Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline 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:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
This is formulation of 49.2% of the test substance in water.
Specific details on test material used for the study:
- Purity: 49.9%

Test animals

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

Administration / exposure

Route of administration:
oral: feed
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
- Exposure period: about 18 wk.
P0 and P1:
Premating exposure period (males): 10 wk.
Premating exposure period (females): 10 wk.
- Duration of test: P0 pre-mating 10 wk, until F2 weaning.
Frequency of treatment:
Continuously
Details on study schedule:
Groups of 25 male and 25 female Sprague-Dawley rats were administered the test substance (purity 49.9%) at levels of 0, 500, 2000 or 4000 ppm in their diet over a period of 10 wk before mating, 2 wk during mating, and until after weaning of the pups (total period 18 wk). From every litter one or two pups were selected to again obtain 25 animals per sex and dose group for the F1 generation. These animals were similarly treated as the parent (P1) animals throughout premating, mating, pregnancy until sacrifice, after weaning of F2 progeny.
Doses / concentrations
Remarks:
Doses / Concentrations:
1000, 2000 and 4000 ppm of test substance
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
- Examination of P0 and P1 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 P1 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 P0 and P1 males.
Litter observations:
Examination of F1/P1 generation:
- On Day 22 post-partum, one male and one female pup per litter were selected to constitute the F1/P1 generation, which comprised 25 males and 25 females per group. The F1/P1 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 P0 and P1 animals:
- After weaning of their respective progeny, P0 and P1 parent males and females were sacrificed. Designated organs were weighed for P0 and P1 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 P0 and P1 males.
- A macroscopic post-mortem examination was performed on all P0 and P1 parent males and females. 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. A microscopic examination was performed on macroscopic lesions, reproductive organs, adrenals, and pituitary glands of all P0 and P1 parents of the control and high dose groups.
- Particularly detailed histopathological examinations were performed for the ovaries and the testes.
Postmortem examinations (offspring):
- A macroscopic post-mortem examination was performed on three pups per sex and per litter of each P0 and P1 (F1 and F2 generation) 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. In all pups, the macroscopic lesions were preserved.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- At 4000 ppm, a slightly lower mean body weight gain was recorded during most of the dosing period in both parental males and females (and was associated with reduced liver weights).
- At 2000 and 500 ppm, a marginally to slightly lower body weight gain was noted over all the dosing period for the males.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- At 4000 ppm, a slightly to moderately lower mean food consumption was recorded during most of the dosing period in both parental males and females.
- At 2000 and 500 ppm, a marginally to slightly lower mean food consumption was noted over all the dosing period for the males.
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):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- At 4000, ppm, reduced liver weights were observed.
- At 2000 ppm, lower liver weights in parental animals were recorded.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- At 4000 ppm, necropsy of these animals revealed dilation of the cecum, colon or ileum in some animals (more marked in P0 parents).
- At 2000 ppm, necropsy of parents revealed dilatation of the cecum in some animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
- At 4000 and 2000 ppm, no effects were noted at histopathological examination of sexual organs.

Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
- At 4000 and 2000 ppm, no effects were noted on sperm parameters.

Reproductive performance:
no effects observed
Description (incidence and severity):
- At 4000 ppm, slightly lower pup body weight was observed (with lower spleen weights).
- At 2000 ppm, no effects were observed on parental fertility as assessed by normal mating, gestation and delivery.
- At 500 ppm, no effects were noted on mating, fertility, gestation, fecundity or delivery of either generation or on development of their progeny.

Details on results (P0)

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/day, should be regarded as NOAEL for P0 and P1generation.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
Remarks on result:
other: the effect level ranged from 16 to 25 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: dose range from 61 to 101 mg/kg bw/day

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
4 000 ppm
System:
other: reproductive performance
Treatment related:
yes
Dose response relationship:
yes

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- At 4000 ppm, a slightly to moderately lower mean body weight gain was recorded during most of the dosing period in both parental males and females (and was associated with reduced liver weights).
- At 2000 ppm, a marginally to slightly lower mean body weight gain was noted.
- At 500 ppm, a marginally to slightly lower mean body weight gain was observed in both sexes. This was associated with lower liver weights of parental males and females.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- At 4000 ppm, a slightly to moderately lower mean food consumption was recorded during most of the dosing period in both parental males and females.
- At 2000 and 500 ppm, a marginally to slightly lower mean food consumption was noted over all the dosing period in both sexes.
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):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- At 4000 ppm, reduced liver weights were observed.
- At 2000 and 500 ppm, lower liver weights in parental animals were recorded.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- At 4000 ppm, necropsy of these animals revealed dilation of the cecum, colon or ileum in some animals (more marked in F0 parents).
- At 2000 ppm, necropsy of parents revealed dilatation of the cecum in a single animal. 
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
- At 4000 and 2000 ppm, no effects were noted at histopathological examination of sexual organs.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P1)

Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
- At 4000 and 2000 ppm, no effects were noted on sperm parameters.

Description (incidence and severity):
- At 4000 ppm, 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 P1 females) and a delay in sexual development. Lower spleen weights were also noted for each progeny.
- At 2000 ppm, no effects were noted on parental fertility as assessed by normal mating, gestation and delivery.
- At 500 ppm, no effects were noted on mating, fertility, gestation, fecundity or delivery and development of their progeny.

Details on results (P1)

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/day, should be regarded as NOAEL for P0 and P1generation.

Effect levels (P1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
Remarks on result:
other: the effect level ranged from 16 to 25 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
Remarks on result:
other: dose range from 61 to 101 mg/kg bw/day

Target system / organ toxicity (P1)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
4 000 ppm
System:
other: reproductive performance
Treatment related:
yes
Dose response relationship:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
- At 4000 ppm, the litter size at birth was reduced. 

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- At 4000 ppm, slightly reduced pup body weight was observed. Pup weight gain was also slightly lower during lactation.
Food consumption and compound intake (if feeding study):
not examined
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
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- At 4000 ppm, lower spleen weights were noted.
- At 2000 ppm, except for a marginally lower spleen weight of the progeny, no other effects were noted on their development.
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Generation:
F1
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios

Target system / organ toxicity (F1)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
4 000 ppm
System:
other: body weight and organs weight
Organ:
spleen
Treatment related:
yes
Dose response relationship:
yes

Results: F2 generation

General toxicity (F2)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
- At 4000 ppm, the litter size at birth were reduced (as a consequence of lower number of implantation sites of F1 parent females).
- At 2000 ppm, no effects were seen in F2 offspring regarding pup survival until weaning.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- At 4000 ppm, slightly lower pup body weight was observed. The pup weight gain was slightly reduced during lactation, 
- At 2000 ppm, no effects were seen regarding pup development.
Food consumption and compound intake (if feeding study):
not examined
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
Sexual maturation:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- At 4000 ppm, lower spleen weights were noted.
- At 2000 ppm, except for a marginally lower spleen weight, no other effects were noted on their development.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At 4000 ppm, dilatation of the caecum with faeces was observed in 4/25 males and 2/25 females.
At 2000 ppm, no effects were seen regarding pup development and after sacrifice at weaning.
Histopathological findings:
not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
general / developmental toxicity
Generation:
F2
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
sexual maturation
mortality
body weight and weight gain
organ weights and organ / body weight ratios
gross pathology
Key result
Dose descriptor:
LOAEL
Remarks:
general / developmental toxicity
Generation:
F2
Effect level:
4 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
sexual maturation
mortality
body weight and weight gain
organ weights and organ / body weight ratios
gross pathology

Target system / organ toxicity (F2)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
4 000 ppm
System:
other: spleen weight
Treatment related:
yes
Dose response relationship:
yes

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
2 000 ppm
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes

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/day, respectively,
- females:

during premating period (Days 1 to 71): 19, 74 and 154 mg/kg bw/day, respectively,
    during pregnancy period (Days 0 to 20 p.c.): 18, 69 and 145 mg/kg bw/day, respectively,
    during lactation period (Days 1 to 21 p.p.): 37, 159 and 326 mg/kg bw/day, respectively.
F1 generation
- males (Days 1 to 120): 24, 96 and 202 mg/kg bw/day, respectively,
- females:
    during premating period (Days 1 to 64): 32, 127 and 269 mg/kg bw/day, respectively,
    during pregnancy period (Days 0 to 20 p.c.): 21, 83 and 164 mg/kg bw/day, respectively,
    during lactation period (Days 1 to 21 p.p.): 41, 162 and 323 mg/kg bw/day, 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/day, respectively  for the F0 generation and 24-31, 96-123 and 202-252 mg/kg bw/day for the F1 generation.

Applicant's summary and conclusion

Conclusions:
BUnder the study conditions, the rat NOEL for parental toxicity was established at 500 ppm for the male and the female animals. The rat NOEL for mating behaviour, fertility and gestation of each generation and for development, growth and survival of each progeny was determined to be 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/day) and 96 to 123 mg/kg bw/day (nominal) (i.e., equivalent to 48 to 61.5 mg a.i./kg bw/day for the F0 and F1 generation respectively)).
Executive summary:

A study was conducted to determine the toxicity to reproduction of the test substance (49.9% purity) according to OECD Guideline 416, in compliance with GLP. In this two-generation study, the substance was administered in the diet to male and female Sprague-Dawley rats at dose levels of 0, 500, 2000 and 4000 ppm (equivalent to 0, 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, 61-101 and 96-123 mg/kg bw/day or 30.5-50 and 48-61.5 mg a.i./kg bw/day in males and females and 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, respectively). Doses were administered before and throughout mating and gestation until the end of the lactation period in both P0 and P1 generations. At 2000 ppm, P0 (males) and P1 (both sexes) 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 4000 ppm, in P0 and P1 generations, number of implantation sites and litter size at birth were reduced. The progenies (F1 and F2) also showed lower pup weights. Pup weight gain was slightly lower during lactation. The weight of the spleen was also reduced. Upon necropsy, dilatation of the caecum with faeces was observed in 4/25 males and 2/25 females in F2. Treatment with the test substance had no effect on mating, fertility and behavioural parameters in P0 and P1 parental Sprague-Dawley rats at treatment levels up to 2000 ppm. No effect was recorded on litter parameters and on pre- and post-natal development of either generation at 2000 ppm. Under the study conditions, the rat NOEL for parental toxicity was determined to be 500 ppm for the male and the female animals. The rat NOEL for mating behaviour, fertility and gestation of each generation and for development, growth and survival of each progeny was established at 2000 ppm (61-101 mg/kg bw/day (nominal) (equivalent to 30.5-50.5 mg a.i./kg bw/day and 96-123 mg/kg bw/day (nominal) (i.e. 48-61.5 mg a.i./kg bw/day for the F0 and F1 generation, respectively)) (Foulon, 2008).