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Effects on fertility

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Referenceopen allclose all

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1989
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Principles of method if other than guideline:
Guideline not mentioned.
GLP compliance:
no
Species:
other: rats and guinea pigs
Sex:
male/female
Route of administration:
oral: gavage
Analytical verification of doses or concentrations:
not specified
Dose / conc.:
25 mg/kg bw/day
No. of animals per sex per dose:
15 rats and 10 guinea pigs
Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Remarks on result:
other: Limited information available
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
25 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Remarks on result:
other: Limited information available
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

A 2 generation study with groups of 15 rats and 10 guinea pigs was conducted with up to 25 mg/kg bw/day by gavage and showed no overt adverse effects on reproduction.

Conclusions:
Under the study conditions, no adverse effects on reproduction were reported in a 2-generation study conducted in groups of 15 rats and 10 guinea pigs, up to the dose 25 mg/kg bw/day (oral gavage).
Executive summary:

A two-generation study was conducted in groups of 15 rats and 10 guinea pigs up to the dose of 25 mg/kg bw/day test substance (administered by stomach tube). Under the study conditions, no overt adverse effects on reproduction were observed (Shelanski, 1989).

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
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Purity: 49.9%
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
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.
Remarks:
Doses / Concentrations:
1000, 2000 and 4000 ppm of test substance
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
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.
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
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: 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.
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.
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
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
4 000 ppm
System:
other: reproductive performance
Treatment related:
yes
Dose response relationship:
yes
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: 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.
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.
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
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
4 000 ppm
System:
other: reproductive performance
Treatment related:
yes
Dose response relationship:
yes
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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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
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
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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
4 000 ppm
System:
other: spleen weight
Treatment related:
yes
Dose response relationship:
yes
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

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.

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).

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPP 83-4 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Test animals
- Source: Sprague-Dawley CD rats were obtained from Charles River Breeding Laboratories, Kingston, NY, USA
- Age at study initiation: Six weeks
- Weight at study initiation: 212.2-213.4 g (males); 148.3-150.2 g (females)
- Housing: Individually in stainless steel, wire mesh cages (22.5x15.5x18.0 cm; mating cages 22.5x31.0x18.0 cm)
- Diet: Certified Ground Rodent Chow # 5002 (Ralstor Purina Co., St. Louis, MO), ad libitum
- Water: Tap water, ad libitum. Water was provided by an automatic watering system with demand control valves mounted on each cage rack.
- Acclimation period: Two weeks

Environmental conditions
- Temperature: 66-73°F
- Humidity: 40-60%
Route of administration:
oral: feed
Vehicle:
other: Certified Ground Rodent Chow # 5002
Details on exposure:
Diet preparation: A concentrated premix was prepared by direct addition of the test substance to ground chow and mixing for approximately an hour. Test diets were prepared by appropriate dilutions of the concentrated premix or higher diet concentrations.
- Rate of preparation of diet (frequency): Fresh diet was prepared weekly.
- Mixing appropriate amounts with (Type of food): Certified Ground Rodent Chow # 5002
- Storage temperature of food: Diets were stored in polyethylene containers at room temperature.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Mating procedure: After pre-breeding exposure the animals were mated on the basis of one male to one female selected randomly within each dose group for a period of 21d to produce F1 generation.
- Proof of pregnancy: Copulation plug and/or vaginal sperm as Day 0 of gestation.
- After the first 7d of the mating period females of unsuccessfully mated pairs were placed with males of other unmated pairs within the same dose group; after an additional 7d, unsuccessfully mated pairs were similarly exchanged again for a period of 7d or until successful mating had occurred, whichever came first, allowing for a total of 21d to mate.
- After successful mating each pregnant female was caged: Pregnant females were housed individually. On Day 20 of gestation each pregnant female was transferred to a shoe-box cage.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Experimental diets were analyzed for stability, homogeneity and concentration of test substance using high pressure liquid chromatography.
- Homogeneity study indicated that the distribution of the test substance in the test diet was uniform.
- Stability study indicated that the dosed feed was stable for at least 14d when stored in open glass feed jars at room temperature. Dosed feed was stable for at least 21d when stored in closed polyethylene containers at room temperature.
- Concentration verification analyses of the dosed feed indicated that the mean concentrations of the test substance in the diet for the 300, 1000 and 2000 ppm dosage levels were 95.3-109.0% of nominal for 300 ppm, 95.6-107.9% of nominal for 1000 ppm and 94.7-108.0% of nominal for 2000 ppm.
Duration of treatment / exposure:
P0 generation: 19 weeks (from 1st prebreed dose to last F0 sacrifice)
F1 generation: 25 weeks (from 1st F1 wean to last F1 sacrifice)
F2 generation: Until weaning
Frequency of treatment:
Daily
Details on study schedule:
- P1 parental animals not mated until 17-18 weeks after selection from the F1 litters.
- Selection of parents from F1 generation when pups were 28d old.
- Number of P1 generation animals selected: 28 males and 28 females

Remarks:
Doses / Concentrations:
0, 300, 1000 or 2000 ppm test substance (i.e., equivalent to 0, 16-31, 51-102, and 100-188 mg/kg bw/day (males) and 0, 21-32, 67-106 and 139-198 mg/kg bw/day (females)).
Basis: nominal in diet
No. of animals per sex per dose:
28
Control animals:
yes, plain diet
Details on study design:
- Rationale for animal assignment: Animals were randomly distributed based on body weight
- Animal identification: By ear tags
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

CLINICAL SIGNS: Yes
- Time schedule: Once daily for overt clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during prebreed and mating for both sexes. For females on gestational Day 0, 6, 15, and 20 and on postnatal Day 0, 7, 14, and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: Weekly during prebreed for both sexes. For females in 3- or 4-day intervals throughout gestation and to postnatal Day 14.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: No

Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible). Excess pups were subjected to detailed external examination and then sacrificed.

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring: Live/Still births on the day of birth (postnatal Day 0), survival indices at Days 0, 4, 7 and 14 after birth and weaning, weight on postnatal Day 1, 4, 7, 14, and at weaning (Day 21), and physical abnormalities for all pups at birth and throughout the pre-weaning period.

GROSS EXAMINATION OF DEAD PUPS: The thoracic and abdominal organs from pups which died after Day 4 were preserved for subsequent histopathological examination.
Postmortem examinations (parental animals):
SACRIFICE: Yes
- How many animals: All animals
- Necropsy method: Animals were anesthetized with methoxyflurane and exsanguinated by severing the brachial vessel.

GROSS NECROPSY: Yes
- How many animals: All animals sacrificed in P0 and P1 parental animals
- Gross necropsy consisted of: Vagina, uterus, ovaries, gross lesions, testes, epididymis, seminal vesicles and prostrate.

HISTOPATHOLOGY : Yes
- How many animals: All animals of control and high dose groups sacrificed in P0 and P1 parental animals
- Tissues evaluated: Vagina, uterus, ovaries, gross lesions, testes, epididymis, seminal vesicles and prostrate and any tissues or organs showing gross alterations from the low and mid dose groups
Postmortem examinations (offspring):
SACRIFICE
- The F1 offsprings not selected as parental animals and all F2 offsprings were sacrificed at 7d of age.

GROSS NECROPSY
- Examination for gross lesions was performed on any pup appearing abnormal or dying on test and for ten randomly selected F1 and F2 weanlings/sex/group.

Statistics:
- The results of the quantitative continuous variables (e.g., body weights, food consumption, etc.) were compared between the three treatment groups and one control group using Levene’s test for equal variances, analysis of variance and (pooled or separate variance) t-test.
- Non-parametric data were statistically evaluated using the Kruskall-Wallis test followed by the Mann-Whitney U test for pairwise comparisons when appropriate.
- Frequency data were compared using the Fisher’s exact test.
Reproductive indices:
Mating index, fertility index and gestational index were determined.
Offspring viability indices:
Live birth index, 4-d survival index, 7-d survival index, 14-d survival index, 21-d survival index and lactation index

Clinical signs:
no effects observed
Description (incidence and severity):
No significant signs of toxicity during the pre-breed, mating, gestation or lactation periods at any dose for either generation were 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):
- During the 10-week pre-breed exposure, P0 males exhibited no reduction in body weight. During the same period, P0 females at 2000 ppm exhibited reduction in body weight during Weeks 5, 6, 9 and 10 of pre-breed treatment. Body weight gain was also reduced at 2000 ppm for one week (Week 8-9) during the pre-breed period.
- On lactation Day 21 mean body weight of P0 dams at 2000 ppm exhibited a significant increase. Increased lactation body weight gain was observed at 2000 ppm throughout lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Food consumption in the P0 females at 2000 ppm was reduced for the first four exposure weeks. Food consumption in P0 males was significantly reduced at 2000 ppm for the first week of treatment only.
- At P0 breed to produce F1 litters, food consumption during gestation and lactation was unaltered by treatment.
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:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating index, fertility index and gestational index
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: equivalent to 51-102 mg/kg bw/day in males and 67-106 mg/kg bw/day in females
Key result
Dose descriptor:
NOEL
Remarks:
reproductive toxicity
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no reproductive toxicity observed
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 ppm
System:
other: body weight and food consumption
Treatment related:
yes
Dose response relationship:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
No significant signs of toxicity during the pre-breed, mating, gestation or lactation periods at any dose for either generation were 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 2000 ppm, only slight reduction were observed in males and females
Food consumption and compound intake (if feeding study):
no effects observed
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:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating index, fertility index and gestational index
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: equivalent to 51-102 mg/kg bw/day in males and 67-106 mg/kg bw/day in females
Key result
Dose descriptor:
NOEL
Remarks:
reproductive toxicity
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no reproductive toxicity observed
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
2 000 ppm
System:
other: body weight
Treatment related:
yes
Dose response relationship:
yes
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The F1 litters exhibited reduced body weight per litter on postnatal Days 21 and 28 at 2000 ppm. F1 pup body weight gain was reduced during lactation Days 14-21 and 21-28.
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:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Generation:
F1
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
2 000 ppm
System:
other: body weight
Treatment related:
yes
Dose response relationship:
yes
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F2 pup weights per litter were reduced at 2000 ppm on postnatal Day 28. Pup weight gain was also reduced at 2000 ppm during lactation Days 14-21 and for Days 21-28.
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:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Generation:
F2
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOEL
Remarks:
developmental toxicity
Generation:
F2
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 ppm
System:
other: body weight
Treatment related:
yes
Dose response relationship:
yes
Key result
Reproductive effects observed:
no
Conclusions:
Under the study conditions, dietary administration of the test substance at dose levels of 0, 300, 1000 and 2000 ppm for two generations to Sprague-Dawley rats was well tolerated and no signs of reproductive toxicity were observed at any dose level. The rat NOAEL (systemic toxicity) for both parental generation (P0 and P1) and offsprings (F1 and F2) was considered to be 1000 ppm (in diet), equivalent to 51-102 and 67-106 mg/kg bw/d for male and female respectively. The rat NOEL (reproductive toxicity) was established at 2000 ppm (the highest dose tested).
Executive summary:

A study was conducted to determine the toxicity to reproduction of the test substance according to US EPA OPP 83 -4, in compliance with GLP. The test substance was orally administered to Sprague-Dawley CD rats (28/sex/group) at dose levels of 0, 300, 1000 or 2000 ppm test substance (equivalent to 0, 16-31, 51-102, and 100-188 mg/kg bw/day (males) and 0, 21-32, 67-106 and 139-198 mg/kg bw/day (females)) in the diet. There was one control group of 28 animals/sex. Following a 10 week pre-breed exposure period, the P0 rats were randomly paired within dose groups for a 3 week mating period to produce the F1 generation. Exposure continued through mating, gestation, parturition and lactation. At weaning, 28 F1 weanling/sex/group were randomly selected and exposed to the same dietary concentration of the test substance as their parents for 10 weeks. After their pre-breed exposure, F1/P1 animals were paired to produce the F2 generation. The animals were observed twice daily and clinical findings, body weights and food consumption were recorded periodically. All P0 and P1 animals were necropsied and examined for gross lesions; selected reproductive tissues from the high dose and control groups were examined histologically as were other tissues with gross lesions. Ten F1 and F2 weanlings/sex/dose were randomly selected and necropsied and examined for gross lesions. P0 female body weights and food consumption were reduced at 2000 ppm during the pre-breeding exposure period. For the pre-breeding period of the P1 animals, only slight reductions in body weight gain were observed for males in the high dose group. No other treatment-related effects were observed on any of the reproductive parameters. Under the study conditions, it can be stated that, dietary administration of the test substance at dose levels of 0, 300, 1000 and 2000 ppm for two generations to Sprague-Dawley rats was well tolerated and no sign of reproductive toxicity was observed at any dose level. Based on the results of the read across study, the rat NOAEL (systemic toxicity) for both parental generation (P and F1) and offspring (F1 and F2) was considered to be 1000 ppm (in diet), equivalent to 51-102 or 41.3-83 mg a.i./kg bw/day in males and 67-106 mg/kg bw/day or 54-86 mg a.i./kg bw/day for female, respectively. The rat NOEL (reproductive toxicity) was established at 2000 ppm (the highest dose tested) (Neeper-Bradley, 1990).

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
30.5 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Information requirement for this tonnage band is sufficiently met with the available data.
Additional information

Study 1. 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).

Study 2. A study was conducted to determine the toxicity to reproduction of the test substance according to US EPA OPP 83 -4, in compliance with GLP. The test substance was orally administered to Sprague-Dawley CD rats (28/sex/group) at dose levels of 0, 300, 1000 or 2000 ppm test substance (equivalent to 0, 16-31, 51-102, and 100-188 mg/kg bw/day (males) and 0, 21-32, 67-106 and 139-198 mg/kg bw/day (females)) in the diet. There was one control group of 28 animals/sex. Following a 10 week pre-breed exposure period, the P0 rats were randomly paired within dose groups for a 3 week mating period to produce the F1 generation. Exposure continued through mating, gestation, parturition and lactation. At weaning, 28 F1 weanling/sex/group were randomly selected and exposed to the same dietary concentration of the test substance as their parents for 10 weeks. After their pre-breed exposure, F1/P1 animals were paired to produce the F2 generation. The animals were observed twice daily and clinical findings, body weights and food consumption were recorded periodically. All P0 and P1 animals were necropsied and examined for gross lesions; selected reproductive tissues from the high dose and control groups were examined histologically as were other tissues with gross lesions. Ten F1 and F2 weanlings/sex/dose were randomly selected and necropsied and examined for gross lesions. P0 female body weights and food consumption were reduced at 2000 ppm during the pre-breeding exposure period. For the pre-breeding period of the P1 animals, only slight reductions in body weight gain were observed for males in the high dose group. No other treatment-related effects were observed on any of the reproductive parameters. Under the study conditions, it can be stated that, dietary administration of the test substance at dose levels of 0, 300, 1000 and 2000 ppm for two generations to Sprague-Dawley rats was well tolerated and no sign of reproductive toxicity was observed at any dose level. Based on the results of the read across study, the rat NOAEL (systemic toxicity) for both parental generation (P and F1) and offspring (F1 and F2) was considered to be 1000 ppm (in diet), equivalent to 51-102 or 41.3-83 mg a.i./kg bw/day in males and 67-106 mg/kg bw/day or 54-86 mg a.i./kg bw/day for female, respectively. The rat NOEL (reproductive toxicity) was established at 2000 ppm (the highest dose tested) (Neeper-Bradley, 1990).

Study 3. A two-generation study was conducted in groups of 15 rats and 10 guinea pigs up to the dose of 25 mg/kg bw/day test substance (administered by stomach tube). Under the study conditions, no overt adverse effects on reproduction were observed (Shelanski, 1989).

Effects on developmental toxicity

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was performed according to the range finding study design of OECD guideline 414.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Route of administration:
oral: gavage
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Day 6 to 28 post coitum
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
3 mg/kg bw/day
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
30 mg/kg bw/day
No. of animals per sex per dose:
6 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Sex: male/female
Details on maternal toxic effects:
At 30 mg/kg bw/day, slightly higher mean number of post-implantation loss associated with slightly lower mean number of live foetuses were recorded. No maternal toxicity or effects on litter data parameters were noted at 3 and 10 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no general maternal toxicity was observed at 3 and 10 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
pre and post implantation loss
dead fetuses
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: post-implantation sites and foetuses viability
Key result
Dose descriptor:
NOAEL
Remarks:
general and developmental toxicity
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: foetuses viability
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes

At 30 mg/kg bw/day, slightly high mean number of post-implantation loss associated with slightly low mean number of live foetuses were recorded. No maternal toxicity or effects on litter data parameters were noted at 3 and 10 mg/kg bw/day.

Based on these results, the 30 mg/kg bw/day dose-level was chosen as the high dose-level in the further developmental toxicity study (CIT Study No. 26148 RSL).

Conclusions:
Under the study conditions (dose range finding study), a slightly higher mean number of post-implantation loss associated with slightly lower mean number of live foetuses was recorded at 30 mg/kg bw/day. No maternal toxicity or effects on litter data parameters were noted at 3 and 10 mg a.i./kg bw/day. Based on these results, the 30 mg/kg bw/d dose-level was chosen as the high dose-level in the further developmental toxicity study.
Executive summary:

A study was conducted to determine the developmental toxicity and teratogenicity of the test substance according to OECD Guideline 414 (range-finding study). Groups of six mated female rabbits received daily oral administration at 0 (vehicle, purified water), 3, 10 or 30 mg/kg bw/day from Day 6 to 28 post-coitum. In the total absence of maternal toxicity at all dose-levels and in order to better define the top dose-level of the main study, it would be better to perform a complementary study with higher dose-levels of 50 and 100 mg/kg bw/day. The test substance from GD 6-28 produced marked maternal toxicity at 50 and 100 mg/kg bw/day shown by high number of deceased females and severe clinical conditions in most animals. At 30 mg/kg bw/day, a slightly higher mean number of post-implantation loss associated with slightly lower mean number of live foetuses was recorded. No maternal toxicity or effects on litter data parameters were noted at 3 and 10 mg/kg bw/d. Based on these results, the 30 mg/kg bw/day dose-level was chosen as the high dose level in the further developmental toxicity study (Gaoua, 2004).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
batch number is not presented for the vehicle (purified water) in the study report; autolysed foetuses were not sexed at the time of hysterectomy and head sections were archived in the same way as brain sections.
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
batch number is not presented for the vehicle (purified water) in the study report; autolysed foetuses were not sexed at the time of hysterectomy and head sections were archived in the same way as brain sections.
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Route of administration:
oral: gavage
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Day 6 to 28 post coitum
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day
Remarks:
vehicle alone
Dose / conc.:
3 mg/kg bw/day
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
30 mg/kg bw/day
No. of animals per sex per dose:
22 mated females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Sex: female
Duration of test: GD 6-28
Maternal examinations:
- Food consumption and body weight were recorded at designated intervals. 
- Clinical signs were checked each day.
Ovaries and uterine content:
- On Day 29 post-coitum, all the surviving dams were sacrificed and subjected to a macroscopic post-mortem examination. A gross examination of placentas was also performed. The fetuses were removed by hysterectomy and the uterus weighed. The net body weight gain was calculated. The litter parameters, namely, the number of corpora lutea, implantation sites, early and late resorptions, dead and live fetuses were recorded. The fetuses were weighed and submitted to external examination.
Fetal examinations:
- The live fetuses were killed and then subjected to a fresh dissection and detailed examination of soft tissue, including body, head and brain. The sex was determined. The carcasses were then fixed and the skeletons (including cartilage) stained and examined.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 30 mg/kg bw/day:
- the relevant clinical signs concerned the deceased females and two prematurely sacrificed females. No other clinical signs were noted in females from this group.
At 10 mg/kg bw/day:
- there were no relevant clinical signs except for two females with blood in the bedding on Days 22 and 23 post-coitum or absence of feces from Day 26 post-coitum
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
At 30 mg/kg bw/day:
- three females died and two females were prematurely sacrificed for ethical reasons or abortion.
At 10 mg/kg bw/day:
- there were no deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 30 mg/kg bw/day:
- body weight gain was transiently reduced (GD 9-12, -70% below the control, p<0.05) but returned to normal values thereafter.
At 10 mg/kg bw/day:
- reduction of maternal body weight gain did not reach statistical significance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
At 10 mg/kg bw/day:
- reduction of food consumption did not reach statistical significance.
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:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
At 30 mg/kg bw/day:
- the necropsies revealed in 8/22 females: accentuated lobular pattern in the liver, pale liver, whitish areas and/or blackish deposits and/or edema in the stomach mucosa, reddish or brownish foci on the lungs, blackish contents in the intestines, dilated intestines and dilated gall bladder.
At 10 mg/kg bw/day:
- the necropsies revealed in 5/22 females: dilated gall bladder, accentuated lobular pattern.
- pale liver, brownish or reddish foci on the lungs, blackish deposit on the stomach mucosa.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The fluctuations recorded for the mean number of resorptions (early or late) and the mean number of dead foetuses and consequently for the percentages of post-implantation loss were also not considered to be treatment-related, as they were minimal and not dose-related.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
The fluctuations recorded for the mean number of resorptions (early or late) and the mean number of dead foetuses and consequently for the percentages of post-implantation loss were also not considered to be treatment-related, as they were minimal and not dose-related.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
The fluctuations recorded for the mean number of resorptions (early or late) and the mean number of dead foetuses and consequently for the percentages of post-implantation loss were also not considered to be treatment-related, as they were minimal and not dose-related.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
The fluctuations noted in the mean number of corpora lutea and implantation sites were slight and not dose-related, they were consequently considered not to be treatment-related.
Details on maternal toxic effects:
At 3 mg/kg bw/day:
- no signs of maternal toxicity
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
3 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
gross pathology
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no reproductive toxicity observed
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: general toxicity, no effects observed on reproductive organs
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The occurrence of some external malformations throughout all treated groups, including the controls, did not suggest any substance-related origin because of their low incidence, absence of dose-relationship and/or statistical significance.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No relevant malformations was noted but the presence of a full supernumerary 13th pair of ribs (as a foetal variation), was markedly increased at 10 and 30 mg/kg bw/day. These differences in foetal or litter incidence, which were within background data, were most probably due to a low control value. The incidence of one other skeletal variation (unossified 5th sternebra) was significantly greater but in the low dose-group only.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The occurrence of some soft tissue malformations throughout all treated groups, including the controls, did not suggest any substance-related origin because of their low incidence, absence of dose-relationship and/or statistical significance.
Details on embryotoxic / teratogenic effects:
Conclusion: 
There were no treatment-related effects on litter data parameters and no treatment-related findings upon external, visceral or skeletal observation in any of the dose groups. The test substance was not teratogenic in rabbits.
Key result
Dose descriptor:
NOAEL
Remarks:
general and developmental toxicity
Effect level:
30 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment related developmental effect up to the highest dose
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Result: 

The test substance was not teratogenic in rabbits.

- LO(A)EL maternal toxic effects:
10 mg/kg bw/d, based on necropsy findings in 5/22 animals. Incidence was increased to 8/22 at 30 mg/kg bw/day (dilated gallbladder 3/22, accentuated lobular pattern liver 3/22).
Foci (reddish/brownish) in the lung was also observed in 2 females, but also in view of findings in range finding study and parallel study with comparable compound, this can be caused by inadvertent presence of substance into the airways and not attributable to systemic toxicity. Incidence was not increased in the top-dose group. There is an indication of lower body weight gain, correlating to a lower food consumption, but that was not statistical significant and in the high-dose goup not different from the mid-dose group. Blackish content in stomack and intestines is indicative of local corrosive effects of test substance.

- NO(A)EL maternal toxic effects:
3 mg/kg bw/day. There seems to be a dose-response related increase in necropsy findings in stomach, intestine and liver. Dilated gallbladder incidence was not increased in highest dose group compared to mid-dose.

- LO(A)EL embryotoxic / teratogenic effects:
Based on the number of foetuses presenting malformations or variations and in the absence of a treatment-related increase of such observation, the embryo-fetal development was not considered to be affected by treatment.
- NO(A)EL embryotoxic / teratogenic effects:
30 mg/kg bw/day, being the highest tested dose.

Conclusions:
Under the study conditions, the rabbit NOAEL for maternal toxicity was 3 mg a.i./kg bw/day while the rabbit NOAEL for embryo-foetal development was considered to be 30 mg a.i./kg bw/day.
Executive summary:

A study was conducted to determine the developmental toxicity and teratogenicity of the test substance according to OECD Guideline 414 and US EPA OPPTS 870.3700, in compliance with GLP. The substance was administered to pregnant rabbits by gavage from Day 6 to 28 post-coitum at the dose-levels of 3, 10 or 30 mg a.i./kg bw/day. The dose of 30 mg a.i./kg bw/day caused the death of three females, severe clinical condition or abortion in two other females and transient, lower maternal body weight gain. Necropsies revealed in 8/22 females accentuated lobular patterns in the liver, whitish areas and/or blackish deposits in the stomach mucosa and dilated intestines. At 10 mg/kg bw/day, relevant necropsy findings were noted in 5/22 females (dilated gall bladder, accentuated lobular pattern, pale liver, brownish or reddish foci on the lungs, blackish deposit on the stomach mucosa). No maternal toxicity or effects on litter data parameters or embryo-foetal development were noted at 3 mg/kg bw/day. Moreover, there were no effects on litter data parameters and no treatment-related findings upon external, visceral or skeletal observation in any of the dose groups up to 30 mg a.i./kg bw/day. Under the study conditions, the rabbit NOAEL for maternal toxicity was 3 mg a.i./kg bw/day while the rabbit NOAEL for embryo-foetal development was considered to be 30 mg a.i./kg bw/day (Gaoua, 2005).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
(Test substance was administered from Days 6-15 of gestation; however, guideline recommends dosing from implantation through the entire period of gestation to the day before caesarean section.)
Qualifier:
equivalent or similar to
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
(Test substance was administered from Days 6-15 of gestation; however, guideline recommends dosing from implantation through the entire period of gestation to the day before caesarean section.)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Physical state: Pale yellow viscous liquid
- Analytical purity: 81.09% active substance in aqueous/ethanol solution.
- Composition of test material, percentage of components: 81.09% Alkyldimethylbenzylammonium Chloride with an alkyl chain length distribution of 40% C12, 50% C14 and 10% C16
- Lot/batch No.: 7293K
- Storage condition of test material: Room temperature
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Test animals:
- Source: Sprague Dawley CD rats (Crl: CD BR) were obtained from Charles River Breeding Laboratories, Portage MI USA.
- Age at study initiation: 11 weeks
- Weight at study initiation: 245.0-246.6 g
- Housing: Animals were housed singly in stainless steel cage, wire mesh caging; dimension, 22.5 x 15.5 x 18.0 cm during the study.
- Diet: Ground Certified Rodent Chow # 5002 (Ralston Purina Company, St. Louis, MO), ad libitum
- Water: Tap water, ad libitum. Water was provided by an automatic watering system with demand control valves mounted on each rack.
- Acclimation period: 2 weeks

Environmental conditions:
- Temperature: 66-77°F
- Humidity: 40-70%
- Air changes: 8/h

In-life dates: From: 14 October 1991 to: 29 November 1991
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(Milli-Q water)
Details on exposure:
Preparations of dosing solutions: Dosing solutions were prepared by dissolving appropriate amount of the test substance with Milli-Q water. Concentrations were adjusted for percent active ingredient of the test substance.
- Rate of dose preparation: Dosing solutions were prepared once.
- Storage of dose formulations: Room temperature

Vehicle
- Concentration in vehicle: 0, 2, 6, and 20 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulations were analysed for concentration, homogeneity and stability by HPLC method.
- The homogeneity study performed on samples from the 10 and 100 mg/kg bw/day concentrations indicated that the test substance was uniformly distributed in the solution.
- The stability study conducted on lowest and highest concentrations indicated that the test substance was stable in the solutions for at least 14d in a glass flask when stored at room temperature.
- Concentration verification analyses of the dosing solutions showed analytical mean values ranging from 94.5 to 104.8% of nominal for all 3 concentrations.
Details on mating procedure:
- Impregnation procedure: Co-housed
- If cohoused: Animals were co-housed in stainless steel wire mesh cages (30.5 x 31.0 x 18.0 cm).
- M/F ratio per cage: 1:1
- Length of cohabitation: Animals were cohoused until evidence of copulation was observed
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: Each male was paired only once in the study.
- Verification of same strain and source of both sexes: Male and female rats were obtained from same source
- Proof of pregnancy: Presence of vaginal copulation plug. This day was designated as Day 0 of gestation
Duration of treatment / exposure:
From Day 6-15 of gestation
Frequency of treatment:
Once daily during exposure period
Duration of test:
16d (From Day 6 to Day 21 post coitum)
Dose / conc.:
0 mg/kg bw/day
Remarks:
vehicle alone
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
30 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosages were selected by the study sponsor on the basis of a range finding study with the test substance (BBRC Project Report 54-613).
- Rationale for animal assignment: Immediately after mating the female rats were randomly allocated to treatment groups and control group using a stratified randomization program based on body weight on gestation Day 0.
- Animal assignment: Mated rats were assigned to the following groups.
Group 1 (vehicle control): 0 mg/kg bw/day
Group 2 (Low dose): 10 mg/kg bw/day
Group 3 (Mid dose): 30 mg/kg bw/day
Group 4 (High dose): 100 mg/kg bw/day
Maternal examinations:
Mortality: Yes
Time schedule: Twice daily

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on Days 0, 6, 9, 12, 15, 18 and 21.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption was recorded for every 3 d intervals from Days 0 to 21 post coitum (Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21)

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on: Day 21 post coitum
- The females were killed by CO2 asphyxiation and the fetuses removed by caesarean section.
- Organs examined: Gravid uterus, ovaries, cervix, vagina, and peritoneal and thoracic cavities

OTHER: Liver and gravid uterine were weighed.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- The fetuses were removed from the uterus, sexed, weighed individually and examined for variations and malformations including cleft palate
- External examinations: Yes (all per litter)
- Soft tissue examinations: Yes (approximately half per litter)
- Skeletal examinations: Yes (approximately half per litter)
- Head examinations: Yes (approximately half per litter)
- Body weight: Yes
Statistics:
- Levene’s test for equal variances, analysis of variance, and a pooled t-tests for pairwise comparisons.
- Nonparametric data were analyzed with Kruskal-Wallis test followed by Mann-Whitney U test when appropriate.
- Incidence data were compared using Fisher’s Exact Test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Perioral wetness in 67% of dams and audible respiration in 3 dams of high dose group. One dam in this group exhibited dehydration, unkempt appearance, loose feces, urine stains, and perioral wetness. Audible respiration in 2 dams of mid dose group. One of these dams also exhibited urine stains, gasping, perinasal encrustation, loose feces and perioral wetness.



Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no effects of treatment on gestational body weight and body weight gain in any dose group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was reduced between Days 6 to 9 in the mid and high dose group.
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:
no effects observed
Description (incidence and severity):
There were no effects of treatment on the gravid uterine weight in any dose group.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Ulceration of stomach and gas-filled intestines, color changes in liver and lymph nodes and small spleen was seen in one dam of high dose group. Swollen liver was observed in one dam of mid dose group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
not examined
Details on maternal toxic effects:
- Maternal toxic effects: Yes
- Developmental toxicity: There were no treatment-related differences in the number of ovarian corpora lutea and in gestational parameters including total number of implantations, number of viable and nonviable implants.
Key result
Dose descriptor:
NOEL
Remarks:
systemic toxicity
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
food consumption and compound intake
gross pathology
Remarks on result:
other: equivalent to 8.1 mg a.i./kg bw/day
Key result
Dose descriptor:
NOEL
Remarks:
reproductive toxicity
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no reproductive toxicity observed
Remarks on result:
other: equivalent to 81 mg a.i./kg bw/day
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: general toxicity
Description (incidence and severity):
Clinical signs, food consumption and gross pathological findings
Fetal body weight changes:
no effects observed
Description (incidence and severity):
No treatment-related effects on fetal body weights were observed in any group.


Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not examined
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No test substance-related relevant effects during the external examination of fetuses were noted in any group.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No treatment-related variations or malformations.
Visceral malformations:
no effects observed
Description (incidence and severity):
No treatment-related variations or malformations.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: No effects
Key result
Dose descriptor:
NOEL
Remarks:
general and developmental toxicity
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Under the study conditions, the test substance was found to produce some adverse effects in the pregnant rats at 30 and 100 mg/kg bw/day; however, no developmental toxicity, including teratogenicity was observed at any of the doses. The rat NOAEL for maternal toxicity was established at 10 mg/kg bw/day (i.e., equivalent to 8.1 mg a.i./kg bw/day) and the rat NOAEL for developmental toxicity was determined to be 100 mg/kg bw/day (i.e., equivalent to 81 mg a.i./kg bw/day).
Executive summary:

A study was conducted to determine the developmental toxicity and teratogenicity of the test substance (81% purity) according to OECD Guideline 414 and US EPA OPP 93 -3, in compliance with GLP. The experiment was performed in pregnant Sprague-Dawley CD rats. The test substance was administered to groups of 25 pregnant rats orally by gavage at dose levels of 0, 10, 30 and 100 mg/kg bw/day (8.1, 24 and 81 mg a.i./kg bw/day), once daily from Days 6 to 15 of gestation inclusive. Control animals were treated with the vehicle alone (Milli-Q water). Clinical observations were made twice daily, and maternal body weights were measured on gestation Days 0, 6, 9, 12, 15, 18 and 21. Maternal food consumption was measured at 3 day intervals from Day 0 to Day 21. All surviving females were sacrificed on Day 21 and the foetuses were examined for visceral and skeletal variations and malformations. No mortality was observed during the study. Treatment-related clinical signs included perioral wetness and audible respiration in high dose group. Audible respiration was also observed in mid dose group. Food consumption was reduced between Days 6 to 9 in mid and high dose groups. There were no effects of treatment on gestational body weight and body weight gain and gravid uterine weight in any dose group. There were also no treatment-related differences in gestational parameters including total number of implantations, number of viable and nonviable implants between the control and the test groups. Further, there were no effects of treatment on fetal body weights per litter, or on the incidences of external, visceral and skeletal malformations and variations. Under the study conditions, the test substance was found to produce some adverse effects in pregnant rats at 30 and 100 mg/kg bw/day, however, no developmental toxicity, including teratogenicity was observed at any of the dosages. The rat NOAEL for maternal toxicity was established 10 mg/kg bw/day (equivalent to 8.1 mg a.i./kg bw/day) and the rat NOAEL for developmental toxicity was determined to be 100 mg/kg bw/day (equivalent to 81 mg a.i./kg bw/day) (Neeper-Bradley, 1992).

Endpoint:
developmental toxicity
Type of information:
other: Literature data
Adequacy of study:
weight of evidence
Study period:
1985 and 1983
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
Guideline not mentioned. Published data as weight of evidence.
GLP compliance:
not specified
Species:
other: rat and rabbit
Strain:
other: albino or Sprague-Dawley for rat and not specified for rabbit
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no effects observed
Remarks on result:
other: oral route in rat
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no effects observed
Remarks on result:
other: oral route in rat
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no effects observed
Remarks on result:
other: oral route in rabbit
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no effects observed
Remarks on result:
other: oral route in rabbit
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no effects observed except for local skin reactions
Remarks on result:
other: dermal route in rat
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no effects observed
Remarks on result:
other: dermal route in rat
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
gross pathology
Remarks on result:
other: vaginal route in rat
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
total litter losses by resorption
early or late resorptions
dead fetuses
other: decrease in foetal growth and pregnancy rate
Remarks on result:
other: vaginal route in rat
Key result
Abnormalities:
no effects observed
Description (incidence and severity):
except following vaginal administration
Key result
Dose descriptor:
NOAEL
Remarks:
general and developmental
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Remarks on result:
other: oral route in rat
Key result
Dose descriptor:
NOAEL
Remarks:
general and developmental
Effect level:
> 30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Remarks on result:
other: oral route in rabbit
Key result
Dose descriptor:
NOAEL
Remarks:
general and developmental
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Remarks on result:
other: dermal route in rat
Key result
Dose descriptor:
NOAEL
Remarks:
general and developmental
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in litter size and weights
skeletal malformations
Remarks on result:
other: vaginal route in rat
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: sternum
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes

Literature data:
-Oral:
Species: Pregnant albino rats
Exposure period: GD 6-15
Doses: 0, 10, 25 or 50 mg/kg bw/day stomach tube. Including a positive (aspirin) control.
Critical effects dams, fetuses: Among the treated groups, neither reproduction performance of the dam nor foetus weights differed from those of the control animals. The incidences of any skeletal abnormality and soft tissue abnormalities were no greater in the test groups than in the control groups. The incidence of both types of abnormalities was significantly greater in the aspirin-treated group. 
NO(A)EL Teratogenicity/Embryotoxicity: 50 mg/kg bw/day
(CIR Final Report on the Safety Assessment of Stearalkonium Chloride. 1977, cited by Bibra, 1989)

Species: pregnant rabbits
Exposure period: GD 7-19
Doses: 30 mg/kg bw/d or more of the test substance by gavage 
Critical effects dams, foetuses: maternal and embryo toxicity (unspecified) reported. No malformations were seen.
NO(A)EL Teratogenicity/Embryotoxicity: >30 mg/kg bw/day (CEC, 1987, cited in Bibra, 1989)

- Dermal:
Species: 20 mated, Sprague Dawley rats
Exposure period: GD 6-15
Doses: 0.5 mL 1.6, 3.3, 6.6% test substance was applied (uncovered) to the shaved skin of rats. (6.6% corresponds to
approximately 150 mg/kg bw/day)
Critical effects dams, fetuses: The doses induced local adverse maternal reaction (skin reactions), but not systemic
toxicity. No effects on litter size, post-implantation loss, litter and mean foetal weights were seen. No signs of
embryotoxicity or foetal abnormalities.

NO(A)EL Teratogenicity/Embryotoxicity: 150 mg/kg bw/day
NO(A)EL Maternal toxicity: 150 mg /kg bw/day (Palmer, 1983)

- Vaginal:
Species: Pregnant rats
Exposure period: Single dose on GD 1
Doses: 0, 25, 50, 100 and 200 mg/kg bw/day
Critical effects dams, fetuses: No adverse effects on pregnancy outcome at the lowest dose level. At 50 mg/kg bw/d
and above, there were decreases in the number of live pups per litter and in litter size and weight. No visceral
anomalies were seen, however abnormal bone development (sternal defects), increases in early embryo/foetal death
(resorptions), reduced foetal growth and slight decreases in pregnancy rate were seen at 100 mg/kg bw/day. In all rats given
100 mg/kg bw/day or more vaginal inflammation was seen at necropsy.
NO(A)EL Teratogenicity/Embryotoxicity: 50 mg/kg bw/day
NO(A)EL Maternal toxicity: 50 mg /kg bw/day (Buttar, 1985)

Conclusions:
Under the study conditions, the test substance presented developmental toxicity effects based on the study conducted in rats via vaginal route. The NOAEL for maternal, reproductive and developmental toxicity of the test substance was determined to 50 mg/kg bw/day.
Executive summary:

Studies were conducted to determine the developmental toxicity / teratogenicity of the test substance in rats and rabbits via oral, dermal and vaginal routes. Under the study conditions, the test substance presented maternal and developmental toxicity effects based on the study conducted in rats via vaginal route. The NOAEL for maternal, reproductive and developmental toxicity of the test substance was determined to 50 mg/kg bw/day (BIBRA, 1989, Buttar, 1985 and Palmer, 1983).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
A range finding study and two full studies conducted in rats and rabbits are available meeting the information requirement for this tonnage band.
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The information requirement for this tonnage band is sufficiently met with the available data.
Additional information

Oral and dermal

Study 1. A study was conducted to determine the developmental toxicity and teratogenicity of the test substance according to OECD Guideline 414 (range-finding study). Groups of six mated female rabbits received daily oral administration at 0 (vehicle, purified water), 3, 10 or 30 mg/kg bw/day from Day 6 to 28 post-coitum. In the total absence of maternal toxicity at all dose-levels and in order to better define the top dose-level of the main study, it would be better to perform a complementary study with higher dose-levels of 50 and 100 mg/kg bw/day. The test substance from GD 6-28 produced marked maternal toxicity at 50 and 100 mg/kg bw/day shown by high number of deceased females and severe clinical conditions in most animals. At 30 mg/kg bw/day, a slightly higher mean number of post-implantation loss associated with slightly lower mean number of live foetuses was recorded. No maternal toxicity or effects on litter data parameters were noted at 3 and 10 mg/kg bw/d. Based on these results, the 30 mg/kg bw/day dose-level was chosen as the high dose level in the further developmental toxicity study (Gaoua, 2004).

Study 2. A study was conducted to determine the developmental toxicity and teratogenicity of the test substance according to OECD Guideline 414 and US EPA OPPTS 870.3700, in compliance with GLP. The substance was administered to pregnant rabbits by gavage from Day 6 to 28 post-coitum at the dose-levels of 3, 10 or 30 mg a.i./kg bw/day. The dose of 30 mg a.i./kg bw/day caused the death of three females, severe clinical condition or abortion in two other females and transient, lower maternal body weight gain. Necropsies revealed in 8/22 females accentuated lobular patterns in the liver, whitish areas and/or blackish deposits in the stomach mucosa and dilated intestines. At 10 mg/kg bw/day, relevant necropsy findings were noted in 5/22 females (dilated gall bladder, accentuated lobular pattern, pale liver, brownish or reddish foci on the lungs, blackish deposit on the stomach mucosa). No maternal toxicity or effects on litter data parameters or embryo-foetal development were noted at 3 mg/kg bw/day. Moreover, there were no effects on litter data parameters and no treatment-related findings upon external, visceral or skeletal observation in any of the dose groups up to 30 mg a.i./kg bw/day. Under the study conditions, the rabbit NOAEL for maternal toxicity was 3 mg a.i./kg bw/day while the rabbit NOAEL for embryo-foetal development was considered to be 30 mg a.i./kg bw/day (Gaoua, 2005).

Study 3. A study was conducted to determine the developmental toxicity and teratogenicity of the test substance (81% purity) according to OECD Guideline 414 and US EPA OPP 93 -3, in compliance with GLP. The experiment was performed in pregnant Sprague-Dawley CD rats. The test substance was administered to groups of 25 pregnant rats orally by gavage at dose levels of 0, 10, 30 and 100 mg/kg bw/day (8.1, 24 and 81 mg a.i./kg bw/day), once daily from Days 6 to 15 of gestation inclusive. Control animals were treated with the vehicle alone (Milli-Q water). Clinical observations were made twice daily, and maternal body weights were measured on gestation Days 0, 6, 9, 12, 15, 18 and 21. Maternal food consumption was measured at 3 day intervals from Day 0 to Day 21. All surviving females were sacrificed on Day 21 and the foetuses were examined for visceral and skeletal variations and malformations. No mortality was observed during the study. Treatment-related clinical signs included perioral wetness and audible respiration in high dose group. Audible respiration was also observed in mid dose group. Food consumption was reduced between Days 6 to 9 in mid and high dose groups. There were no effects of treatment on gestational body weight and body weight gain and gravid uterine weight in any dose group. There were also no treatment-related differences in gestational parameters including total number of implantations, number of viable and nonviable implants between the control and the test groups. Further, there were no effects of treatment on fetal body weights per litter, or on the incidences of external, visceral and skeletal malformations and variations. Under the study conditions, the test substance was found to produce some adverse effects in pregnant rats at 30 and 100 mg/kg bw/day, however, no developmental toxicity, including teratogenicity was observed at any of the dosages. The rat NOAEL for maternal toxicity was established 10 mg/kg bw/day (equivalent to 8.1 mg a.i./kg bw/day) and the rat NOAEL for developmental toxicity was determined to be 100 mg/kg bw/day (equivalent to 81 mg a.i./kg bw/day) (Neeper-Bradley, 1992).

Study 4. Studies were conducted to determine the developmental toxicity / teratogenicity of the test substance in rats and rabbits via oral, dermal and vaginal routes. Under the study conditions, the test substance presented maternal and developmental toxicity effects based on the study conducted in rats via vaginal route. The NOAEL for maternal, reproductive and developmental toxicity of the test substance was determined to 50 mg/kg bw/day (BIBRA, 1989, Buttar, 1985 and Palmer, 1983).

Justification for classification or non-classification

The available data suggests that the test substance has no effects on fertility or development. Therefore, no classification for these endpoints is required according to CLP (EC 1272/2008) criteria.