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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 Oct, 2003 - 21 May, 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Deficiencies: Yes F0 animals: the day 11 samples taken for stability in closed bags and open feeders, were actually sampled on day 12 by error, males D26143 and D26154 given 1500 ppm were weighed again on day 17 due to aberrant values on day 15, for females D26364, pup No. 5 and D26393, pup No. 10 (given respectively 500 and 1500 ppm), macroscopic examination was performed although not scheduled in the study plan (data not presented in the study report), Male D26146 (given 1500 ppm): left testis and epididymis were mislaid before microscopic examination. F1 animals: according to the Study plan, the dosing period was 20 weeks. However, the study was finished in week 18, the dietary admixtures were analyzed in week 18 rather than week 20, as specified in the Study plan, because the study finished early, female D26509 given 4000 ppm and females D26439 and D26446 given 0 ppm were mated one extra day by error (the females had been positive for mating the previous day), during the mating periods (F0 and F1 animals), the food consumption was not recorded (typing error in the final study plan), organ weights with aberrant values have been excluded from mean calculation: thyroid glands of male D26210 (given 500 ppm), adrenal glands of male D26232 (given 1500 ppm) and pituitary gland of male D26234 (given 1500 ppm), male D26226 (given 500 ppm): dilated renal pelvis was observed for left kidney at macroscopic examination but no microscopic examination was performed, male D26262 (given 4000 ppm): adrenal glands were not found for the preparation of slides, female D26457 (given 500 ppm): pituitary gland was mislaid after weighing, female D26493, pup No. 4 (given 1500 ppm) found dead on day 2 post-partum was mislaid before macroscopic examination. These deviations were considered not to have compromised the validity or integrity of the study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Didecyldimethylammonium chloride
EC Number:
230-525-2
EC Name:
Didecyldimethylammonium chloride
Cas Number:
7173-51-5
Molecular formula:
C22H48N Cl
IUPAC Name:
N,N-Didecyl-N,N-dimethylammonium chloride
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Species: Rat
Strain: Sprague-Dawley, Crl CD® (SD) IGS BR
Source: Charles River Laboratories France, L’Arbresle, France.
Sex: 100 males and 100 females
Age/weight at study initiation: At the beginning of the treatment period, the animals were approximately 5 to 6 weeks old and had a mean body
weight of 137 g (range: 104 g to 168 g) for the males and 118 g (range: 96 g to 139 g) for the females.
Number of animals per group: 25 males and 25 females
Duration of mating: 2 weeks
Deviations from standard protocol: Female D26509 given 4000 ppm and females D26439 and D26446 given 0 ppm were mated one extra day
by error (the females had been positive for mating the previous day).
Control animal: Yes

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Administration/ Exposure: Oral
Duration of exposure before mating: 10 weeks
Duration of exposure in general P, F1, F2 males, females From beginning of the study until sacrifice of Parent, F1, F2-generation
Type: oral In food
Concentration Food consumption per day: 0, 500, 1500 and 4000 ppm ad libitum, for groups 1, 2, 3 and 4, respectively.
Vehicle: The test item was mixed in the powdered maintenance diet UAR A04C P2.5 (UAR, Villemoisson, Epinay-sur-Orge, France).
Controls: Vehicle
Details on mating procedure:
Males and females were paired for, at most, a 2-week period, until mating was obtained.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Doses were verified for test substance concentrations in regular intervals during the study.
Duration of treatment / exposure:
Exposure: Premating exposure period (males): 10 weeks
Premating exposure period (females): 10 weeks
Duration of test: F0 pre-mating 10 weeks, until F2 weaning. (Continuously)
Frequency of treatment:
Continuously
Details on study schedule:
F0 generation: 10 weeks before mating, during the mating period until sacrifice (weaning of pups) for the males, 10 weeks before mating, during the mating, pregnancy and lactation periods (until day 21 post-partum) for the females.

A group of 25 males and 25 females received untreated diet alone, under the same experimental conditions, and acted as a reference control group. 

F1 generation: At weaning of the F1 generation, on day 22 post-partum, three groups of 25 male and 25 female Sprague-Dawley rats received the same test item, under the same experimental conditions as above, during their growth, adulthood, mating, pregnancy and lactation, until weaning of the pups (F2 generation).

A group of 25 males and 25 females received untreated diet alone under the same experimental conditions and acted as a reference control group. 
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
500, 1500, and 4000 ppm of test substance (40% active DDAC) (i.e., corresponding to 203, 608 and 1620 ppm of DDAC)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
14, 39 and 109 mg a.i./kg bw/day for males and 18, 51 and 137 mg a.i./kg bw/day in females
Basis:
nominal in diet
No. of animals per sex per dose:
25 per sex per group
Control animals:
yes, concurrent vehicle
Details on study design:
Refer to study design above
Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
Examination of F0 generation: Clinical signs and mortality were checked daily. Food consumption and body weight were recorded at designated intervals.  Males and females were paired for, at most, a 2-week period, until mating was obtained.  The F0 females were allowed to deliver normally, and rear their progeny. Pregnancy and litter parameters were recorded. The F0 parent males and females were sacrificed after weaning of their progeny. During lactation, the pups from the F1 generation were observed daily for survival and clinical signs. Body weight was measured at designated intervals and the sex-ratio was recorded. On day 4 post partum, the size of each litter was adjusted to obtain eight pups per litter (four males and four females). Reflex development was assessed at designated endpoints.
Oestrous cyclicity (parental animals):
Refer to study design
Sperm parameters (parental animals):
Refer to study design
Litter observations:
Examination of F1 generation; On day 22 post-partum, one male and one female pup per litter were selected to constitute the F1 generation, which comprised 25 males and 25 females per group. The F1 animals were observed daily for clinical signs and mortality. Body weight and food consumption were recorded once a week. Sexual development of both males and females was assessed. Neurobehavioral tests were conducted at designated intervals to assess auditory and visual function. Spontaneous locomotor activity was also evaluated twice over a 60-min interval when the animals were 7 and 8 weeks old. When the animals were 12 weeks old, F1 males and F1 females were paired.
The F1 females were allowed to deliver normally, and rear their progeny. Pregnancy and litter parameters were recorded. During lactation, the pups from F2 generation were observed daily for survival and clinical signs; body weight was recorded at designated intervals; the sex-ratio was recorded. On day 4 post partum, the size of each litter was adjusted to obtain eight pups per litter (four males and four females).
Reflex development was assessed at designated endpoints.
Postmortem examinations (parental animals):
Terminal examination of F0 and F1 animals: After weaning of their respective progeny, F0 and F1 parent males and females were sacrificed. Designated organs were weighed for F0 and F1 parents as well as brain, spleen and thymus of one pup per sex per litter of each generation. Epididymal and testicular sperm parameters were evaluated in both F0 and F1 males.
A macroscopic post-mortem examination was performed on all F0 and F1 parent males and females and on three pups per sex and per litter of each F0 and F1 female killed at weaning.  Any pups which died or were killed prematurely during the lactation period were also submitted for macroscopic post-mortem examination.  Macroscopic lesions, reproductive organs, adrenals and pituitary glands were sampled in all parent animals. In all pups, the macroscopic lesions were preserved. A microscopic examination was performed on macroscopic lesions, reproductive organs, adrenals, and pituitary glands of all F0 and F1 parents of the control and high dose groups. A detailed histopathological examination was performed on the ovaries and the testes.
Postmortem examinations (offspring):
Terminal examination of F0 and F1 animals: After weaning of their respective progeny, F0 and F1 parent males and females were sacrificed. Designated organs were weighed for F0 and F1 parents as well as brain, spleen and thymus of one pup per sex per litter of each generation. Epididymal and testicular sperm parameters were evaluated in both F0 and F1 males.
A macroscopic post-mortem examination was performed on all F0 and F1 parent males and females and on three pups per sex and per litter of each F0 and F1 female killed at weaning.  Any pups which died or were killed prematurely during the lactation period were also submitted for macroscopic post-mortem examination.  Macroscopic lesions, reproductive organs, adrenals and pituitary glands were sampled in all parent animals. In all pups, the macroscopic lesions were preserved. A microscopic examination was performed on macroscopic lesions, reproductive organs, adrenals, and pituitary glands of all F0 and F1 parents of the control and high dose groups. A detailed histopathological examination was performed on the ovaries and the testes.
Statistics:
No data
Reproductive indices:
Please refer to study design for F0 animals
Offspring viability indices:
Please refer to study design for F1 animals

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
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:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

F0 generation
Test group 4 (4000 ppm): - no treatment-related deaths and no remarkable clinical signs,  - for the males, a lower body weight gain during the premating period (days 1-15), and food consumption was affected throughout the study,  - for the females, a lower body weight gain was noted during the first week of the premating period, during the gestation and the lactation, and food consumption was affected from week 2 of the study, but no effect was detected during the lactation, - there were no apparent effects on mating, fertility, fecundity or delivery,  - the pup body weight gain was significantly lower than controls from day 4 p.n.,  - no disturbances of the reflex development in the pups,  - none of the seminology parameters evaluated in F0 parents males were affected,  - distension of cecum with feces was seen at the autopsy in 13/25 F0 males, with no histopathological findings in this organ,  - higher adrenal weights (p<0.01) in F0 females correlated with enlargement of the gland and cortical cell hypertrophy seen at histopathology, - lower spleen weights in male and female pups sacrificed at weaning (p<0.01),  - no treatment-related findings were observed in male and female genital organs, and the accessory organs.          
Test group 3 (1500 ppm): - no treatment-related deaths, - no remarkable clinical signs, - neither body weight gain nor food consumption of parent animals were affected, - there were no apparent effects on mating, fertility, fecundity or delivery, - no effect on the pup body weight gain, - no disturbances of the reflex development in the pups, - none of the seminology parameters evaluated in F0 parents males were affected, - no treatment-related differences in organ weights were recorded, - neither macroscopic nor microscopic findings were observed.
Test group 2 (500 ppm): - no treatment-related deaths, - no remarkable clinical signs, - neither body weight gain nor food consumption of parent animals were affected, - there were no apparent effects on mating, fertility, fecundity or delivery, - no effect on the pup body weight gain, - no disturbances of the reflex development in the pups, - none of the seminology parameters evaluated in F0 parents males were affected, - no treatment-related differences in organ weights were recorded, - neither macroscopic nor microscopic findings were observed.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
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:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

F1 generation
Test group 4 (4000 ppm): - no treatment-related deaths, - no remarkable clinical signs, - for the males, there was a lower body weight gain until day 43 of the premating period and food consumption was affected, mainly during the same period of the study, - for the females, a lower body weight gain was noted during the first 2 weeks of the premating period, from GD 7 (GD: gestation day) and during the lactation. The food consumption was affected during the premating period, during GD 0-14 and during days 7 14 p.p., - there were no apparent effects on mating, fertility, fecundity or the progress of delivery,  - the pup body weight gain was significantly lower than controls during the lactation, - no disturbances of the reflex development in the pups, - none of the seminology parameters evaluated in F1 parents males were affected, - none of the organ weight differences recorded in F1 parents were directly treatment-related, - lower spleen weights were recorded in F1 pups, - no relevant findings were seen at necropsy of parents or pups, - neither macroscopic nor microscopic findings were observed.
Test group 3 (1500 ppm): - no treatment-related deaths, - no remarkable clinical signs, - neither body weight gain nor food consumption of parent animals were affected, - there were no apparent effects on mating, fertility, fecundity or the progress of delivery, - increase in the male ratio, - no effect on the pup body weight gain, - no disturbances of the reflex development in the pups, - none of the seminology parameters evaluated in F1 parents males were affected. - no treatment-related differences in organ weights were recorded, - neither macroscopic nor microscopic findings were observed.
Test group 2 (500 ppm): - no treatment-related deaths, - no remarkable clinical signs, - neither body weight gain nor food consumption of parent animals were affected, - there were no apparent effects on mating, fertility, fecundity or the progress of delivery, - increase in the male ratio, - no effect on the pup body weight gain, - no disturbances of the reflex development in the pups, - none of the seminology parameters evaluated in F1 parents males were affected. - no treatment-related differences in organ weights were recorded, - neither macroscopic nor microscopic findings were observed.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 1 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Results: F2 generation

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
ca. 4 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects on mating behaviour, fertility and gestation, of each generation and for development, growth and survival of each progeny

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Result: Not toxic to reproduction
concentrations of 500, 1500 or 4000 ppm test substance,
corresponding to 203, 608 or 1620 ppm of DDAC.

The estimation of the achieved dosages of test item
(expressed as mg of DDAC/kg/day) are summarized in the
following table:
Mean achieved dosages (in mg DDAC/kg/day)
Concentration (ppm)         500         1500         4000
F0 generation
Males
. premating (days 1-71)         17         49         137
. post-mating (days 85-120)     10         30         84
Females
. premating (days 1-71)         20         58         157
. gestation (GD 0-20)          15         45         52
. lactation (days 1-21 p.n.)    31         93         261
F1 generation
Males
. premating (days 1-64)         22         65         183
. post-mating (days 85-120)     10         31         94
Females
. premating (days 1-64)         23         69         198
. gestation (GD 0-20)          17         48         57
. lactation (days 1-21 p.n.)    36         93         263




Applicant's summary and conclusion

Conclusions:
Under the study conditions, the NOAEL for parental toxicity was 1500 ppm (39-51 mg a.i./kg bw/day) for the male and female animals. The NOAEL for mating behaviour, fertility and gestation of each generation and for development, growth and survival of each progeny was 4000 ppm (109-137 mg a.i./kg bw/day).
Executive summary:

A two-generation study was conducted to determine the toxicity to reproduction of the test substance, DDAC (40% active in water) according to OECD Guideline 416, in compliance with GLP. In this study, the substance was administered in the diet to male and female Sprague-Dawley rats at dose levels of 0, 500, 1500 and 4000 ppm (equivalent to an average of 14, 39 and 109 mg a.i./kg bw/day for males and 18, 51 and 137 mg a.i./kg bw/day in females, calculated from the mean achieved dose levels in the parental generation). Doses were administered before and throughout mating and gestation until the end of the lactation period in both F0 and F1 generations. At 4000 ppm, F0 and F1 parents showed significantly lower body weight gains and reduced food consumption. At 1500 or 500 ppm, no relevant changes were noted. Treatment with the test substance had no effect on the reproductive parameters in F0 and F1 parental rats at treatment levels up to 4000 ppm. No effect was observed on mating, fertility, gestation, fecundity or delivery at any concentration for either generation. No effect was recorded on litter parameters and on pre- and post-natal development of either generation at any concentration. Apart from cortical cell hypertrophy found in the adrenal glands of F0 females treated at 4000 ppm and lower spleen weight in F1 pups of the same group, no other treatment-related findings were seen upon microscopic examination of the concerned tissues. Consequently, under the study conditions, the NOAEL for parental toxicity was 1500 ppm (39-51 mg a.i./kg bw/day) for the male and female animals. The NOAEL for mating behaviour, fertility and gestation of each generation and for development, growth and survival of each progeny was 4000 ppm (109-137 mg a.i./kg bw/day) (Chevalier, 2008).