Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
concentration-driven

Effects on fertility

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 2008 - August 2009
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)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Deutschland, Sulzfeld, Germany
- Age at study initiation: (P - 6-7 weeks)
- Weight at study initiation: (P) Males: 173-206 g; Females: 140-175 g; (F1) Males: 36-72 g; Females: 40-75 g
- Housing: Macralon cages with wood shavings as bedding and strips of paper for environmental enrichment, during premating animals were housed by sex 4/cage, during mating - 1 male plus 1 female, following mating - females were housed individually and with her litter following delivery.
- Diet: Ad libitum except during exposure
- Water: Ad libitum except during exposure
- Acclimation period: Approximately 1-2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 40-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: March - November 2008
Route of administration:
inhalation
Type of inhalation exposure (if applicable):
other: Nose only except during lactation when whole body exposure is used.
Vehicle:
other: unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
A) Nose-only exposure unit
- Exposure apparatus: Nose only -cylindrical PVC column with a volume of ca. 70 L, surrounded by a transparent hood.
- Method of holding animals in test chamber: Plastic animal holders
- Humidified compressed air
- System of generating particulates/aerosols: Flow of gas will be regulated by volumetric flow meters to achieve the desired concentrations in the exposure apparatus
- Temperature, humidity, : 22 ± 2ºC, 30-77%

B) Whole body exposure unit
- Exposure apparatus: Whole body, 2 m3 whole body inhalation chambers made of stainless steel.
- Method of holding animals in test chamber: Housed in individual cages
- System of generating atmosphere: Flow of gas will be regulated by volumetric flow meters to achieve the desired concentrations in the exposure apparatus
- Temperature, humidity: 22 ± 2ºC, 30-77%

TEST ATMOSPHERE
- Brief description of analytical method used: Total carbon content by flame ionization GC
- Samples taken from breathing zone: Yes

Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Up to 2 weeks
- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 of pregnancy
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Total carbon analysis
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
F0 generation animals were exposed to the test material in air for 6 hours a day, 5 days a week for at least 10 weeks prior to mating and daily during mating. Daily exposure continued for F0 females through gestation day (GD) 19 for 6 hours/day (nose-only). From lactation day 5 - 21, females were exposed daily (6 hours/day) to the test item by whole body exposure.

The F1-generation male and female pups were exposed by whole body exposure(6 hours/day, 5 days/week) from postnatal (PN) day 22 up to ca. 6 weeks of age. Subsequently, the animals were exposed in a similar manner as that described for the F-0 generation.
Details on study schedule:
- Selection of parents from F1 generation when pups were 21 days of age.
- F1 parental animals not mated until 10 weeks after selection from the F1 litters.

Dose / conc.:
5 000 ppm
Remarks:
Group 2: Low dose.
Dose / conc.:
15 000 ppm
Remarks:
Group 3: Mid dose.
Dose / conc.:
50 000 ppm
Remarks:
Group 4: High dose.
No. of animals per sex per dose:
28
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: High dose same as used for repeated dose toxicity studies. Concentrations higher than 50000 ppm can result in secondary effects due to oxygen deprivation.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: daily
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: daily

BODY WEIGHT:
- Time schedule for examinations: 2 days prior to exposure initiation, day 0, weekly (males, premating females), gestation days 1,7,14,21, lactation days 1,4,7,14, 21
Oestrous cyclicity (parental animals):
Vaginal smears daily for 3 weeks prior to mating.
Sperm parameters (parental animals):
Parameters examined in all male parental generations:
- Testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible)

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 or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
- Yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals as soon as possible after the last litters in each generation were produced.
- Maternal animals: All surviving animals after the last litter of each generation was weaned.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination and weighed, respectively.
- adrenals
- brain
- epididymides *(left cauda which will be used for sperm analysis
- kidneys
- liver
- ovaries including oviduct*
- pituitary gland*
- prostate*
- seminal vesicles and coagulating glands*
- spleen
- testes*(right testis will be preserved in Bouin’s fixative, the left one will be used for sperm analysis
- thyroid
- uterus* (after counting of the implantation sites) (Salewski 1964)
- vagina*
- organs and tissues showing macroscopic abnormalities
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations (macroscopic a)

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination and weighed, respectively.
- brain
- spleen
- thymus
- organs and tissues showing macroscopic abnormalities

Skeletal examinations were carried out on F1 pups
Statistics:
Other statistical tests may be performed when considered appropriate. P < 0.05 will be considered as a level of significance.
- Clinical findings will be evaluated by Fisher's exact probability test.
- Body weight, body weight gain, organ weights and food consumption data will be subjected to one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests.
- Fisher's exact probability test will be used to evaluate the number of mated and pregnant females and females with live pups.
- Number of implantation sites, live and dead fetuses or pups will be evaluated by Kruskal-Wallis nonparametric analysis of variance followed by the Mann Whitney U-test.
- Mortality data and data of the pathology of parent females will be evaluated by the Fisher’s exact probability test.
Reproductive indices:
- Pre-coital time = time between the start of mating and successful copulation
- Duration of gestation = time between gestation day 0 and day of delivery
- Mating index= (number of females mated/number of females placed with males) x 100
- Male fertility index = (number of males that became sire/number of males placed with females) x 100
- Female fertility index = (number of pregnant females/number of females placed with males) x 100
- Female fecundity index = (number of pregnant females/number of females mated) x 100
- Gestation index = (number of females with live pups or pups/number of females pregnant) x 100
- Live birth index = (number of pups born alive/number of pups born) x 100
- Viability index day 4-21= (number of pup surviving 21 days/number of liveborn after culling day 4) x100
- Pup mortality day n = (number of dead pups on day n/total number of pups on day n) x 100
- Sex ratio day n = (number of live male fetuses or pups on day n/ number of live fetuses or pups on day n) x 100
- Number of lost implantations = number of implantations sites - number of pups born alive
- Post-implantation loss = [(number of implantation sites - number of pups born alive)/number of implantation sites] x 100
Offspring viability indices:
- Number of pups delivered (live- and stillborn)
- Number of live pups at day 1, 4, 7, 14, 21
- Number of pups lost measured as above
- Number of litters lost entirely
- Number of male pups at day 1, 4, 7, 14, 21
- Number of implantation sites
- Number of lost implantations
- Litter size
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The clinical observations observed in the animals of the F0 parental generation are common findings in rats of this strain and age or occurred as individual fortuitous findings. Furthermore, they were about equally distributed amongst the different treatment groups or occurred in only one or a few animals. Therefore, they were not considered to be related to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decreases of body weight and body weight change were observed in the test substance exposed groups during the premating period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decreases of food consumption was observed in the test substance exposed groups during the premating period.
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, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No statistically significant changes were observed. A few changes were noted but they were common findings for this strain of rat, were equally distributed amonst the different treatment groups or only occured in one or few animals.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Although a statistically significant increase of the mean cycle length was observed in the females of the mid-concentration group when compared to the control group and the length of the longest cycle was statistically significantly decreased in the females of the low- and mid dose groups of the F0-generation, this effect was not considered to be a test substance related effect as no effect was observed in the high-concentration group of the F0-generation and no effect on estrus cycle was observed in the test substance-exposed groups of the F1-generation (see attached tables).
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
Duration of the gestation period was slightly increased in the test substance-exposed groups and was statistically significant in the low and high dose group of the F0 generation. The gestation period did not exceeded the 22 days, which is a normal duration of gestation period for this strain of rats, in the control or treatment related dams. Therefore, this effect on mean gestation length is not considered as an adverse effect.
Key result
Dose descriptor:
NOAEC
Effect level:
> 50 000 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The clinical observations observed in the animals of the F1 parental generation are common findings in rats of this strain and age or occurred as individual fortuitous findings. Furthermore, they were about equally distributed amongst the different treatment groups or occurred in only one or a few animals. Therefore, they were not considered to be related to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In F1-females, there were test substance-related reductions in mean body weights at all doses tested during the first three weeks of the premating period. Mean body weights on approximately post-natal days 28 , 35, and 42 (week 0, 1 and 2 of the F1-generation, respectively) were up to 13, 15, and 14% lower than controls at 5000, 15000, and 50000 ppm, respectively. This period of time corresponded with the initiation of direct exposure. Although test substance-related and toxicologically relevant to the onset of puberty, these reductions were not considered adverse because they were transient and by the end of the premating period, mean body weights for all groups were within 3% of the control mean. Lack of a strong dose-related response, the relative low magnitude of the change, and the fact that the body weight data were consistent with the food consumption data supported that these effects were not considered to be adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See 'body weight and weight changes' field.
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, non-treatment-related
Description (incidence and severity):
Although some statistically significant changes were observed, they were not related to dose.The decrease in organ weights and the decrease and increase in relative organ weights was related to the reducted body weights of the exposed groups and was not considered adverse.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination at necropsy did not reveal treatment related gross changes.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No effect on estrus cycle was observed in the test substance-exposed groups of the F1-generation.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Duration of the gestation period was slightly increased in the test substance-exposed groups and was statistically significant in the mid- and high concentration groups of the F1 generation. The gestation period did not exceeded the 22 days, which is a normal duration of gestation period for this strain of rats, in the control or treatment related dams. Therefore, this effect on mean gestation length is not considered as an adverse effect.
Key result
Dose descriptor:
NOAEC
Effect level:
> 50 000 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The statistically significant increase of pup mortality (8.5%) as observed in the high-concentration group of the F1-generation on PN 4 was within the historical range (0-20.7%) and therefore not considered to be an adverse effect.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Pup body weights and body weight changes were considered not to be affected by exposure to the test substance. In F1-females, there were test substance-related reductions in mean body weights at all doses tested during the first three weeks of the premating period for this generation. Mean body weights on post-natal days 28, 35, and 42 were up to 13, 15, and 14% lower than controls at 5000, 15000, and 50000 ppm, respectively. This period of time corresponded with the initiation of direct exposure. Although test substance-related and toxicologically relevant to the onset of puberty, these reductions were not considered adverse because they were transient and by the end of the premating period, mean body weights for all groups were within 3% of the control mean. In addition, and similar to the effects seen in males, there was not a strong dose-related response and the data was consistent with food consumption data for these females.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See 'body weight and weight changes' field.
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
Description (incidence and severity):
see details below
Anogenital distance (AGD):
effects observed, treatment-related
Description (incidence and severity):
In F1-females, there was an apparent delay in the onset of puberty evident as a delay in days to achievement of vaginal opening. These apparent delays were not considered to reflect a direct effect on this endpoint but rather, were considered secondary to previously described test substance-related reductions in body weight and food consumption parameters that were evident during the first three weeks of exposures and concurrent with the onset of vaginal patency. The relationship between onset of puberty in rats and body weight and food consumption data was described in a published feed restriction study in which it was demonstrated that pubertal delays of up to 6 days were produced in rats with weight gain reductions that were induced by feed restriction (Carney et al., 2004 - article attached).
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The decrease in absolute and relative thymus weight and in relative thymus weightof the low-concentration group were considered not to be treatment-related as no relation with concentration was observed. In addition, no histopathology findings were noted.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Two dams of the high-concentration group gave birth to 3 pups with malformations. One pup showed acauda and anal atresia and another pup of the same dam showed polypodia of the right hind limb. A pup of another dam showed acauda. As no malformations were observed in the prenatal developmental toxicity study with the test substance, this finding was not considered to be a treatment-related effect
Histopathological findings:
no effects observed
Description (incidence and severity):
Microscopic observation of the thymus of the control and high-concentration groups of the F1-generation did not reveal any treatment-related effects. For that reason the decrease detected in absolute and relative thymus weight of the high-concentration group was not considered to be a relevant effect. No statistically significant findings were observed. The changes observed were common findings in rats of this strain and age or occured as indicifual fortiutous findings. They were also equally distributed amongst different treatment groups of in only one or few animals and were not considered to be treatment related.
No skeletal abnormalities, such as wavy ribs (observed in the rat developmental toxicity study) were noted in the F1 pups.
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
> 50 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'
Key result
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
> 50 000 ppm
Based on:
test mat.
Sex:
male
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not specified
Body weight and weight changes:
not specified
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
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The decrease in relative thymus weight of the low-concentration group were considered not to be treatment-related as no relation with concentration was observed. In addition, no histopathology findings were noted.
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEC
Generation:
F2
Effect level:
> 50 000 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
Based on the data of this two-generation reproduction toxicity study in rats, the high-concentration group (50000 ppm) is considered to be NOAEL (No Observed Adverse Effect Level) for systemic toxicity and reproductive toxicity.
Executive summary:

No reproductive or systemic toxicity directly attributed to the test material were noted. While in the F1-generation females a delay in the onset of puberty was seen in the high exposure level group, this was attributed to a reduction of body weight gain seen in these animals early in exposure. It is well-established that the onset of puberty in rats is a body weight-dependent event. In feed restriction studies designed to examine the relationship between body weight and the onset of puberty evident as vaginal patency and preputial separation, it has been demonstrated that lower body weights and weights gains can delay the onset of puberty in rats by up to 6 days (Carney et al., 2004). In the current study, vaginal patency onset generally occurred during the third week of exposures for the F1-generation females (first week of exposure was designated week 0, see report Table 9). At the end of the second and third weeks of exposures (weeks 1 and 2, Table 9), mean body weights in treated F1-generation females were up to 14% lower than controls. On the basis of these data and considering the lack of any other corroborative evidence of either developmental or reproductive toxicity, these apparent delays in vaginal patency are considered secondary to lower body weights. There were no effects on mating, fertility, or other structural or functional indicators of reproductive performance in either generation. In compliance with the testing guidelines for this study, measurement of anogenital distance in F2-generation offspring was triggered by the observed apparent delays in the F1-generation females and no test substance-related changes were observed for this endpoint. Therefore, the apparent delay in onset of puberty in F1-generation females is not considered indicative of specific developmental or reproductive toxicity but rather is considered a consequence of test substance-related reductions in body weights. Considering this conclusion and taking into consideration the lack of any other evidence of test substance-related effect on either the structure or function of the reproductive system or on the offspring exposed in utero and via milk intake during lactation, the NOAEC for reproductive and systemic toxicity for the current study is considered 50000 ppm (233000 mg/m3), the highest exposure concentration tested.

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
233 000 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
The study is a guideline study in compliance with GLP.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A 2-generation inhalation toxicity study was conducted at exposure levels of 5000, 15000 and 50000 ppm (equivalent to 23300, 69900, and 233000 mg/m3, respectively) in Wistar rats according to OECD guideline 416. Exposures were 6 hours a day, 5 days a week except during mating, gestation and lactation periods when exposures were 7 days/week.  A delayed vaginal opening was observed in female pups at 50000 ppm. However, this was a secondary effect to test substance-related reductions in body weight and food consumption parameters that were evident during the first three weeks of exposures and concurrent with the onset of vaginal patency. Although test substance-related and toxicologically relevant to the onset of puberty, these reductions were not considered adverse because they were transient and by the end of the premating period, mean body weights for all groups were within 3% of the control mean. Lack of a strong dose-related response, the relative low magnitude of the change, and the fact that the body weight data were consistent with the food consumption data supported that these effects were not considered to be adverse. There were no dose-related changes in estrus cycle. The number of pregnant animals was comparable in all groups. In both generations, the number of live and dead pups at delivery, the viability of the pups, the sex ratio, pup body weights and body weight changes were all considered not to be affected by exposure to the test substance. No adverse effects were seen in sexual development of the male or female rats. The no-observed-adverse-effect-concentration for this study was 50000 ppm (233000 mg/m3).


Short description of key information:
In the 2-generation reproductive toxicity study in rats, the only effect noted was a delayed vaginal opening in the female pups at 50000 ppm (233000 mg/m3). These apparent delays were not considered to reflect a direct effect on this endpoint but rather, were considered secondary test substance-related reductions in body weight and food consumption parameters that were evident during the first three weeks of exposures and concurrent with the onset of vaginal patency. No adverse effects were observed in any other reproductive or developmental parameters. The no-observed-adverse-effect-concentration (NOAEC) for reproductive and systemic toxicity was 50000 ppm (233000 mg/m3) for this study.

Effects on developmental toxicity

Description of key information

No maternal or fetal toxicity was observed in rats exposed by inhalation to concentrations up to 50000 ppm during gestation. Maternal mortality was observed in rabbits exposed to levels of 5500 ppm (25630 mg/m3) and above. Non-statistically significant visceral malformations of the heart and/or great vessels were observed in a few pups but only at concentrations resulting in maternal mortality. In addition, cardiac lesions of unknown significance were seen in maternal animals exposed to 2500 ppm and higher. Based on repeated inhalation, cardiotoxicity mechanism studies in the minipig, the cardiotoxicity observed in the rabbits is not appropriate for use in human health risk assessments.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan - May 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Covance Research, Kalamazoo, MI
- Age at study initiation: 5-9 months on gestation day 0
- Weight at study initiation: 2.9 - 4.5 kg on gestation day 0
- Housing: Individual in suspended stainless steel cages elevated above ground corncob bedding
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 4-5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-22
- Humidity (%): 30-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light):12

IN-LIFE DATES: From: Jan 2008 To: June 2008
Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel and glass whole body exposure chambers (1500 to 2000 liters)
- Method of holding animals in test chamber: Individual cages
- Source and rate of air: HEPA and charcoal filtered
- System of generating test substance atmosphere: Metering of gas from headspace of storage cylinder controlled by appropriate valves and flow meters
- Temperature, humidity, in air chamber: 65.3°F to 66.1°F (18.5°C to 19.0°C), mean daily relative humidity ranged from 50.0% to 58.5% during the study
- Air change rate: At least 10/hour

TEST ATMOSPHERE
- Brief description of analytical method used: Samples taken at least hourly for analysis by gas chromatography
- Samples taken from breathing zone: Yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Gas chromatography
Details on mating procedure:
- Impregnation procedure: Purchased timed pregnant
Duration of treatment / exposure:
Gestation days 6-28
Frequency of treatment:
6 hours a day
Dose / conc.:
4 000 ppm
Remarks:
Group 2: Low dose.
Dose / conc.:
5 500 ppm
Remarks:
Group 3: Mid dose.
Dose / conc.:
7 500 ppm
Remarks:
Group 4: High dose.
No. of animals per sex per dose:
24
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: Based on rabbit teratogenicity range finding study which indicated significant maternal mortality at 10000 ppm and 50000 ppm.
Maternal examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Daily

BODY WEIGHT:
- Time schedule for examinations: Daily

FOOD CONSUMPTION:
- Food consumption for each animal was determined and mean daily diet consumption was calculated as g food/kg body weight/day.

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day # 29
- Organs examined: Heart, lungs, liver, kidney, brain

CAGE SIDE OBSERVATIONS:
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Daily

BODY WEIGHT:
- Time schedule for examinations: GD 0, 4, 6-29 (daily)

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day # 29
- Organs examined: Uterus, liver, kidney, brain, heart, lungs, all gross lesions
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, all per litter
Statistics:
Two tailed for a minimum significance of 5% and 1% with means and standard deviations presented. All statistical tests were done by computer with appropriate programming. Data obtained from nongravid animals were excluded from statistical analyses. Due to the different rounding conventions inherent in the types of software used, the means, standard deviations and standard errors on the summary and individual tables may differ by ±1 in the last significant figure. Where applicable, the litter was used as the experimental unit.

Mean maternal body weights (absolute and net), body weight changes (absolute and net) and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites and viable fetuses and fetal body weights (separately by sex and combined) were subjected to a parametric one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-exposed groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal and combined) and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance-exposed groups to the control group.
Historical control data:
Included in report
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Some of the animals which were found dead or killed in extremis (2 of 4 at 5500 ppm and and 3 of 7 at 7500 ppm) also exhibited labored and/or decreased respiration and/or hypoactivity prior to death or euthanasia.
No test substance related clinical findings were noted in females that survived to scheduled necropsy.
Dermal irritation (if dermal study):
no effects observed
Mortality:
mortality observed, treatment-related
Description (incidence):
In total, 4 and 7 females in the 5500 and 7500 ppm groups, respectively, were found dead or euthanized in extremis. All other females survived to the scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Lower mean body weight gain was noted in the 7500 ppm group during gestation days 12-20 with corresponding occasional reductions in mean daily food consumption. Because several females died or were euthanized prior to the scheduled necropsy, mean net body weight and net body weight change in this group were not significantly different from the control group. Mean body weights in the 7500 ppm group were similar to the control group throughout the study. A slightly lower mean body weight gain and a mean body weight loss were observed in the 5500 ppm group during gestation days 12-20 and 20-29, resulting in a lower mean body weight gain when the entire exposure period (gestation days 6-29) was evaluated. A large mean net body weight loss was observed in this group.
Correspondingly lower mean food consumption was observed in this group during gestation days 20-29. However, mean body weights throughout the study and mean net body weight in the 5500 ppm group were similar to the control group. Slightly lower mean body weight gains were observed in the 4000 ppm group during gestation days 12-20 and 20-29, resulting in a lower mean body weight gain when the entire exposure period (gestation days 6-29) was evaluated.
However, because there were no test substance-related effects on mean food consumption, mean net body weight or net body weight change, and there were no clinical or macroscopic findings indicative of systemic toxicity observed in this group, the lower mean body weight gain noted in the 4000 ppm group was not considered adverse.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
See 'body weight and weight changes' field.
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:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Mean gravid uterine weights were unaffected by exposure to the test substance at all concentrations.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All females found dead or euthanized in extremis were gravid with normally developing implantations or fetuses in utero.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histological examination revealed these deaths were of undetermined causation (4/5 at 5500 ppm, 6/10 at 7500 ppm), due to subacute inflammation of the heart (1/5 at 5500 ppm and 3/10 at 7500 ppm) or a result of edema/hemorrhage of the lung (1/10 at 7500 ppm). Test substance exposure was also associated with subacute inflammation in the heart in the 2500 (n=8), 4000 (n=12), 5500 (n=10), and 7500 (n=15) ppm groups, coagulation necrosis of the heart in the 7500 (n=1) ppm group, and renal tubular necrosis in the 5500 n=3) and 7500 (n=1) ppm groups. At 5500 ppm, renal necrosis was noted in 2 animals that died or were euthanized. Although not dose related, these changes were considered adverse.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
One and 3 females in the 5500 and 7500 ppm groups, respectively, aborted on gestation day 26, 28 or 29. The abortions in the 5500 and 7500 ppm groups were attributed to the test substance; no test substance-related macroscopic findings were observed in these females.
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
One female at 5500 ppm had a late resorption and one female at 7500 ppm had an early resorption in utero.
Changes in pregnancy duration:
effects observed, treatment-related
Description (incidence and severity):
One female in the 7500 ppm group delivered on gestation day 29. The premature delivery in the 7500 ppm groups were attributed to the test substance; no test substance-related macroscopic findings were observed in these females.
Other effects:
no effects observed
Description (incidence and severity):
Corpea lutea were unaffected by exposure to the test substance at all concentrations.
Key result
Dose descriptor:
LOAEC
Remarks:
General toxicity
Effect level:
> 2 500 ppm
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic
mortality
Key result
Dose descriptor:
LOAEC
Remarks:
Maternal developmental toxicity
Effect level:
> 2 500 ppm
Based on:
test mat.
Basis for effect level:
effects on pregnancy duration
number of abortions
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Mean fetal/pup weight by sex and with sexes combined were not affected by test substance exposure at any concentration.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio were not affected by test substance exposure at any concentration.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Mean number and percent of live offspring were not affected by test substance exposure at any concentration.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No test substance-related developmental variations were observed at any exposure level.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No test substance-related developmental variations were observed at any exposure level.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related visceral malformations of the heart and/or great vessels were observed at frequencies above the WIL historical control values in the maternally toxic 5500 (2 fetus total; 2 litters) and 7500 (3 fetus total; 2 litters) ppm groups. These findings, were not statistically different from the concurrent study control group, consisted of bulbous aorta, stenotic pulmonary arch, interventricular septal defect (absent septa), absent tricuspid valve and/or interrupted aortic arch. No test substance-related developmental variations were observed at any exposure level.
Other effects:
not examined
Key result
Dose descriptor:
NOAEC
Effect level:
750 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
visceral malformations
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
visceral/soft tissue: cardiovascular
Description (incidence and severity):
Exposure related visceral malformations in the heart and/or great vessels were observed in 2 fetus at 5500 and 3 fetus at 7500 ppm groups. These effects were not statistically significant and occurred at exposure levels that resulted in maternal lethality.
Key result
Developmental effects observed:
no

Table 1.  Animals found dead, euthanized in extermis, aborted or delivered prior to scheduled necropsy

Dose (ppm) 0 2500 4000 5500 7500
found dead 0 0 0 3 (GD13, 14,27) 6 (GD12(1),14(3),18(1),29(1))
euthanized in extremis 0 0 0 1 (GD28) 1 (GD16)
Aborted 0 0 0 1 (GD28) 3 (GD26(2), 29(1))
Delivered  0 0 0 0 1 (GD29)

Attachment with body weight changes and food comsumption are included in the background information section below

Table 2. Incidence of selected histopathologic findicngs and cause of death in the rabbit

Exposure Level (ppm):
   
0
2500
4000
5500
7500
 
Sample size
24
24
24
24
24
 
# animals adverse effects
0
8
12
14
22

 

Heart

 

 

 

 

 

 
  Inflammation, subacute
0
8
12
10*
15*
 
      Minimal
-
3
1
2
2
 
      Mild
-
2
2
5
6
 
      Moderate
-
3
8
3
6
 
      Severe
-
0
1
0
1
 

 

 

 

 

 

 

 
Number of Deaths
0
0
0
5
10
 
Cause of Death

 

 

 

 

 

 
  Undetermined
-
-
-
4
6
 
  Heart Inflammation
-
-
-
1
3
 
  Lung hemorrhage and
            edema
-
-
-
0
1
 
Conclusions:
Based on mortality, moribundity, abortions, premature delivery, lower mean body weight gain, mean body weight loss and/or lower food consumption observed at 5500 and 7500 ppm (25630 and 34950 mg/m3), an exposure concentration of 4000 ppm (18650 mg/m3) was considered to be the no-observed-adverse-effect level (NOAEC) for maternal lethality. Non statistically significant test substance-related visceral malformations in the heart and/or great vessels were observed in the 5500 (2 fetus) and 7500 ppm (3 fetus) groups only in the presence of maternal toxicity (mortality). Therefore, an exposure concentration of 7500 ppm was considered to be the NOAEL for embryo/fetal development when pregnant New Zealand White rabbits were exposed to the test substance via whole-body inhalation. These conclusions are drawn from the underlying data from this study which meets the requirements cited in OECD 414.

Histological evaluation of selected tissues (brain, heart, liver, lungs, kidneys) failed to reveal the cause of death in the majority of animals. However, cardiac inflammation were observed at 2500 ppm (11620 mg/m3) and higher. The significance of these findings is not understood.

Executive summary:

Administration of filtered air or test substance atmospheres to pregnant female New Zealand White [Hra:(NZW)SPF] rabbits, approximately 5.5 to 6 months of age, as 6-hour, daily whole-body exposures during the period of major organogenesis (gestation days 6 through 28) at concentrations of 2500, 4000, 5500, and 7500 ppm was associated with early death in the 5500 (n=5) and 7500 (n=10) ppm groups, and these deaths were of undetermined causation (4/5 at 5500 ppm, 6/10 at 7500 ppm), due to subacute inflammation of the heart (1/5 at 5500 ppm and 3/10 at 7500 ppm) or a result of edema/hemorrhage of the lung (1/10 at 7500 ppm). Test substance exposure was also associated with subacute inflammation in the heart in the 2500 (n=8), 4000 (n=12), 5500 (n=10), and 7500 (n=15) ppm groups, coagulation necrosis of the heart in the 7500 (n=1) ppm group, and renal tubular necrosis in the 5500 n=3) and 7500 (n=1) ppm groups. At 5500 ppm, renal necrosis was noted in 2 animals that died or were euthanized. Although not dose related, these changes were considered adverse.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
11 620 mg/m³
Study duration:
subacute
Species:
rabbit
Quality of whole database:
The study is a guideline study in compliance with GLP.
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In a rat developmental toxicity study, no maternal or developmental toxicity was observed in rats following exposures up to 50000 ppm (233000 mg/m3).

Pregnant rabbits were exposed to the test substance at 2500, 4000, 5500, and 7500 ppm (11620, 18640, 25630, and 34950 mg/m3), 6 hours a day during gestation days 6 -28. Mortality was observed in a dose dependent manner at 5500 and 7500 ppm. A small number of malformations (visceral malformations of the heart and/or great vessels) occurred at a frequency above the historical control levels but only at maternally lethal exposure levels of 5500 ppm and higher (2 fetus at 5500 and 3 fetus at 7500 ppm). These findings were not statistically different from the concurrent study control group. No test substance-related developmental variations were observed at any exposure level. The NOAEC for maternal lethality and for developmental toxicity was 4000 ppm.

In addition, the LOEC for maternal systemic toxicity was 2500 ppm (11620 mg/m3) based on cardiac lesions of unknown significance seen in maternal animals exposed to 2500 ppm and higher.

Justification for classification or non-classification

There was no reproductive or pre-natal developmental toxicity observed. No maternal toxicity, pre-natal developmental toxicity or teratogenicity was observed in rats exposed to up to 50000 ppm (233000 mg/m3). In rabbits a small number of malformations (visceral malformations of the heart and/or great vessels) occurred at a frequency above the historical control levels but only at maternally lethal exposure levels of 5500 ppm and higher (2 fetus at 5500 and 3 fetus at 7500 ppm). These findings were not statistically different from the concurrent study control group. No test substance-related developmental variations were observed at any exposure level. The NOAEC for maternal lethality was 4000 ppm and for developmental toxicity was 7500 ppm. In addition, cardiac lesions of unknown significance were seen in maternal animals exposed to 2500 ppm and higher.

 

In the rat 2-generation reproductive toxicity study, there was an apparent delay in the onset of puberty evident as a delay in days to achievement of vaginal opening in F-1 females. These apparent delays were not considered to reflect a direct effect on this endpoint, but rather, were considered secondary to test substance-related reductions in body weight and food consumption parameters that were evident during the first three weeks of exposures and concurrent with the onset of vaginal patency (Carney et al, 2004). Therefore the substance does not need to be classified for reproductive or developmental toxicity in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008 and its amendments.

Additional information