day 14 were subjected to a gross necropsy, in which the cranial, thoracic and abdominal contents were examined macroscopically. From each litter of F1 and F2 pups, 2 male and 2 female pups were necropsied at weaning. The necropsy consisted of an external examination, followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities. The remaining pups (except F1 weanlings selected for rearing to produce the next generation) were killed after external examination and the carcasses discarded without necropsy.
Statistics:
Where considered appropriate to assist with interpretation, statistical analysis was applied to determine the statistical significance of differences from control. Organ weight data were analysed by analysis of variance. Testes and epididymides weights were also analysed by analysis of covariance using the terminal body weight as a single covariate. For prostate and seminal vesicles weights, tests for linearity of relationship to body weight and for homogeneity of slopes indicated that analysis of covariance was inappropriate for these organs. Treatment means were compared using an F-protected Least Significant Difference procedure (Snedecor and Cochran, 1980). For other parameters, interpretation was based on examination of the individual and group values.
Reproductive indices:
The following reproductive indices were calculated for each group: Fertility index and gestation index
Offspring viability indices:
The following offspring viability indices were calculated for each litter and group: Birth index, live birth index, viability index, lactation index and overall survival index.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

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

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
At 27 mg/kg bw/day, most animals of both generations showed signs of reaction to treatment, consisting of dyspnea, piloerection and hunched posture, and many animals also had episodes of post-dosing salivation. For occasional animals, the outline of the spine was prominent. A total of 8 animals died or were killed after showing marked signs of reaction, and a 9th death may also have been related to treatment.
At 9 mg/kg bw/day occasional animals in both generations showed post dosing salivation; this observation was probably associated with treatment.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In both generations, at 27 mg/kg/day, mean weight gain was markedly lower than control; this effect was apparent for males, and for females in the premating period and during gestation.

FOOD CONSUMPTION (PARENTAL ANIMALS)
Food consumption in both generations was slightly lower than in controls at 27 mg/kg bw/day.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating performance, fertility, duration of gestation, litter size and pup survival were considered to be similar in all groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Mean seminal vesicle weights at 27 mg/kg bw/day in both generations were significantly lower than controls, however, it was considered that this reduction was an indirect effect of the lower body weights, rather than a direct effect on the seminal vesicles.
At 27 mg/kg bw/day, mean epidymides weights in both generations were lower than control, with the value for F0 animals attaining statistical significance. However, after adjustment for bodyweight (covariance analysis) the epididymides weights were similar to control in both generations. Mean epididymides weights at the lower levels were not obviously affected by treatment.
Mean absolute testes weights in both generations at 90 mg/kg/day were slightly lower than control, with the value for F1 males attaining statistical significance. These lower values were considered to reflect the lower body weights, and after adjustment for bodyweight (covariance analysis) mean values were similar to control. Testes weights at 3 and 9 mg/kg/day were similar to control.
Mean prostate weights were essentially similar in all groups of both generations.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
9 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Based on observed signs of toxicity and a marked reduction in food consumption and body weight gain of males and of females during the premating and gestation periods at 27 mg/kg bw/day.
Remarks on result:
other: Generation: P and F1 (migrated information)
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
27 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No adverse effects on fertility up to the highest tested dose.
Remarks on result:
other: Generation: P and F1 (migrated information)

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

CLINICAL SIGNS (OFFSPRING)
Two pups at 27 mg/kg bw/day showed body tremors in late lactation; these tremors may have been associated with treatment.

BODY WEIGHT (OFFSPRING)
At 27 mg/kg bw/day, mean litter and pup weights of the F2 pups were slightly lower than controls. Although these differences were probably incidental, the possibility that they were related to treatment could not be entirely discounted. The litter and pup weights of the F1 pups at 27 mg/kg bw/day and of both generations at 3 and 9 mg/kg bw/day were similar to controls.

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity / systemic toxicity
Generation:
F2
Effect level:
9 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Based on slightly reduced pup and litter weights and observed clinical signs (2 pups with body tremors) at 27 mg/kg bw/day.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The study was performed in accordance with the guideline specified.
The performance of the 2 generations was very similar.
Under the conditions of the study 30 mg/kg/day produced only minor parental toxicity but no reproductive effects.
At 90 mg/kg/day there was very marked parental toxicity, a possible minor effect on pup and litter weights, and 2 pups with body tremors. There were no obvious adverse effects on mating and littering performance at any of the levels tested.
Executive summary:

An oral two-generation study was conducted to assess the reproductive toxicity potential of N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine in rats according to the OECD Guideline 416 and in compliance with GLP.

 

Sprague Dawley rats were dosed orally by gavage, once daily at dose levels, 0, 10, 30 and 90 mg/kg/day (i.e., equivalent to 0, 3, 9 and 27 mg a.i./kg bw/day). F0 animals were randomised into the 4 treatment groups, each containing 28 males and 28 females. From each treatment group, 24 male and 24 female F1 weanlings were selected for rearing to maturity and mating to produce the F2 generation. The F0 animals were dosed for 10 weeks prior to mating, and then throughout the mating, gestation and lactation periods until sacrifice at the time of weaning of the F1 animals. The F1 animals were exposed to possible effect of the test substance from conception through to weaning. Clinical observations were performed daily. Body weight and food consumption were recorded at various intervals throughout the study. Females were allowed to litter normally and observations on the females and litters were recorded. All F0 and F1 animals were subjected to necropsy, consisting of an external examination, followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities. Gross lesions were monitored and applicable organs were weighed. Only 2 male and 2 female pups that were weaned from each litter underwent necropsy. No histopathology was performed as this was available from a 90-day repeat oral dose study.

 

Toxicity to parent (adult) animals at 27 mg a.i./kg bw/day was indicated in both generations by a marked reduction in body weight gain of males, and of females during the pre-mating and gestation periods. Food consumption at this level was slightly lower than control. Additionally most animals in both generations receiving 27 mga.i./kg bw/day showed clinical signs of reaction to treatment (principally dyspnoea, piloerection and hunched posture, many animals also had episodes of post dose salivation, and for occasional animals the outline of the spine was prominent). A total of 8 animals (2 F0 males, 3 F0 females, 1 F1 male and 2 F1 females) died or were killed after showing marked signs of reaction, and a ninth death (of an F1 female) may also have been related to treatment. At 9 mga.i./kg bw/day, the only finding that was considered to have been probably associated with treatment was occasional animals in both generations with post dosing salivation; this finding might not be indicative of systemic toxicity.

Mean seminal vesicle weights in both generations at 27 mga.i./kg bw/day were significantly lower than control; it was considered that this reduction was an indirect effect of the lower body weights rather than a direct effect on the seminal vesicles.Mating performance, fertility, duration of gestation, litter size and pup survival were considered to be similar in all groups of both generations. At 27 mga.i./kg bw/day, mean litter weight and pup weights of the F2 pups were slightly lower than control; although these differences were probably incidental, the possibility that they indicated a slight effect of treatment could not be entirely discounted. The litter and pup weights of the F1 pups at 27 mga.i./kg bw/day, and of both generations at 3 and 9 mga.i./kg bw/day, were similar to control. Two pups at 27 mga.i./kg bw/day showed body tremors in late lactation; these may have been associated with treatment. At the 3 mga.i./kg bw/day dose level no marked toxicity was noted.

 

Hence, under the conditions of the study, the NOAEL for reproductive toxicity was established at 27 mg a.i./kg bw/day and the NOAEL for parental and developmental toxicity was established at the next tested lower dose level of 9 mg a.i./kg bw/day.