Polyhaloalkene

Endpoint summary

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.:

2 500 ppm

Remarks:

Group 2

Dose / conc.:

4 000 ppm

Remarks:

Group 3 and 5. Phase I. Two test substances manufactured by different supplier

Dose / conc.:

5 500 ppm

Remarks:

Group 3 and 5. Phase II. Two test substances manufactured by different supplier

Dose / conc.:

7 500 ppm

Remarks:

Group 4

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):

No test substance-related clinical findings were noted females that survived to the scheduled necropsy at the daily examinations, during mid-point of exposure or 1 hour following exposure at any exposure concentration. Findings noted in the test substance-treated groups, including hair loss on various body surfaces, occurred infrequently or, at similar frequencies in the control group, and/or were observed prior to the exposure period or in a manner that was not dose-related.

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.
On the day of or prior to death or euthanasia, 1 and 3 females in the 5500 and 7500 ppm groups, respectively, had labored and/or decreased respiration, 2 females in the 5500 ppm group were hypoactive, 1 female in the 7500 ppm group had clear material around the nose and 1 female in the 7500 ppm group had red material on the left hindlimb. In addition, the female in the 5500 ppm group had red material on the urogenital area on the day prior to euthanasia and 1 occurrence of labored respiration several days prior to death. Aside from a single occurrence of decreased defecation in 1 female in the 7500 ppm group, no other noteworthy clinical findings were observed during the exposure period for females that were found dead or euthanized in extremis. The only noteworthy finding for the aborted females was 1 or 11 occurrences of decreased defecation noted in 3 females in the 7500 ppm group. The abortions and premature delivery were attributed to the test substance. All other females survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean maternal body weight gain in the 7500 ppm group was similar to the control group during gestation days 6-9 and 9-12. A significantly (p<0.01) lower mean body weight gain was observed in this group during gestation days 12-20 relative to the control group. Mean body weight gain in the 7500 ppm group was similar to the control group during gestation days 20-29, presumably because the most sensitive animals had died. As a result of the lower mean body weight gain during gestation days 12-20, mean body weight gain in the 7500 ppm group was slightly lower (not statistically significant) when the entire exposure period (gestation days 6-29) was evaluated. The decrements in mean body weight gain in this group were not of sufficient magnitude to result in lower mean body weights. Mean net body weight and gravid uterine weight in this group were similar to the control group.
Mean body weight gains in the 5500 ppm group were similar to the control group during gestation days 6-9 and 9-12. A slightly lower mean body weight gain was observed in this group during gestation days 12-20 (significant, p<0.05, during gestation days 12-13 only), and a significant (p<0.01) mean body weight loss was noted during gestation days 20-29. The decrements in mean body weight gain in the 5500 ppm group resulted in a significantly (p<0.01) lower mean body weight gain when the entire exposure period (gestation days 6-29) was evaluated. A significant (p<0.01) mean net body weight loss (174.8 g) was also observed in this group when compared to the control group. Although there was no apparent exposure-related trend, the effects on mean body weights during the last week of exposure and mean net body weight loss were considered test substance-related because the most sensitive population in the 7500 ppm group had been eliminated by this point. However, the mean body weight loss and lower mean body weight gain in the 5500 ppm group were not of sufficient magnitude to result in lower mean body weights or a lower mean net body weight. Mean gravid uterine weight in this group was similar to the control group value.
Mean body weight gain in the 4000 ppm group was similar to the control group during gestation days 6-9 and 9-12. Slightly (not statistically significant) lower mean body weight gains were noted in this group during gestation days 12-20 and 20-29, resulting in a significantly lower (p<0.05) mean body weight gain when the entire exposure period (gestation days 6-29) was evaluated. Although the slight reduction in mean body weight gain in the 4000 ppm group was considered test substance-related, mean body weights in this group were similar to the control group throughout the exposure period, mean net body weight was similar to the control group value, and only a slight (not statistically significant) mean net body weight loss was noted. Therefore, the slightly lower mean body weight gain in the 4000 ppm group was not considered adverse.
Mean body weights, body weight gains, net body weight, net body weight gain and gravid uterine weight in the 2500 ppm group were unaffected by exposure to the test substance. Differences from the control group were slight and not statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Mean food consumption, evaluated as g/animal/day and g/kg/day, in 7500 ppm group was generally similar to the control group throughout the study. However, significantly (p<0.05 or p<0.01) lower mean food consumption was observed in this group relative to the control group during several daily intervals (gestation days 13-14 [g/kg/day only], 18-19, 22-23 and 23-24).
Mean food consumption in the 5500 ppm group was similar to the control group during gestation days 6-9, 9-12 and 12-20, with the following exception. Significantly (p<0.05 or p<0.01) lower mean food consumption was noted compared to the control group during gestation days 18-19. During gestation days 20-29, food consumption in the 5500 ppm group was significantly (p<0.01) lower compared to the control group; the lower food consumption corresponded to a mean body weight loss noted during this same period.
Mean food consumption in the 2500 and 4000 ppm groups was similar to that in the control group throughout the exposure period. No statistically significant differences were noted.

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:

effects observed, treatment-related

Description (incidence and severity):

Four and 7 females in the 5500 and 7500 ppm groups, respectively, were found dead or euthanized in extremis during the study. Dark red areas or dark red discoloration in the lungs were noted in 3 and 2 females in these respective groups, and 1 female each in the 5500 and 7500 ppm groups had dark red areas in the thymus gland. In addition to these findings, 1 female in the 5500 ppm group had green contents in the uterus and 1 female in the 7500 ppm group had dark red areas in the spleen. Another female in the 7500 ppm group had brown discoloration in the lungs, red fluid contents and white material in the thoracic cavity, a small lobe of the liver and dark red areas in the kidneys.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There was subacute inflammation in the heart of animals exposed to ≥ 2500 ppm with increased incidence and severity with increasing dose. Subacute inflammation was not present in control group animals. The inflammation was characterized by a mixture of infiltration of lymphocytes and heterophils, degeneration of cardiac myofibers, expansion of endomyseal basophilic ground substance, and fibrosis. The lesion was multifocal throughout the left and right ventricles, and occasionally present in papillary muscles and atrial walls.
One 7500 ppm group female had moderate locally extensive necrosis of a papillary muscle of the heart, which was characterized by coagulation necrosis without inflammation.
The kidney had multifocal discrete foci of necrosis of the cortical renal tubules in the 5500 and 7500 ppm dose groups. The incidence of this change did not follow a dose response profile. Necrotic tubules had intact basement membrane lined by remnants of eosinophilic cytoplasm within the lumen. There was no remaining cellular architecture. Rarely, there were pyknotic nuclei and/or cellular debris. The affected tubules were interpreted to be proximal convoluted tubules, but in the absence of recognizable architecture, this identification of tubule type is not definitive. Tubules lying adjacent to necrotic tubules consisted mostly of distal tubules, and were intact and normal. Necrosis of kidney tubules appeared similar to the necrosis of the kidney tubules in early death animals, and this change was not present in control group animals.
There were no test substance-related findings in the lungs. There were a variety of findings including congestion, alveolar hemorrhage, and subacute inflammation in all treatment groups, however, these findings did not show a clear dose response profile and were also present in control animals. Subacute inflammation was characterized by multifocal discrete foci with intra-alveolar neutrophils and alveolar macrophages, and thickened hypercellular alveolar walls with occasional prominence of alveolar epithelial cells. The slight increase in incidence and/or severity of these changes in treated animals when compared to the control group represented normal biologic variability, and the changes were considered incidental.
Collections of pigmented and non-pigmented alveolar macrophages were present in the control and all test substance-exposed groups, and there was no clear test substance or dose-related response profile for this particular finding. There was an increase in severity and incidence of these macrophages in the 2500 and 4000 ppm groups only. The finding was characterized by individualized and well-demarcated clusters of densely packed macrophages, ranging in numbers from 15-50, residing within distended alveoli. In many of the macrophages, there was golden brown pigment typical of hemosiderin.
There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than exposure to the test substance. There was no test substance related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

See Table 2 in 'Any other information on results incl. tables'.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

One and 4 females in the 5500 and 7500 ppm groups, respectively, aborted or delivered during the study. The female in the 5500 ppm group aborted 2 viable and 5 dead fetuses with no apparent malformations and 1 viable and 2 dead fetuses that were partially cannibalized; 2 viable fetuses were noted in utero. This female also had dark red areas in the lungs. Three females in the 7500 ppm group aborted late resorptions or 1 dead fetus; 2 late resorptions in the same litter had open eyelids. The female that aborted 1 dead fetus also had 9 viable fetuses in utero. A swollen spleen and mottled liver were observed for another of the females in this group that aborted.

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

All of the females that were found dead or euthanized in extremis were gravid with normally developing implantations or fetuses in utero. In addition, 1 female in the 5500 ppm group had a late resorption, and 1 female in the 7500 ppm had an early resorption in utero.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

effects observed, treatment-related

Description (incidence and severity):

One female in the 7500 ppm group delivered on gestation day 29 and had dark red areas in the lungs. Other macroscopic findings (accessory spleen, small gallbladder or cystic oviducts) noted in females that were found dead, euthanized in extremis, aborted or delivered are common in this species and strain.

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

All other females survived to the scheduled necropsy. At the scheduled necropsy on gestation day 29, no test substance-related internal findings were observed at any exposure level. Macroscopic findings observed in the test substance-treated groups occurred infrequently, at similar frequencies in the control group and/or in a manner that was not dose-related.

Intrauterine growth and survival were unaffected by test substance administration at exposure concentrations of 2500, 4000, 5500 and 7500 ppm. Parameters evaluated included postimplantation loss, live litter size, mean fetal body weights and fetal sex ratios. No statistically significant differences from the concurrent control group were noted, and values were within the ranges of the historical control data for definitive studies. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of preimplantation loss were similar across all groups.

Key result

Dose descriptor:

NOAEC

Remarks:

General toxicity

Effect level:

< 2 500 ppm

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEC

Remarks:

Maternal developmental toxicity

Effect level:

4 000 ppm

Based on:

test mat.

Basis for effect level:

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

Description (incidence and severity):

The numbers of fetuses (litters) available for morphological evaluation were 206(24), 191(23), 206(24), 153(18) and 132(14) in the control, 2500, 4000, 5500 and 7500 ppm groups, respectively.

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):

External malformations were observed in 1(1), 2(1) and 1(1) fetuses (litters) in control, 2500 and 7500 ppm groups, respectively. Fetus no. 53771-01 in the 7500 ppm group had carpal flexure (bilateral) and hyperflexion of the hindpaw (bilateral); no skeletal origin as apparent for these malformations. Fetus nos. 53745-03 and 53745-10 in the 2500 ppm group had microphthalmia (unilateral). Skeletally, the orbit was smaller than normal for fetus no. 53745-10; however, there was no apparent skeletal origin for microphthalmia in the other fetus. These findings were not considered test substance-related because they occurred infrequently, not in an exposure related manner, the mean litter proportions were not statistically significantly different from the concurrent control group and/or the values were within the ranges of the historical control data. In the control group, fetus no. 54153-08 had a short tail that consisted of only 12 caudal vertebrae present and misshapen and fused caudal vertebrae.
No external developmental variations were noted at any exposure level. Four fetuses in 1 litter in the 2500 ppm group had white areas in the skin (dorsal neck or entire external surface). This finding was not classified as a malformation or developmental variation and was not included in any tabulation.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Skeletal malformations were observed in 3(2), 4(4), 0(0), 3(2) and 1(1) fetuses (litters) in
the control, 2500, 4000, 5500 and 7500 ppm groups, respectively. Vertebral anomalies
with or without associated rib anomalies were noted in 1(1), 2(2), 0(0), 2(1) and 1(1) fetuses (litters) in the same respective groups. For 1 fetus in each in the 2500, 5500 and 7500 ppm groups, this finding consisted of an extra arch, rib and/or half of centrum, forked and/or fused ribs (5500 and 7500 ppm groups), malpositioned arches and halves of centra (2500 and 5500 ppm groups) and costal cartilage from the extra rib associated with the sternum (2500 and 7500 ppm groups) resulting in malpositioning of subsequent costal cartilages (2500 ppm group). The fetus in the 7500 ppm group also had a costal cartilage anomaly consisting of costal cartilages that fused, then bifurcated prior to associating with the sternum normally. One fetus each in the control and 5500 ppm group had vertebral anomalies that consisted of an absent half of a centrum and malpositioned arches and halves of centra; the 5500 ppm group fetus also had a malproportioned arch.
One fetus in the 2500 ppm group had a vertebral anomaly consisting of a malproportioned centrum attached to an adjacent centrum, malpositioned arches and halves of centra and fused ribs. Also in the 2500 ppm group, 1 fetus had a vertebral centra anomaly consisting of centra or halves of centra that were absent, attached to an adjacent centrum or malpositioned and another fetus had a severely malaligned sternebra resulting in fused sternebrae. Two (2) and 1(1) fetuses (litters) in the control and 5500 ppm groups, respectively, had rib anomalies consisting of bifurcated and/or fused ribs and/or extra rib, costal cartilage from extra rib or anterior fork of bifurcated ribs that associated with the sternum and/or malpositioned costal cartilage. Because skeletal malformations occurred similarly in the control group, there was no exposure-response relationship, the mean litter proportions were not statistically significantly different from the concurrent control group and/or the values were within the ranges of the historical control data, the malformations observed in the test substance-exposed groups were considered spontaneous.
Skeletal developmental variations observed in all groups, including the control group, consisted of 13th rudimentary rib(s), 13th full rib(s), sternebra(e) nos. 5 and/or 6 unossified, bent hyoid arch(es) and 27 presacral vertebrae. Other skeletal developmental variations occurred similarly in the control group, were not observed in an exposure-related manner, the mean litter proportions were not statistically significantly different from the concurrent control group and/or the values were within the range of the historical control data. No relationship to the test substance was evident.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

Soft tissue malformations were observed in 2(2), 0(0), 2(1), 5(4) and 3(2) fetuses (litters)
in the control, 2500, 4000, 5500 and 7500 ppm groups, respectively. In the 7500 ppm group, fetus nos. 54108-08, 54108-10 and 54135-08 had a bulbous aorta (ascending and/or arch), stenotic pulmonary trunk and an absent interventricular septum; fetus nos. 54108-08 and 54135-08 also had an absent tricuspid valve. Fetus no. 54145-01 in the 5500 ppm group also had a bulbous aorta (ascending and arch), a stenotic pulmonary trunk and an absent interventricular septum. Fetus no. 54127-05 in the same group had an interrupted aortic arch (the right and left subclavian and carotid arteries arose from the ascending aorta, with no brachiocephalic trunk). The mean litter proportions of these findings were not statistically significant compared to the concurrent control group; however, the values for bulbous aorta (0.8% and 2.4% per litter) and stenotic pulmonary trunk (0.8% and 2.4% per litter) in the 5500 and 7500 ppm groups, respectively, exceeded the maximum mean values in the historical control data (0.5% per litter for both findings). Absent tricuspid valve and interrupted aortic arch have not been observed in the historical control data. Based on the mean litter proportion and the similarity of the multiple findings (cardiovascular), the heart and great vessel anomalies in the 5500 and 7500 ppm groups were attributed to the test substance. Fetus no. 54170-01 in the 5500 ppm group had a diaphragmatic hernia (a portion of the liver protruded into the thoracic cavity through an opening in the diaphragm). Lobular agenesis of the lungs (absent right accessory lobe) was noted in 1(1), 2(1) and 2(1) fetuses (litters) in the control, 4000 and 5500 ppm groups, respectively. These malformations were not considered test substance-related because there was no exposure- response relationship and/or the finding was observed similarly in the control group. A malpositioned kidney (located more posterior than normal) was observed in fetus no. 53735-02 in the control group. No other visceral malformations were noted.
Soft tissue developmental variations noted in all groups, including the control group, consisted of retrocaval ureter, extra papillary muscle in the heart, major blood vessel variations (the left carotid artery arose from the brachiocephalic trunk or retroesophageal right subclavian artery) and accessory spleen. Other visceral developmental variations occurred infrequently, similarly in the control group and/or not in an exposure-related manner. No relationship to the test substance was evident.
Several visceral findings were not classified as either a malformation or developmental variation and were not included in any tabulation. Other findings were not attributed to the test substance because they occurred infrequently, were noted similarly in the control group or did not occur in an exposure- related manner.
See Table 3 in 'Any other information on results incl. tables'.

Other effects:

not examined

Key result

Dose descriptor:

NOAEC

Effect level:

4 000 ppm

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:

yes

Lowest effective dose / conc.:

5 500 ppm

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

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 findings and cause of death in the rabbit

			Females
Exposure Level (ppm):	0	2500	4000	5500	7500
Heart ^a	24	24	24	24	24
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
Kidneys ^a	24	24	24	24	24
Necrosis	0	0	0	3	1
Mild	-           -           -			2	0
Moderate	-           -           -			1	0
Severe	-           -           -			0	1
Lungs ^a	24	24	24	24	24
Congestion	0	2	5	3	3
Mild	-	1	1	2	2
Moderate	-	1	4	1	1
Hemorrhage	2	6	3	3	1
Minimal	2	2	0	1	0
Mild	0	4	3	1	0
Moderate	0	0	0	1	1
Inflammation	4	8	8	3	4
Minimal	4	5	6	3	2
Mild	0	3	2	0	2
Alveolar macrophages ^b	2	10	7	2	3
Minimal	1	6	6	2	3
Mild	1	4	0	0	0
Moderate	0	0	1	0	0
Cause of Death	0	0	0	5	10
Undetermined	-	-	-	4	6
Heart Inflammation        -		-	-	1	3
Lung hemorrhage and   -		-	-	0	1

            edema 

^a = Number of tissues examined from each group.

^b = Included the incidence of pigmented and non-pigmented macrophages

Table 3. Visceral Findings Not Classified As Malformations Or Developmental Variations

	Number of Fetuses Affected
Finding	0 ppm	2500 ppm	4000 ppm	5500 ppm	7500 ppm
Renal papilla not fully developed (Woo and Hoar grade 1)	1	1	-	-	-
Cyst(s)	1a	1b	-	2c	-
Thymus gland - dark red areas or dark red discoloration	-	3	-	-	-
Distended stomach	-	1	-	-	-
Thoracic cavity - dark red contents	1	1	-	-	-
Thoracic cavity - fluid contents or fluid-filled	-	3	-	1d	1d
Abdominal cavity - fluid contents or fluid-filled	-	4	-	2d	1d
- = Not observed. ^a = On gallbladder. ^b = On diaphragm. ^c = On oviduct(s). ^d = One fetus with heart and great vessel malformations.

 

Conclusions:

Based on the results of this study, mortality, moribundity, abortions, premature delivery, lower mean body weight gain, mean body weight loss and/or lower food consumption were observed at 5500 and 7500 ppm and test substance-related, adverse microscopic findings were noted in all exposure groups that consisted of subacute inflammation of the heart at ≥ 2500 ppm, coagulation necrosis of the heart at 7500 ppm, and renal tubular necrosis at ≥ 5500 ppm. Therefore, an exposure concentration of < 2500 ppm was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Based on the abortions observed at 5500 and 7500 ppm, the maternal developmental NOAEL has been set at 4000 ppm. Because test substance-related visceral malformations in the heart and/or great vessels were observed in the 5500 and 7500 ppm groups in the presence of maternal toxicity, an exposure concentration of 4000 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.

Executive summary:

The objectives of the study were to determine the potential of the test article to induce developmental toxicity after maternal exposure during the critical period of organogenesis, to characterize maternal toxicity at the exposure levels tested and to determine a no-observed-adverse-effect level (NOAEL) for maternal toxicity and developmental toxicity. Five groups of time-mated female New Zealand White rabbits (12/group per phase; approximately 5.5-6 months of age at initiation of exposure) were exposed to either clean filtered air or vapor atmospheres of the test substance for approximately 6 hours daily in whole-body inhalation chambers during gestation days 6-28. Because of the limited amount of space available in the exposure chambers, each group was divided into 2 phases, with the following exception. The 4000 and 5500 ppm test substance concentrations were administered only during Phase I and Phase II, respectively. Target test substance concentrations were 0, 2500, 4000, 7500 and 4000 parts per million (ppm) for each phase, with the following exception. Because no toxicity was noted at 4000 ppm in Phase I, the sponsor elected to increase the mid-exposure concentration to 5500 ppm for Phase II. In order to determine if there were any differences in the test substance manufactured by a different supplier, the 4000 and 5500 ppm groups were divided into 2 subgroups each, exposed to the test substance supplied by either the sponsor or the other manufacturer. Overall mean measured exposure concentrations were 2504, 3982, 7512 and 4013 ppm for the 2500, 4000, 7500 and 4000 ppm groups, respectively, in Phase I and 2479, 5408, 7441 and 5479 ppm for the 2500, 5500, 7500 and 5500 ppm groups, respectively, in Phase II.
All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights and food consumption were recorded at appropriate intervals. On gestation day 29, a laparohysterectomy was performed on each surviving female. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. In addition, microscopic examination of the brain, heart, kidneys, liver, and lungs was performed for all females. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations.
Four and 7 females in the 5500 and 7500 ppm groups, respectively, were found dead or euthanized in extremis. Labored and/or decreased respiration and/or hypoactivity were observed prior to death or euthanasia for 2 and 3 females in the same respective groups. One and 3 females in the 5500 and 7500 ppm groups, respectively, aborted on gestation day 26, 28 or 29, and 1 female in the 7500 ppm group delivered on gestation day 29; these deaths were of undetermined causation, due to subacute inflammation of the heart, or a result of edema/hemorrhage of the lung. The mortality, moribundity, abortions and premature delivery in the 5500 and 7500 ppm groups were attributed to the test substance; no test substance-related macroscopic findings were observed in these females. All other females survived to the scheduled necropsy.
No test substance-related clinical findings were noted at the daily examinations or at mid-point of or 1 hour following exposure at any concentration.
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 the most sensitive 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. Mean gravid uterine weights were unaffected by exposure to the test substance at all concentrations.
Intrauterine growth and survival were not affected by test substance exposure at any concentration. However, test substance-related (not statistically significant) visceral malformations of the heart and/or great vessels were observed at frequencies above the historical control values in the maternally toxic 5500 and 7500 ppm groups. These findings, 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.
The histopathological evaluation and interpretation of these data were completed after issuance of the final report. Microscopic examination revealed subacute inflammation in the heart in the 2500, 4000, 5500 and 7500 ppm groups, coagulation necrosis of the heart in the 7500 ppm group, and renal tubular necrosis in the 5500 and 7500 ppm groups. All of these changes were considered related to test substance administration and considered adverse.
Based on the results of this study, mortality, moribundity, abortions, premature delivery, lower mean body weight gain, mean body weight loss and/or lower food consumption were observed at 5500 and 7500 ppm and test substance-related, adverse microscopic findings were noted in all exposure groups that consisted of subacute inflammation of the heart at ≥ 2500 ppm, coagulation necrosis of the heart at 7500 ppm, and renal tubular necrosis at ≥ 5500 ppm. Therefore, an exposure concentration of < 2500 ppm was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Because test substance-related visceral malformations in the heart and/or great vessels were observed in the 5500 and 7500 ppm groups in the presence of maternal toxicity, an exposure concentration of 4000 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.

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). Because of the increase compared to historical control and the similarity of the findings, the malformations in the cardiovascular system of fetuses in the 5500 and 7500 ppm group were considered test substance-related. Therefore, the NOAEL for developmental toxicity was set at 4000 ppm. The NOAEC for maternal lethality and for developmental toxicity was 4000 ppm. However, based on the cardiac lesions of unknown significance seen in maternal animals exposed to 2500 ppm and higher, the NOAEL for general toxicity was set at < 2500 ppm (11620 mg/m3).

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 4000 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.