N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Reference 1

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 22, 1993 to August 01, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

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

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

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

VEHICLE
- Concentration in vehicle: 0, 1, 3 and 8 mg/mL (for 30.2% aqueous solution of the test substance)
- Amount of vehicle (if gavage): 10 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Until mating or 7 nights elapsed
- Proof of pregnancy: Vaginal plug or sperm in vaginal smear, referred to as Day 0 of pregnancy
- After 7 d of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged (how): Individual solid bottomed cages (42 x 27 x 20 cm)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On 6 occasions during the study, triplicate samples of dosing solutions were taken and analysed for concentration and homogeneity. The method of analysis was reported the provision of IRI Project 370340.

Duration of treatment / exposure:

Parental: 10 weeks prior to mating to weaning of F1 generation
F1: From Day 25 after birth to weaning of F2 generation.

Frequency of treatment:

Daily

Details on study schedule:

F1 parental animals not mated until 11 weeks after weaning

Remarks:

Doses / Concentrations:
0, 10, 30 and 90 mg/kg bw/day (30.2% aqueous solution)
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
0, 3, 9 and 27 mg/kg bw/day (pure substance)
Basis:
actual ingested

No. of animals per sex per dose:

28/sex for parental generation and 24/sex for F1 generation

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on evaluation of existing data.
- Rationale for animal assignment (if not random): Computer generated randomly sequenced number, but ensuring that siblings were not placed in the same treatment group.

Parental animals: Observations and examinations:

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

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

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

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED
The following parameters were examined in F1/F2 offspring: Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical abnormalities.
GROSS EXAMINATION OF DEAD PUPS: Yes where appropriate, for external and internal abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, termination after weaning of the litters
- Maternal animals: All surviving animals, termination after weaning of the litters

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were weighed: Ovaries, uterus, cervix and vagina, testes (weighed individually), epididymides (weighed individually), seminal vesicle, coagulating gland, prostate gland, pituitary gland. The female reproductive tract was examined for signs of pregnancy and the number of visible implantation sites was recorded.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 7 weeks of age without necropsy.

GROSS NECROPSY
Offspring found dead or killed before Day 14 of lactation were sexed, examined for the externally visible abnormalities and for the presence of milk in the stomach. Offspring dying on or after Day 14 were subjected to a gross necropsy, in which the cranial, thoracic and abdominal contents were examined macroscopically. From each litter of F1 and F2 pups, 2 male and 2 female pups were necropsied at weaning. The necropsy consisted of an external examination, followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities. The remaining pups (except F1 weanlings selected for rearing to produce the next generation) were killed after external examination and the carcasses discarded without necropsy.

Statistics:

Where considered appropriate to assist with interpretation, statistical analysis was applied to determine the statistical significance of differences from control. Organ weight data were analysed by analysis of variance. Testes and epididymides weights were also analysed by analysis of covariance using the terminal body weight as a single covariate. For prostate and seminal vesicles weights, tests for linearity of relationship to body weight and for homogeneity of slopes indicated that analysis of covariance was inappropriate for these organs. Treatment means were compared using an F-protected Least Significant Difference procedure (Snedecor and Cochran, 1980). For other parameters, interpretation was based on examination of the individual and group values.

Reproductive indices:

The following reproductive indices were calculated for each group: Fertility index and gestation index

Offspring viability indices:

The following offspring viability indices were calculated for each litter and group: Birth index, live birth index, viability index, lactation index and overall survival index.

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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

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

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

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

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

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

9 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Based on observed signs of toxicity and a marked reduction in food consumption and body weight gain of males and of females during the premating and gestation periods at 27 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

27 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: No adverse effects on fertility up to the highest tested dose.

Key result

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

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

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

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

9 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity / systemic toxicity

Generation:

F2

Effect level:

9 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Based on slightly reduced pup and litter weights and observed clinical signs (2 pups with body tremors) at 27 mg/kg bw/day.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

Under the study conditions, 30 mg/kg/day produced only minor parental toxicity but no reproductive effects. At 90 mg/kg/day there was very marked parental toxicity, a possible minor effect on pup and litter weights, and 2 pups with body tremors. There were no obvious adverse effects on mating and littering performance at any of the levels tested. The NOAEL for reproductive toxicity was established at 27 mg a.i./kg bw/day and the NOAEL for parental and developmental toxicity was established at the next tested lower dose level of 9 mg a.i./kg bw/day.

Executive summary:

An oral two-generation study was conducted to determine the reproductive toxicity potential of the test substance in rats according to the OECD 416 Guideline, in compliance with GLP. Sprague Dawley rats were dosed orally by gavage, once daily at dose levels, 0, 10, 30 and 90 mg/kg/day (equivalent to 0, 3, 9 and 27 mg a.i./kg bw/day). F0 animals were randomised into the 4 treatment groups, each containing 28 males and 28 females. From each treatment group, 24 male and 24 female F1 weanlings were selected for rearing to maturity and mating to produce the F2 generation. The F0 animals were dosed for 10 weeks prior to mating, and then throughout the mating, gestation and lactation periods until sacrifice at the time of weaning of the F1 animals. The F1 animals were exposed to possible effect of the test substance from conception through to weaning. Clinical observations were performed daily. Body weight and food consumption were recorded at various intervals throughout the study. Females were allowed to litter normally and observations on the females and litters were recorded. All F0 and F1 animals were subjected to necropsy, consisting of an external examination, followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities. Gross lesions were monitored and applicable organs were weighed. Only 2 male and 2 female pups that were weaned from each litter underwent necropsy. No histopathology was performed as this was available from a 90 d repeat oral dose study.

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

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

Under the study conditions, 30 mg/kg/day produced only minor parental toxicity but no reproductive effects. At 90 mg/kg/day there was very marked parental toxicity, a possible minor effect on pup and litter weights, and 2 pups with body tremors. There were no obvious adverse effects on mating and littering performance at any of the levels tested. The NOAEL for reproductive toxicity was established at 27 mg a.i./kg bw/day and the NOAEL for parental and developmental toxicity was established at the next tested lower dose level of 9 mg a.i./kg bw/day (Barton, 1995).

 

In the current version of the OECD Guideline 416, histopathology of the pups is part of the standard procedure. The above study was performed in accordance with the 1983 version of the guideline in which these investigations were not required. However, in the subchronic 90 d study in rats (Mhedhbi, 2003) histopathological examinations were performed. The lack of effects on reproductive performance in the 2-generation study along with the supporting data from the 90 d study confidently allows the conclusion that the substance is not a selective reproductive toxicant.

Reference 2

Endpoint:

multi-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

Guideline not applicable, additional investigations to study A6.8.2(1) Barton 1995.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Doses / Concentrations:
0 and 90 mg test substance/kg bw, corresponding to 0 and 27 mg a.i./kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

28 for parental generation (F0) and 24 for F1 generation

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

All histology findings were typical of the background pathology of the testes seen in this laboratory, and there were no findings that were related to the administration of 90 mg test substance/kg bw/day. It was considered that the original study conclusions remained unchanged.

Key result

Dose descriptor:

NOEL

Remarks:

reproductive toxicity

Effect level:

27 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male

Basis for effect level:

other: no effects on testes

Remarks on result:

other: All generations

Key result

Critical effects observed:

no

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

All histology findings were typical of the background pathology of the testes seen in this laboratory, and there were no findings that were related to the administration of 90 mg test substance/kg bw/day. It was considered that the original study conclusions remained unchanged.

Key result

Dose descriptor:

NOEL

Effect level:

27 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male

Basis for effect level:

other: no effects on testes

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

Under the study conditions, no abnormality was detected in testes up to a test substance concentration of 27 mg a.i/kg bw/day; the reproductive NOEL of 9 mg a.i./kg bw/day for all generations remained the same.

Executive summary:

A study was conducted to determine the effects caused by the test substance on the testes of two generations of Sprague-Dawley rats according to OECD Guideline 416, in compliance with GLP. This experiment is an extension of the previous study which investigated the effects on reproduction (Barton, 1995). The number of animals was 28 for the parental generation (P0) and 24 for P1 generation. The test substance was administered at dose levels of 0 and 90 mg/kg bw/day (corresponding to 0 and 27 mg a.i./kg bw/day) in water daily by oral gavage. The parental generation was treated for the 10 weeks prior to mating until weaning of F1 generation. P1 generation was treated from Day 25 after birth until weaning of the F2 generation. All histology findings were typical of the background pathology of the testes seen in this laboratory and there were no findings that were related to the administration of 27 mg a.i./kg bw/day. It was considered that the original study conclusions remained unchanged. Under the study conditions, no abnormality was detected in testes up to a test substance concentration of 27 mg a.i/kg bw/day; the reproductive NOEL of 9 mg a.i./kg bw/day for all generations remained the same (Barton, 2007).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

27 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

An oral two-generation study was conducted to determine the reproductive toxicity potential of the test substance in rats according to the OECD 416 Guideline, in compliance with GLP. Sprague Dawley rats were dosed orally by gavage, once daily at dose levels, 0, 10, 30 and 90 mg/kg/day (equivalent to 0, 3, 9 and 27 mg a.i./kg bw/day). F0 animals were randomised into the 4 treatment groups, each containing 28 males and 28 females. From each treatment group, 24 male and 24 female F1 weanlings were selected for rearing to maturity and mating to produce the F2 generation. The F0 animals were dosed for 10 weeks prior to mating, and then throughout the mating, gestation and lactation periods until sacrifice at the time of weaning of the F1 animals. The F1 animals were exposed to possible effect of the test substance from conception through to weaning. Clinical observations were performed daily. Body weight and food consumption were recorded at various intervals throughout the study. Females were allowed to litter normally and observations on the females and litters were recorded. All F0 and F1 animals were subjected to necropsy, consisting of an external examination, followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities. Gross lesions were monitored and applicable organs were weighed. Only 2 male and 2 female pups that were weaned from each litter underwent necropsy. No histopathology was performed as this was available from a 90 d repeat oral dose study.

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

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

Under the study conditions, 30 mg/kg/day produced only minor parental toxicity but no reproductive effects. At 90 mg/kg/day there was very marked parental toxicity, a possible minor effect on pup and litter weights, and 2 pups with body tremors. There were no obvious adverse effects on mating and littering performance at any of the levels tested. The NOAEL for reproductive toxicity was established at 27 mg a.i./kg bw/day and the NOAEL for parental and developmental toxicity was established at the next tested lower dose level of 9 mg a.i./kg bw/day (Barton, 1995).

 

In the current version of the OECD Guideline 416, histopathology of the pups is part of the standard procedure. The above study was performed in accordance with the 1983 version of the guideline in which these investigations were not required. However, in the subchronic 90 d study in rats (Mhedhbi, 2003) histopathological examinations were performed. The lack of effects on reproductive performance in the 2-generation study along with the supporting data from the 90 d study confidently allows the conclusion that the substance is not a selective reproductive toxicant.

 

A study was conducted to determine the effects caused by the test substance on the testes of two generations of Sprague-Dawley rats according to OECD Guideline 416, in compliance with GLP. This experiment is an extension of the previous study which investigated the effects on reproduction (Barton, 1995). The number of animals was 28 for the parental generation (P0) and 24 for P1 generation. The test substance was administered at dose levels of 0 and 90 mg/kg bw/day (corresponding to 0 and 27 mg a.i./kg bw/day) in water daily by oral gavage. The parental generation was treated for the 10 weeks prior to mating until weaning of F1 generation. P1 generation was treated from Day 25 after birth until weaning of the F2 generation. All histology findings were typical of the background pathology of the testes seen in this laboratory and there were no findings that were related to the administration of 27 mg a.i./kg bw/day. It was considered that the original study conclusions remained unchanged. Under the study conditions, no abnormality was detected in testes up to a test substance concentration of 27 mg a.i/kg bw/day; the reproductive NOEL of 9 mg a.i./kg bw/day for all generations remained the same (Barton, 2007).

Effects on developmental toxicity

Description of key information

Based on the results of two developmental toxicity studies with rats and rabbits, the test substance is not considered to be a developmental toxicant. The only observed embryotoxic effects (i.e., early embryonic deaths) in both these studies occurred secondary to maternal toxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 07, 2004 to February 02, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: LPT Laboratory of Pharmacology and Toxicology KG, Hamburg, Germany; branch facility: LPT Löhndorf
- Age at study initiation: 4-5 months
- Weight at study initiation: 2.37-3.26 kg
- Housing: Individually in breeding cages with wire floor (surface of ca. 0.2 m2)
- Diet: Ssniff K-Z V2323, ad libitum
- Water: Ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 55 ± 15
- Photoperiod (hrs dark / hrs light): 12/ 12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance preparations were prepared freshly every day. The test substance was suspended in the vehicle to appropriate dose levels.

VEHICLE
- Amount of vehicle (if gavage): 2 mL/kg bw
- Concentration in vehicle: 3, 4.5 and 10 mg/mL

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical determinations of the test substance in aqueous solution and in test substance-carrier were performed.

Details on mating procedure:

- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: Until copulation was ascertained by observation
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy: Signs of copulation referred to as Day 0 of pregnancy

Duration of treatment / exposure:

Days 6-28 of gestation

Frequency of treatment:

Daily

Duration of test:

Until Day 29 of gestation

Remarks:

Doses / Concentrations:
0, 6, 9 and 20 mg/kg bw/day (pure substance)
Basis:
actual ingested

No. of animals per sex per dose:

20 animals per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a dose-range finding study (NOEL of 7.5 mg/kg bw/day, based on reduced food intake and local intolerance reactions).

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily
- Cage side observations included: Behavioral changes, reaction to treatment or illness. In addition animals were observed for viability early in the morning and in the afternoon.
BODY WEIGHT: Yes
- Time schedule for examinations: Daily, starting at Day 0 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day # 29
- Organs examined: Internal organs

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
- Other: Placenta weight was determined.

Fetal examinations:

- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, all per litter
- Skeletal examinations: Yes, all per litter
- Head examinations: Yes, all per litter

Statistics:

The following statistical analysis was used:
For numeric values: Bartlett chi-square test, Dunnett test (for homogeneous variances), Student's t-test (for heterogeneous variances).
For the comparison of classification measurements the Fisher's exact test was employed.
Analytical determinations of Lonzabac in aqueous solution and in test item-carrier were performed.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Maternal toxicity was indicated at 20 mg/kg/day by the death of two dams, by marginally reduced body weight and food intake during the last days of pregnancy. At 20 mg/kg/day, the body weight was marginally reduced during the last gestion days. There was a reduction in food consumption at 20 mg/kg/day from gestation Day 16 onwards, which was up to 36% below the control value (p = 0.05).
Necropsy revealed an irritation of the gastrointestinal tract in 5 dams and an increased incidence of resorptions.

Key result

Dose descriptor:

NOAEL

Effect level:

9 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

Key result

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- At 20 mg/kg bw/day there was a marginal increased incidence of resorptions suggesting early embryonic deaths secondary to maternal toxicity.
- No test substance-related influence was detected on the prenatal foetal development. The incidences of foetal malformations, variations and retardations were similar in all groups. Under the conditions of the study no teratogenic effects (as indicated by the incidences of foetal malformations, variations and retardations) were found up to 20 mg/kg bw/day of the test substance.

Key result

Dose descriptor:

NOAEL

Effect level:

9 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Basis for effect level:

other: embryotoxicity

Key result

Dose descriptor:

NOAEL

Effect level:

> 20 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Basis for effect level:

other: teratogenicity

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Under the study conditions, the test substance was not considered to be a developmental toxicant. The NOAELs for maternal effects and embryotoxicity was set at 9 mg a.i./kg bw/day and the NOAEL for teratogenicity was set at 20 mg a.i./kg bw/day respectively.

Executive summary:

A pre-natal developmental toxicity study was conducted to determine the developmental or teratogenic potential of the test substance to Himalayan rabbits according to the OECD 414 Guideline, in compliance with GLP. Groups of 20 pregnant females received the substance by gavage at dose levels of 0, 6, 9 and 20 mg a.i./kg bw/day during days 6-28 of gestation. Animals were sacrificed on gestation day 29.

Maternal toxicity was indicated at 20 mg a.i./kg bw/day by the death of two dams and by marginally reduced body weight and food intake during the last days of pregnancy. Necropsy revealed an irritation of the gastrointestinal tract in 5 dams and an increased incidence of resorptions. Marginal increased incidence of resorptions suggesting early embryonic deaths secondary to maternal toxicity was observed at 20 mg a.i./kg bw/day. No substance-related influence was detected on the pre-natal foetal development. The incidences of foetal malformations, variations and retardations were similar in all groups. As a result, the NOAELs for both maternal and embryotoxicity were set at 9 mg a.i./kg bw/day and the NOAEL for teratogenicity was set at 20 mg a.i./kg bw/day.

Under the study conditions, the test substance was not considered to be a developmental toxicant. The NOAELs for maternal effects and embryotoxicity was set at 9 mg a.i./kg bw/day and the NOAEL for teratogenicity was set at 20 mg a.i./kg bw/day respectively (Leuschner, 2005).

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 02, 1993 to January 12, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, England
- Age at study initiation: ca. 9 weeks
- Weight at study initiation: ca. 245 g (males), ca. 9 weeks (females)
- Housing: females: Individually in polypropylene cages with stainless steel grid bottoms and mesh tops, measuring 42 x 27 x 20 cm. Prior to mating females were housed in pairs in this type of cage. Males: singly in cages of similar design, measuring 58 x 38.5 x 20 cm.
- Diet: Rat and Mouse Breeder Diet No. 3 (Expanded) SQC, ad libitum
- Water: Domestic mains water, ad libitum
- Acclimation period: 6 d

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

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Fresh solutions were prepared daily. For high concentration group, the requisite quantity of the test substance was weighed into a labelled container. The necessary volume of vehicle was added, and mixing was by inversion, as required. For lower concentrations aliquots of the high dose solution were diluted with the vehicle to give required concentrations.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw
- Concentration in vehicle: 2.5, 7.7, 20 mg/mL (calculated for pure substance)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A single 10 mL sample was taken from each concentration on the 1st and 8th d of dosing and triplicate sub-samples were analyzed for concentration and homogeneity in the IRI Analytical Chemistry Laboratory by method # 7034.

Details on mating procedure:

- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: Continuous until mating was detected
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy: Vaginal plug or sperm in vaginal smear referred to as Day 0

Duration of treatment / exposure:

Gestations Day 6-16

Frequency of treatment:

daily

Duration of test:

Until Day 20 of gestation

Remarks:

Doses / Concentrations:
0, 25, 75, 200 mg/kg bw/day (test substance as 30.2% aqueous solution)
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
0, 7.5, 22.5 and 60 mg/kg bw/day (pure substance)
Basis:
actual ingested

No. of animals per sex per dose:

25 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on a separate dose range finding study
- Rationale for animal assignment (if not random): Computer generated series of randomly sequenced numbers, ensuring that siblings and females mated by the same male were not placed in the same treatment group.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily. In addition animals were checked for viability at the beginning of each day and as late as possible on each day.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 6, 9, 13, 17 and 20 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Daily, commencing on Day 3 of gestation

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day # 20
- Organs examined: The contents of thoracic and abdominal cavity

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:

- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, all per litter
- Skeletal examinations: Yes, half per litter
- Head examinations: No

Statistics:

Weight gain was analysed using the Kruskal-Wallis test, with comparisons with the control using a modified Dunn's procedure. Food consumption was analysed by parametric analysis of variance, with homogeneity of variance checked using Levene's test and normality of data by the Shapiro-Wilk test. Comparisons with the control were made using Dunnett's t-test. For all other parameters, interpretation was based on examination of the individual and group mean values.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
- All animals at 60 mg/kg bw/day and many at 22.5 mg/kg bw/day showed reaction to treatment. The principle findings were dyspnoea, salivation with associated brown staining, piloerection and indications of reduced activity (such as hunched, ataxic and cold to touch). Two animals at 60 mg/kg bw/day were killed prematurely. Necropsy findings included intestinal distension, and therefore the similar distension observed for a further animal was also considered to be associated with treatment. The other clinical observations and necropsy findings were considered to be either associated with the above findings or to be incidental.

- There was a dose related reduction in weight gain over the dosing period at 22.5 and 60 mg/kg bw/day, which had become apparent by Day 9 of gestation. At 60 mg/kg/day, weight gain over Days 6-17 was significantly lower than control (P<0.0 1) and 5 animals at this level showed weight loss between Days 9 and 13 of gestation. Gain over Days 6-17 at 22.5 mg/kg/day, although lower than control, was not statistically significant (P> 0.05). Body weight performance at 7.5 mg/kg bw/day was essentially similar to control.

- There was a reduction in food consumption at 60 mg/kg/day, which persisted throughout the dosing period. At 22.5 mg/kg/day, food consumption was marginally reduced. At both these levels, food consumption over the dosing period was significantly lower than control (P<0.01). Food consumption at 7.5 mg/kg/day was similar to Control.

Key result

Dose descriptor:

NOAEL

Effect level:

7.5 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

Key result

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- The mean number of implants at 7.5 and 22.5 mg/kg/day was slightly lower than control or at 60 mg/kg/day. However, this could not be attributed to treatment because the number of implants would have been established prior to commencement of treatment.

- The incidence of early embryonic deaths, and hence of total dead implants was greater at 60 mg/kg bw/day than in control. This increase was largely associated with three animals which had large numbers of early deaths. The incidence of dead implants at 7.5 and 22.5 mg/kg/day was similar to control.

- Mean foetal weight at 60 mg/kg bw/day was slightly lower than control, with the difference approaching but not achieving statistical significance(P>0.05).

-The number and type of major foetal abnormalities were considered to be typical of those seen in historical data. Taken together, the incidences of minor abnormalities and variants were typical of those that would be expected from historical data and were similar in all groups. The group values indicating the state of skeletal ossification were similar in all groups. Slight differences were considered too small to be attributed to treatment.

Dose descriptor:

NOAEL

Effect level:

22.5 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Basis for effect level:

other: embryotoxicity

Dose descriptor:

NOAEL

Effect level:

> 60 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Basis for effect level:

other: teratogenicity

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

Conclusions:

Under the study conditions, the test substance was not considered to be a developmental toxicant. The NOAELs for maternal effects, embryotoxicity and teratogenicity was set at 7.5, 22.5 and 60 mg a.i./kg bw/day respectively.

Executive summary:

A pre-natal developmental toxicity study was conducted to determine the developmental or teratogenic potential of the test substance in Sprague Dawley rats according to the OECD 414 Guideline, in compliance with GLP. Groups of 25 female rats received the test substance (as 30.2 % aqueous solution) by gavage at dose levels of 0, 25, 75, 200 mg/kg bw/day (i.e., corresponding to 0, 7.5, 22.5 and 60 mg a.i./kg bw/day) during gestation day 6 -16. Animals were sacrificed on gestation day 20 and assessed for the number of implantation sites, number of corpora lutea, early and late resorptions and gravid uterus weight. Foetuses were counted, sexed, weighed and analysed for external and internal (soft tissue and skeletal) abnormalities.

The maternal animals showed decreased food consumption and body weight gain at the medium and highest dose level. The NOAEL for maternal effects was set at 7.5 mg a.i./kg bw/day. At the highest dose level an increase in incidence of early embryonic deaths and slightly decreased (not statistically significant) mean foetal weight was observed, which was considered to be secondary to maternal toxicity. No further developmental toxicity was observed and hence the NOAEL for embryotoxicity was set at 22.5 mg a.i./kg bw/day. Further, in the absence of any foetal malformations, anomalies and variants, the NOAEL for teratogenicity can be considered to be at the highest tested dose level 60 mg a.i./kg bw/day. Under the study conditions, the test substance was not considered to be a developmental toxicant. The NOAELs for maternal effects, embryotoxicity and teratogenicity was set at 7.5, 22.5 and 60 mg a.i./kg bw/day respectively (Barton, 1994).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

9 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

Reliable guideline-compliant studies were available meeting the tonnage information requirements.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A pre-natal developmental toxicity study was conducted to determine the developmental or teratogenic potential of the test substanceinSprague Dawley rats according to the OECD 414 Guideline, in compliance with GLP.Groups of 25 female rats received the test substance (as 30.2 % aqueous solution) by gavage at dose levels of 0, 25, 75, 200 mg/kg bw/day (i.e., corresponding to 0, 7.5, 22.5 and 60 mg a.i./kg bw/day) during gestation day 6 -16. Animals were sacrificed on gestation day 20 and assessed for the number of implantation sites, number of corpora lutea, early and late resorptions and gravid uterus weight. Foetuses were counted, sexed, weighed and analysed for external and internal (soft tissue and skeletal) abnormalities. The maternal animals showed decreased food consumption and body weight gain at the medium and highest dose level. The NOAEL for maternal effects was set at 7.5 mg a.i./kg bw/day. At the highest dose level an increase in incidence of early embryonic deaths and slightly decreased (not statistically significant) mean foetal weight was observed, which was considered to be secondary to maternal toxicity. No further developmental toxicity was observed and hence the NOAEL for embryotoxicity was set at 22.5 mg a.i./kg bw/day. Further, in the absence of any foetal malformations, anomalies and variants, the NOAEL for teratogenicity can be considered to be at the highest tested dose level 60 mg a.i./kg bw/day. Under the study conditions, the test substance was not considered to be a developmental toxicant. The NOAELs for maternal effects, embryotoxicity and teratogenicity was set at 7.5, 22.5 and 60 mg a.i./kg bw/day respectively (Barton, 1994).

A pre-natal developmental toxicity study was conducted to determine the developmental or teratogenic potential of the test substance toHimalayan rabbits according to the OECD 414 Guideline, in compliance with GLP.Groups of 20 pregnant females received the substance by gavage at dose levels of 0, 6, 9 and 20 mg a.i./kg bw/day during days 6-28 of gestation. Animals were sacrificed on gestation day 29. Maternal toxicity was indicated at 20 mg a.i./kg bw/day by the death of two dams and by marginally reduced body weight and food intake during the last days of pregnancy. Necropsy revealed an irritation of the gastrointestinal tract in 5 dams and an increased incidence of resorptions. Marginal increased incidence of resorptions suggesting early embryonic deaths secondary to maternal toxicity was observed at 20 mg a.i./kg bw/day. No substance-related influence was detected on the pre-natal foetal development. The incidences of foetal malformations, variations and retardations were similar in all groups. As a result, theNOAELs for both maternal and embryotoxicity were set at 9 mg a.i./kg bw/day and the NOAEL for teratogenicity was set at 20 mg a.i./kg bw/day. Under the study conditions, the test substance was not considered to be a developmental toxicant. The NOAELs for maternal effects and embryotoxicity was set at 9 mg a.i./kg bw/day and the NOAEL for teratogenicity was set at 20 mg a.i./kg bw/day respectively (Leuschner, 2005).

Justification for classification or non-classification

Based on the results of the reproductive and developmental toxicity studies, the substance does not need to be classified for these endpoints according to CLP (EC 1272/2008) criteria.