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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was performed accoding to the OECD 416 in compliance with GLP, with few deviations that 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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
containing ca. 50% C12-16-benzyldimethylammonium chloride (CAS no.: 68424-85-1) in water only.
Specification: C12-16 BKC (C12: 72.0%; C14: 27.7%; C16: 0.3%)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female

Administration / exposure

Route of administration:
oral: feed
Duration of treatment / exposure:
Exposure period: about 18 wk.
(F0 and F1) Premating exposure period (males): 10 wk.
Premating exposure period (females): 10 wk.
Duration of test: F0 pre-mating 10 wk, until F2 weaning.
Frequency of treatment:
Continuously
Doses / concentrations
Remarks:
Doses / Concentrations:
1000, 2000 and 4000 ppm of test substance.
Basis:

No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle

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 a 2-wk period, until mating was obtained. The F0 females were allowed to deliver normally, and rear their progeny. Pregnancy and litter parameters were recorded.
During lactation, the pups (F1 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
time-points.
Sperm parameters (parental animals):
Epididymal and testicular sperm parameters were evaluated for both F0 and F1 males.
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.
Neurobehavioural tests were conducted at designated intervals to assess auditory and visual functions. Spontaneous
locomotor activity was also evaluated when the animals were between 7 and 8 wk old. After sexual maturity, F1 male and F1 female animals were
paired. The F1 females were allowed to deliver normally, and rear their progeny. Pregnancy and litter parameters were recorded.
During lactation, the pups (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 time-points.
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 for 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 females killed at weaning. Any pups which died or were killed prematurely during the lactation period were also submitted for a 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.
Particularly detailed histopathological examinations were performed for the ovaries and the testes.

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

Result: Not toxic to reproduction
F0 and F1 generations. At 4000 ppm (corresponding to approximately 123-208 mg/kg bw/d for F0 males and females and to 202-252 mg/kg bw/d for F1 males and females, respectively), a slightly to moderately lower mean food consumption and mean body weight gain were recorded during most of the dosing period in both parental males and females of the two generations and was associated with reduced liver weights. Necropsy of these animals (parents of both generations) revealed dilation of the cecum, colon or ileum in some animals (more marked in F0 parents). No effects were noted on sperm parameters or on histopathological examination of sexual organs. Slightly lower pup body weight was observed for each progeny and was associated, for the F2 generation pups, with a reduction in litter size (as a consequence of lower number of implantation sites of F1 parent females) and a delay in sexual development. Lower spleen weights were also noted for each progeny.
At 2000 ppm (corresponding to approximately 61-101 mg/kg bw/d for F0 males and females and 96-123 mg/kg bw/d for F1 males and females, respectively), a marginally to slightly lower mean food consumption and body weight gain were noted over all the dosing period for the males of the F0 generation and in both sexes for the F1 generation. Necropsy of parents of both generations revealed dilatation of the cecum in some animals of the F0 generation and in a single animal of the F1 generation. This was associated with lower liver weights in parental animals of both generations. No effects were noted on parental fertility as assessed by normal mating, gestation and delivery and, particularly, there were no effects on sperm parameters or at histopathological examination of sexual organs. Except for a marginally lower spleen weight of the progeny of each generation, no other effects were noted on their development.
At 500 ppm (corresponding to approximately 16-25 mg/kg bw/d for F0 males and females and 24-31 mg/kg bw/d for F1 males and females, respectively), a marginally to slightly lower mean food consumption and body weight gain were noted over all the dosing period for the males of the F0 generation and in both sexes for the F1 generation. This was associated in the F1 generation with lower liver weights of parental males and females. No effects were noted on mating, fertility, gestation, fecundity or delivery of either generation or on development of their progeny. As lower food consumption is known to occur due to palatability of compound. As no other substance related effects were seen than can be attributed to lower food intake, the level of 500 ppm test substance in the diet, corresponding to 16-25 mg/kg bw/d, should be regarded as NOAEL for parent and F1generation.

At 4000 ppm, number of implantation sites and litter size at birth were reduced. The progeny (F2) also showed lower pup weights. No effects were seen in F2 offspring at 2000 ppm regarding pup development and survival until weaning, and macroscopic examination after sacrifice at weaning. At the highest level of 4000 ppm pup weight gain was slightly lower during lactation, and upon necropsy dilatation of the caecum with faeces was observed in 4/25 males and 2/25 females. Therefore, 4000 ppm can be regarded as LOAEL, and 2000 ppm as NOAEL for F2.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
16 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The effect level ranged from 16 to 25, due to lower body weight gains and reduced food consumption and/or necropsy findings at higher doses.
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Effect level:
61 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the effect observed on mating, behaviour, fertility at the highest dose. The effective dose ranged from 61 to 101 mg/kg bw/day (nominal) (i.e., equivalent to 30.5 to 50.5 mg a.i./kg bw/d.

Results: F1 generation

Details on results (F1)

Result: Not toxic to reproduction
F0 and F1 generations. At 4000 ppm (corresponding to approximately 123-208 mg/kg bw/d for F0 males and females and to 202-252 mg/kg bw/d for F1 males and females, respectively), a slightly to moderately lower mean food consumption and mean body weight gain were recorded during most of the dosing period in both parental males and females of the two generations and was associated with reduced liver weights. Necropsy of these animals (parents of both generations) revealed dilation of the cecum, colon or ileum in some animals (more marked in F0 parents). No effects were noted on sperm parameters or on histopathological examination of sexual organs. Slightly lower pup body weight was observed for each progeny and was associated, for the F2 generation pups, with a reduction in litter size (as a consequence of lower number of implantation sites of F1 parent females) and a delay in sexual development. Lower spleen weights were also noted for each progeny. At 2000 ppm (corresponding to approximately 61-101 mg/kg bw/d for F0 males and females and 96-123 mg/kg bw/d for F1 males and females, respectively), a marginally to slightly lower mean food consumption and body weight gain were noted over all the dosing period for the males of the F0 generation and in both sexes for the F1 generation. Necropsy of parents of both generations revealed dilatation of the cecum in some animals of the F0 generation and in a single animal of the F1 generation. This was associated with lower liver weights in parental animals of both generations. No effects were noted on parental fertility as assessed by normal mating, gestation and delivery and, particularly, there were no effects on sperm parameters or at histopathological examination of sexual organs. Except for a marginally lower spleen weight of the progeny of each generation, no other effects were noted on their development. At 500 ppm (corresponding to approximately 16-25 mg/kg bw/d for F0 males and females and 24-31 mg/kg bw/d for F1 males and females, respectively), a marginally to slightly lower mean food consumption and body weight gain were noted over all the dosing period for the males of the F0 generation and in both sexes for the F1 generation. This was associated in the F1 generation with lower liver weights of parental males and females. No effects were noted on mating, fertility, gestation, fecundity or delivery of either generation or on development of their progeny. As lower food consumption is known to occur due to palatability of compound. As no other substance related effects were seen than can be attributed to lower food intake, the level of 500 ppm test substance in the diet, corresponding to 16-25 mg/kg bw/d, should be regarded as NOAEL for parent and F1generation. At 4000 ppm, number of implantation sites and litter size at birth were reduced. The progeny (F2) also showed lower pup weights. No effects were seen in F2 offspring at 2000 ppm regarding pup development and survival until weaning, and macroscopic examination after sacrifice at weaning. At the highest level of 4000 ppm pup weight gain was slightly lower during lactation, and upon necropsy dilatation of the caecum with faeces was observed in 4/25 males and 2/25 females. Therefore, 4000 ppm can be regarded as LOAEL, and 2000 ppm as NOAEL for F2.

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
24 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The effect level ranged from 16 to 25, due to lower body weight gains and reduced food consumption and/or necropsy findings at higher doses.
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Generation:
F1
Effect level:
96 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the effect observed on mating, behaviour, fertility at the highest dose. The effective dose ranged from 96 to 123 mg/kg bw/d (nominal) (i.e., equivalent to 48 to 61.5 mg a.i./kg bw/d

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

The mean achieved dosages of the test substance for the dose-levels of 500, 2000 and 4000 ppm of test substance were as follows:
F0 generation
- males (Days 1 to 106): 16, 61 and 123 mg/kg bw/d, respectively,
- females: during premating period (Days 1 to 71): 19, 74 and 154 mg/kg bw/d, respectively,
    during pregnancy period (Days 0 to 20 p.c.): 18, 69 and 145 mg/kg bw/d, respectively,
    during lactation period (Days 1 to 21 p.p.): 37, 159 and 326 mg/kg bw/d, respectively.
F1 generation
- males (Days 1 to 120): 24, 96 and 202 mg/kg bw/d, respectively,
- females:
    during premating period (Days 1 to 64): 32, 127 and 269 mg/kg bw/d, respectively,
    during pregnancy period (Days 0 to 20 p.c.): 21, 83 and 164 mg/kg bw/d, respectively,
    during lactation period (Days 1 to 21 p.p.): 41, 162 and 323 mg/kg bw/d, respectively.

The actual intake of test substance for both males and females given 500, 2000 and 4000 ppm throughout the study is approximately
16-25, 61-101 and 123-208 mg/kg bw/d, respectively  for the F0 generation and 24-31, 96-123 and 202-252 mg/kg bw/d for the F1 generation.

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of this study, the No Observed Effect Level (NOEL) for parental toxicity is 500 ppm for the male and the female animals. The No Observed Effect Level (NOEL) for mating behaviour, fertility and gestation of each generation and for development, growth and survival of each progeny is 2000 ppm (i.e., 61 to 101 mg/kg bw/day (nominal) (i.e., equivalent to 30.5 to 50.5 mg a.i./kg bw/d and 96 to 123 mg/kg bw/d (nominal) (i.e., equivalent to 48 to 61.5 mg a.i./kg bw/d for the F0 and F1 generation respectively).
Executive summary:

In a two-generation study, C12-C16 ADBAC was administered in the diet to male and female Sprague Dawley rats at 0, 500 (i.e.,16-25 and 24-31 mg/kg bw/day or 8-12.5 and 12-15.5 mg a.i./kg bw/day in males and females); 2,000 (i.e., 61-101and 96-123 mg/kg bw/day or 30.5-50 and 48 to 61.5 mg a.i./kg bw/day in males and females) or 4,000 ppm (i.e., 123-208 and 202-252 mg/kg bw/day or 61-104 and 101-126 mg a.i./kg bw/day in males and females, before and through mating and gestation until the end of the lactation period in both F0 and F1 generations. At 2,000 ppm, F0 (males) and F1 parents showed marginally to slightly lower body weight gains and reduced food consumption. Necropsy of parents of both generations revealed dilatation of the caecum in some animals. This was associated with lower liver weights in parental animals of both generations. At 4,000 ppm, in F0 and F1 parents, number of implantation sites and litter size at birth were reduced. The progeny (F2) also showed lower pup weights. Pup weight gain was slightly lower during lactation. Upon necropsy, dilatation of the caecum with faeces was observed in 4/25 males and 2/25 females. Treatment with the test substance had no effect on mating, fertility and behavioural parameters in F0 and F1 parental Sprague-Dawley rats at treatment levels up to 2,000 ppm. No effect was recorded on litter parameters and on pre- and post-natal development of either generation at 2,000 ppm. Consequently, under the experimental conditions of this study, the No Observed Effect Level (NOEL) for parental toxicity was 500 ppm for the male and the female animals. The No Observed Effect Level (NOEL) for mating behaviour, fertility and gestation of each generation and for development, growth and survival of each progeny was 2,000 ppm.

Under the experimental conditions of this study, the No Observed Effect Level (NOEL) for parental toxicity is 500 ppm for the male and the female animals. The No Observed Effect Level (NOEL) for mating behaviour, fertility and gestation of each generation and for development, growth and survival of each progeny is 2000 ppm (i.e., 61 to 101 mg/kg bw/day (nominal) (i.e., equivalent to 30.5 to 50.5 mg a.i./kg bw/d and 96 to 123 mg/kg bw/d (nominal) (i.e., equivalent to 48 to 61.5 mg a.i./kg bw/d for the F0 and F1 generation respectively) (Foulon O, 2008).