Trifluoroiodomethane

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec 1996 to Jan 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996 to 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

A combined repeated dose and reproductive toxicity screening study was performed in Sprague-Dawley Crl:CD(SD)BR rats. The study had 16 male and 16 female rats/group to yield at least 12 pregnant females at term. The animals were assigned to one of four groups (0, 0.2, 0.7, and 2.0%) and exposed to substance vapor for 4 wk (6 h/day, 5 days/wk) prior to mating. An additional six male and three female rats were assigned to a fifth group only to serve as positive controls in the micronuclei assay. Animals were exposed 6 h/day, 7 days/wk, during the mating, gestation, and lactation phases of the study. However, dams were not exposed from gestation day 21 through lactation day 4 to allow for parturition and early lactation. Pups were not exposed to and remained in their home cages under observation. Following the 14-day mating period, eight male rats/group were terminated (study wk 7). The remaining 8 male rats/group and 16 female rats/group were terminated over a 5-day period subsequent to when the last female rat on study reached lactation day 21. Animals were killed by CO2 inhalation overdose followed by exsanguination.
Examinations included clinical observations (twice daily), bodyweight, haematology, clinical chemistry, serum thyroid hormone levels and bone marrow erythrocyte micronucleus test (summarized in section 7.6.2). At termination a gross examination was performed, organ weights were determined and selected tissues from male and female rats of the control and high-dose groups were subjected to histopathologic examination. Reproductive indices and parameters were assessed for all dose groups.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

Supplier: Ajay North America, LLC, Powder Springs, GA
Purity > 99.78%
Appearance: Colourless gas

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, MA.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 11 weeks at the start of exposure
- Weight at study initiation: around 225 g at SD 0. Body weights of males are not reported.
- Housing: The rats were housed in wire-bottom cages during exposure (one per cage except during the mating period) and in plastic shoebox cages with bedding (two males per cage, one female per cage, except during the mating period) during nonexposure periods. Exposure cages were rotated in a clockwise manner within the chamber each exposure day.
- Diet: certified rodent diet (PMI Feeds, Inc., St. Louis, MO) was available ad libitum during nonexposure periods.
- Water: water was available ad libitum during nonexposure periods.
- Acclimatization period: 2 to 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): between 21 and 26
- Humidity (%): between 35 and 65
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Whole-body inhalation exposures were performed in 690-L chambers made of stainless steel and glass.
- Source and rate of air: The substance and air for dilution were controlled through flow meters
- Temperature, humidity, oxygen and ammonia in air chamber: Relative humidity and temperature of the exposure atmosphere were constantly monitored and recorded. Oxygen concentration of the high concentration chamber was analyzed on occasion using a Hudson O2 sensor model 82T (MDA, Lincolnshire, IL). Ammonia levels during exposure were estimated not to be of biological importance
- Air flow and air change rate: To conserve the use of test material and minimize cost, total chamber air flow was reduced to 60 L/min (approximately 5 air changes/h).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Continuous analysis of the chamber air for test substance was performed using infrared absorption spectrometers. Instrumental calibration was performed using known concentrations of freshly prepared test substance in air contained in Tedlar sample bags. Calibration checks were performed at appropriate intervals.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

- 6 hours/day, 5 days/wk prior to mating
- 6 hours/day, 7 days/ wk during the mating, gestation, and lactation phases of the study.
- Dams were not exposed from gestation day 21 through lactation day 4 to allow for parturition and early lactation.
- Pups were not exposed

Dose / conc.:

2 000 ppm

Remarks:

equivalent to 0.2%

Dose / conc.:

7 000 ppm

Remarks:

equivalent to 0.7%

Dose / conc.:

20 000 ppm

Remarks:

equivalent to 2.0%

No. of animals per sex per dose:

16. An additional six male and three female rats were assigned to a fifth group only to serve as positive controls in the micronuclei assay (section 7.6.2).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: 2 and 13 wk repeated dose toxicity studies are available. Exposure concentrations selected for this study extended beyond those of the 13-wk study (Dodd 1997)

Positive control:

Single dose of cyclophosphamide (7.5 mg/kg) for the micronucleus assay (please refer to section 7.6.2).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS
The animals were observed twice daily (morning and afternoon), including inhalation exposure periods. Signs of toxicity were recorded.

BODY WEIGHT
The body weight of the male rats were determined prior to the first exposure and weekly thereafter. The body weights of female rats were determined and recorded in the same manner until confirmation of mating. During gestation, female rats were weighed on gestational days 0, 7, 14, and 20. Dams producing litters were weighed on days 0, 4, 7, 12, and 21 postpartum.

HAEMATOLOGY
Routine haematology evaluations were conducted on blood samples taken immediately prior to termination from all animals. The blood was sampled via the posterior vena cava. Haematologic parameters were determined according to established procedures, utilizing a Cell-Dyn 3500 (Abbott Diagnostics, Chicago). Assessed parameters included blood cell count, haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, hematocrit, total and differential leukocyte count and platelet count.

CLINICAL CHEMISTRY
Routine clinical chemistry evaluations were conducted on blood samples taken immediately prior to termination from all animals. The blood was sampled via the posterior vena cava. Serum chemistry parameters were determined according to established procedures, utilizing a Vitros 250XR (Johnson and Johnson, Rochester, NY). Assessed parameters included total protein, albumin, globulin, alanine aminotransaminase (ALT), aspartate aminotransaminase (AST), alkaline phosphatase, urea nitrogen, creatinine, calcium, glucose, potassium, phosphorus, sodium, magnesium, trigylcerides and cholesterol.

SERUM THYROID LEVELS
The following serum thyroid hormone levels were determined in control and exposed rats: thyroxine (T4), triiodothyronine (T3), reverse T3 (rT3), and thyroid-stimulating hormone (TSH).
Assays for T4, T3, rT3, and TSH were performed using radioimmunoassay (RIA) kits and were carried out according to manufacturer’s instructions. For all thyroid hormone or TSH measurements, assay kits were prepared for each animal termination period (study wk 7 or 14) with the same batch number and the same expiration date. Tracer (125I) radioactivity was measured with a Packard gamma counter (Packard Instrument Co., Meriden, CT). For T3, the RIA assay kit was purchased from Diagnostic Product Corp. (Los Angeles, CA), and canine T3 antibody-coated tubes were used. For T4, the RIA assay kit was purchased from Diagnostic Product Corp. (Los Angeles, CA), and T4 antibody coated tubes were used. For rT3, the RIA assay kit was purchased from Wein Laboratories (Succasunna, NJ), and rT3 antiserum raised in the rabbit was used. For TSH, the RIA assay kit was purchased from Amersham Corp. (Arlington Heights, IL), and both lyophilized rabbit anti-rat TSH serum and Amerlex-M second antibody (donkey anti-rabbit serum coated onto magnetized polymer particles containing sodium azide) were used.

Sacrifice and pathology:

SACRIFICE
Following the 14-day mating period, eight male rats/group were terminated (study wk 7). The remaining 8 male rats/group and 16 female rats/group were terminated over a 5-day period subsequent to when the last female rat on study reached lactation day 21. Animals were not fasted prior to termination, since fasting might affect thyroid hormone levels.

GROSS NECROPSY
A gross examination was performed at necropsy.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organs weighed included adrenal glands, heart, lungs, liver, kidneys, ovaries, testes, epididymides, brain, spleen, and thymus. Thyroid glands (including parathyroid glands) were removed with the trachea, fixed in buffered formalin, then dissected free of the trachea, and weighed. A single technician performed the dissection of the thyroid glands. These organs and the pituitary gland, nasal cavity, trachea, bronchi, bone marrow (sternal and femoral sections and smear), pancreas, urinary bladder, stomach, duodenum, ileum, colon, prostate, scrotum, seminal vesicles, vagina, uterus, and other tissues with gross lesions were removed from all animals and preserved in 10% neutral-buffered Formalin for possible histopathological examination. Select tissues (lungs, ovaries, testes, epididymides, Thyroid glands (including parathyroid glands), nasal cavity, trachea, bronchi, bone marrow (sternal and femoral sections and smear), prostate, scrotum, seminal vesicles, vagina, uterus, and other tissues with gross lesions from male and female rats of the control and high-dose groups were subjected to histopathologic examination. Testes and epididymides were fixed in Bouin’s fixative; sections were stained with periodic acid and Schiff’s (PAS) and counterstained with hematoxylin.

For animals dying during the study, a complete gross necropsy and histopathologic examination were conducted to determine the possible cause of death.

Statistics:

For body weight data, a repeated-measures analysis of variance (ANOVA) was conducted. An ANOVA with Bonferroni multiple comparisons was conducted on the thyroid hormone data. Fisher’s exact test was used on the reproductive data that are calculated on a group basis (e.g., gestational index). For reproductive data calculated on an individual basis (e.g., live birth index), micronuclei data, haematology and serum chemistry data, and organ weight data, the Wilcoxon rank sum test was used to increase the statistical sensitivity when comparing control group values to treated group values.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related clinical findings. Areas of alopecia were sporadic and considered incidental.

Mortality:

no mortality observed

Description (incidence):

No deads reported

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Though not statistically significant, except for the final body weight values in female rats (day 93–95), there was a marginal decrease in mean body weights of the 2.0% group compared to the control group. The mild decrease in absolute body weights for this group of rats is due primarily to a depression in body weight gain during the first 2–3 wk of the study. Body weight values were normal for male and female rats of the 0.2 and 0.7% groups.

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:

effects observed, treatment-related

Description (incidence and severity):

For male and female rats, statistical significant differences in haematology parameters were few in number and not considered treatment related, due to (1) lack of consistency with time (7-wk compared to 14- wk values for male rats), (2) lack of dose response, or (3) the difference being of small magnitude (clinically insignificant). Decreases in total leukocyte count (14% lower than the control mean), haemoglobin (5%), and haematocrit (4%) were observed in the female rats of the 2.0% group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

For male and female rats, statistical significant differences in serum chemistry parameters (except serum thyroid hormones) were few in number and not considered treatment related, due to (1) lack of consistency with time (7-wk compared to 14- wk values for male rats), (2) lack of dose response, or (3) the difference being of small magnitude (clinically insignificant). Increases in cholesterol (~30% higher than the control mean), total protein (5%), and albumin (9%) were observed in both the 0.7% and 2.0% groups of female rats.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Mean relative (to body weight) liver weights were mildly increased (6–10% above control values) in the male rats of the 2.0% group at both the 7-wk and 14-wk sacrifice periods. Mean absolute and relative epididymides weights were also increased in male rats of the 0.7% and 2.0% groups at 7 wk, but not at 14 wk. The increase in epididymides weights was not concentration dependent.
- For female rats, decreases in mean absolute organ weights included the brain (0.7% and 2.0%), ovaries (0.7% and 2.0%), and heart (2.0%). Mean relative brain (2.0%), kidney (2.0%), and liver (0.7%) weights were increased. Mean relative ovaries weights were decreased (0.7% and 2.0%). Note that the mean body weight was statistically significantly decreased in the 2.0% female rats compared to the control female rats.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no clinically significant or exposure-related gross lesions in any of the study animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no lesions of clinical significance in any treated (2.0% group) or control group animals. Tissues from animals in the 0.7% or 0.2% groups were not examined microscopically, due to the lack of microscopic findings in rats of the control and 2.0% groups.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant and concentration-related increases in T4 and rT3 were observed in male and female rats after 7 or 14 wk of exposure. T3 levels were decreased in a concentration-related manner in these animals. For TSH, an increase was observed in exposed male rats after 7 wk of exposure, but there was no concentration relationship. After 14 wk of exposure, male and female rats of the 2.0% group had statistically significant increases in TSH levels compared to control rats. A marginal and statistically significant increase in TSH was observed in female rats of the 0.2% group, but this effect was not statistically significant in female rats at 0.7%.

Key result

Dose descriptor:

NOAEC

Effect level:

20 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest dose tested

Dose descriptor:

NOEC

Effect level:

7 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Serum TSH concentrations

Key result

Critical effects observed:

no

Analytical verification of chamber atmosphere

The test material was 99.7+%, which remained stable throughout the study period. Analytical chamber concentrations matched the target exposure concentrations of 0 (control), 0.2, 0.7, and 2.0%.

The nominal mean concentrations (standard deviation) were 2.08% (0.09), 0.72% (0.03), and 0.24% (0.015) for the target concentrations of 2.0%, 0.70%, and 0.20%. The ratio between analytical/nominal concentrations as calculated from the rotometer settings for the air and test substance were 0.97, 0.98 and 0.83 for the 2%, 0.7%, and 0.2% target concentrations, respectively.

Chamber temperature and relative humidity means for all exposure groups ranged from 73.5 to 74.2°F and 43.4 to 54.8%, respectively. The oxygen concentration of the 2.0% chamber was approximately 19%. The deviation in this study from the accepted 10–14 air changes per hour during animal exposure to 5 air changes per hour did not have an impact on exposure temperature, relative humidity, oxygen concentration, or substance concentration.

Conversion of NOAEC

2% test substance is corresponding to 20.000 ppm or 160254 mg/m3 (based on a molecular weight of 195.91)

Conclusions:

Exposure of 2.0% test substance vapor for approximately 14 weeks produced minimal general toxicity in Sprague-Dawley rats.

Executive summary:

A combined repeated dose/ reproductive screening study was performed. The purpose of this study was to determine and evaluate the potential of the substance to produce reproductive toxicity and to provide additional information on the effect of test substance exposure on the thyroid. Groups of 16 male and 16 female rats were exposed (6hr/day) to substance vapor at concentrations of 0 (control), 0.2, 0.7, and 2.0% using whole body inhalation chambers. Prior to mating, rats were exposed for 4 weeks (5 days/wk). Exposures were 7 days/wk during the periods of mating (2 wk), gestation (3 wk), and lactation (3 wk). First generation pups were not exposed to substance vapor. In parental animals, there were no clinical signs of toxicity except for a minimal decrease in mean body weight in female rats at 2.0%. At necropsy, gross findings, mean serum chemistry levels, mean haematology values mean bone marrow micronuclei scores (detailled in section 7.6.2), and mean organ weights were similar for all exposure groups, including the air control group. Statistically significant differences were considered incidental. There were no treatment-related histopathologic tissue findings, including the thyroid organ. Analysis of reproductive indices and parameters indicate that the substance is not a reproductive toxicant. Results of serum thyroid hormone levels (e.g., T3, T4, rT3, and TSH), indicated concentration-related increases in TSH, T4, and rT3. T3 levels were decreased. First generation pup survival and mean body weights were similar in all exposure groups, including the control. Exposure of 2.0% vapor for approximately 14 weeks produced minimal general toxicity in Sprague-Dawley rats. On the basis of serum TSH concentrations, the no-observable-effect-level (NOEL) is 0.7%. The NOAEC for systemic toxicity was set at 2% (or 160254 mg/m3 based on a molecular weight of 195.91).

.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec 1996 to Jan 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

A combined repeated dose and reproductive toxicity screening study was performed in Sprague-Dawley Crl:CD(SD)BR rats. The study had 16 male and 16 female rats/group to yield at least 12 pregnant females at term. The animals were assigned to one of four groups (0, 0.2, 0.7, and 2.0%) and exposed to substance vapor for 4 wk (6 h/day, 5 days/wk) prior to mating. An additional six male and three female rats were assigned to a fifth group only to serve as positive controls in the micronuclei assay. Animals were exposed 6 h/day, 7 days/wk, during the mating, gestation, and lactation phases of the study. However, dams were not exposed from gestation day 21 through lactation day 4 to allow for parturition and early lactation. Pups were not exposed to and remained in their home cages under observation. Following the 14-day mating period, eight male rats/group were terminated (study wk 7). The remaining 8 male rats/group and 16 female rats/group were terminated over a 5-day period subsequent to when the last female rat on study reached lactation day 21. Animals were killed by CO2 inhalation overdose followed by exsanguination.
Examinations included clinical observations (twice daily), bodyweight, haematology, clinical chemistry, serum thyroid hormone levels and bone marrow erythrocyte micronucleus test (summarized in section 7.6.2). At termination a gross examination was performed, organ weights were determined and selected tissues from male and female rats of the control and high-dose groups were subjected to histopathologic examination. Reproductive indices and parameters were assessed for all dose groups.
All pups were examined as soon as possible after birth to determine number of viable and stillborn members of each litter. Survival indices were calculated at 0, 4, 7, 14, and 21 days after birth. Live pups were counted, sexed, and examined grossly at birth (postnatal day 0) and weighed individually on days 1, 4, 7, 14, and 21 after birth. Standardization of litter sizes, 4 per sex selected randomly when possible, occurred on postnatal day 4. Pups were terminated at weaning and subjected to a gross pathologic examination.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

Supplier: Ajay North America, LLC, Powder Springs, GA
Purity > 99.78%
Appearance: Colourless gas

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, MA.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 11 weeks at the start of exposure
- Weight at study initiation: around 225 g at SD 0. Body weights of males are not reported.
- Housing: The rats were housed in wire-bottom cages during exposure (one per cage except during the mating period) and in plastic shoebox cages with bedding (two males per cage, one female per cage, except during the mating period) during nonexposure periods. Exposure cages were rotated in a clockwise manner within the chamber each exposure day.
- Diet: certified rodent diet (PMI Feeds, Inc., St. Louis, MO) was available ad libitum during nonexposure periods.
- Water: water was available ad libitum during nonexposure periods.
- Acclimatization period: 2 to 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): between 21 and 26
- Humidity (%): between 35 and 65
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Whole-body inhalation exposures were performed in 690-L chambers made of stainless steel and glass.
- Source and rate of air: The substance and air for dilution were controlled through flow meters
- Temperature, humidity, oxygen and ammonia in air chamber: Relative humidity and temperature of the exposure atmosphere were constantly monitored and recorded. Oxygen concentration of the high concentration chamber was analyzed on occasion using a Hudson O2 sensor model 82T (MDA, Lincolnshire, IL). Ammonia levels during exposure were estimated not to be of biological importance
- Air flow and air change rate: To conserve the use of test material and minimize cost, total chamber air flow was reduced to 60 L/min (approximately 5 air changes/h).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Continuous analysis of the chamber air for test substance was performed using infrared absorption spectrometers. Instrumental calibration was performed using known concentrations of freshly prepared test substance in air contained in Tedlar sample bags. Calibration checks were performed at appropriate intervals.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: a maximum of 14 days
- Proof of pregnancy: The observation of vaginal or dropped copulatory plugs and/or vaginal sperm was considered evidence of successful mating (reffered to as GD 0)
- There was no replacement of males if mating did not occur. For any female that did not show evidence of successful mating after 14 days of cohabitation, the last scheduled mating day was considered gd 0 for that female and the animal was treated similarly to the other dams for subsequent events.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

- 6 hours/day, 5 days/wk prior to mating
- 6 hours/day, 7 days/ wk during the mating, gestation, and lactation phases of the study.
- Dams were not exposed from gestation day 21 through lactation day 4 to allow for parturition and early lactation.
- Pups were not exposed

Duration of test:

14 weeks

Dose / conc.:

2 000 ppm

Remarks:

equivalent to 0.2%

Dose / conc.:

7 000 ppm

Remarks:

equivalent to 0.7%

Dose / conc.:

20 000 ppm

Remarks:

equivalent to 2.0%

Control animals:

yes, concurrent vehicle

Details on study design:

16 (to yield at least 12 pregnant females). An additional three female rats were assigned to a fifth group only to serve as positive controls in the micronuclei assay (section 7.6.2).

Maternal examinations:

CAGE SIDE OBSERVATIONS
The animals were observed twice daily (morning and afternoon), including inhalation exposure periods. Signs of toxicity were recorded. Females were observed twice daily beginning on gd 20 for evidence of littering. The dams were allowed to rear their young to day 21 postpartum.

BODY WEIGHT
The body weight of the male rats were determined prior to the first exposure and weekly thereafter. The body weights of female rats were determined and recorded in the same manner until confirmation of mating. During gestation, female rats were weighed on gestational days 0, 7, 14, and 20. Dams producing litters were weighed on days 0, 4, 7, 12, and 21 postpartum.

HAEMATOLOGY
Routine haematology evaluations were conducted on blood samples taken immediately prior to termination from all animals. The blood was sampled via the posterior vena cava. Haematologic parameters were determined according to established procedures, utilizing a Cell-Dyn 3500 (Abbott Diagnostics, Chicago). Assessed parameters included blood cell count, haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, hematocrit, total and differential leukocyte count and platelet count.

CLINICAL CHEMISTRY
Routine clinical chemistry evaluations were conducted on blood samples taken immediately prior to termination from all animals. The blood was sampled via the posterior vena cava. Serum chemistry parameters were determined according to established procedures, utilizing a Vitros 250XR (Johnson and Johnson, Rochester, NY). Assessed parameters included total protein, albumin, globulin, alanine aminotransaminase (ALT), aspartate aminotransaminase (AST), alkaline phosphatase, urea nitrogen, creatinine, calcium, glucose, potassium, phosphorus, sodium, magnesium, trigylcerides and cholesterol.

SERUM THYROID LEVELS
The following serum thyroid hormone levels were determined in control and exposed rats: thyroxine (T4), triiodothyronine (T3), reverse T3 (rT3), and thyroid-stimulating hormone (TSH).
Assays for T4, T3, rT3, and TSH were performed using radioimmunoassay (RIA) kits and were carried out according to manufacturer’s instructions. For all thyroid hormone or TSH measurements, assay kits were prepared for each animal termination period (study wk 7 or 14) with the same batch number and the same expiration date. Tracer (125I) radioactivity was measured with a Packard gamma counter (Packard Instrument Co., Meriden, CT). For T3, the RIA assay kit was purchased from Diagnostic Product Corp. (Los Angeles, CA), and canine T3 antibody-coated tubes were used. For T4, the RIA assay kit was purchased from Diagnostic Product Corp. (Los Angeles, CA), and T4 antibody coated tubes were used. For rT3, the RIA assay kit was purchased from Wein Laboratories (Succasunna, NJ), and rT3 antiserum raised in the rabbit was used. For TSH, the RIA assay kit was purchased from Amersham Corp. (Arlington Heights, IL), and both lyophilized rabbit anti-rat TSH serum and Amerlex-M second antibody (donkey anti-rabbit serum coated onto magnetized polymer particles containing sodium azide) were used.

SACRIFICE
The 16 female rats/group were terminated over a 5-day period subsequent to when the last female rat on study reached lactation day 21. Animals were not fasted prior to termination, since fasting might affect thyroid hormone levels.

GROSS NECROPSY
A gross examination was performed at necropsy.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organs weighed included adrenal glands, heart, lungs, liver, kidneys, ovaries, brain, spleen, and thymus. Thyroid glands (including parathyroid glands) were removed with the trachea, fixed in buffered formalin, then dissected free of the trachea, and weighed. A single technician performed the dissection of the thyroid glands. These organs and the pituitary gland, nasal cavity, trachea, bronchi, bone marrow (sternal and femoral sections and smear), pancreas, urinary bladder, stomach, duodenum, ileum, colon, vagina, uterus, and other tissues with gross lesions were removed from all animals and preserved in 10% neutral-buffered Formalin for possible histopathological examination. Select tissues (lungs, ovaries, Thyroid glands (including parathyroid glands), nasal cavity, trachea, bronchi, bone marrow (sternal and femoral sections and smear), vagina, uterus, and other tissues with gross lesions from female rats of the control and high-dose groups were subjected to histopathologic examination.

For animals dying during the study, a complete gross necropsy and histopathologic examination were conducted to determine the possible cause of death.

Ovaries and uterine content:

Select tissues including ovaries and the uterus from female rats of the control and high-dose groups were subjected to histopathologic examination.

Fetal examinations:

Pups were terminated at weaning and subjected to a gross pathologic examination.

Statistics:

For body weight data, a repeated-measures analysis of variance (ANOVA) was conducted. An ANOVA with Bonferroni multiple comparisons was conducted on the thyroid hormone data. Fisher’s exact test was used on the reproductive data that are calculated on a group basis (e.g., gestational index). For reproductive data calculated on an individual basis (e.g., live birth index), micronuclei data, haematology and serum chemistry data, and organ weight data, the Wilcoxon rank sum test was used to increase the statistical sensitivity when comparing control group values to treated group values.

Indices:

- Number of females paired (placed with a male)
- Mating index= (Number of females with plug or sperm positive × 100)/(number of females paired)
- Fecundity index= (Number of females delivering a litter × 100)/(number of females with plug or sperm positive)
- Fertility index= (Number of females delivering a litter × 100)/(number of females paired)
- Mean gestation length
- Gestation index (%)= (Number of females with live litters × 100)/(number of females delivering a litter)
- Mean number of pups per litter
- Pup sex ratio= (Number of male pups per group)/(number of female pups per group)
- Live birth index= (Number of live pups at birth × 100)/(number of pups born)
- 4-Day survival index= (Number of pups surviving 4 days × 100)/(number of live pups at birth)
- 7-Day survival index= (Number of pups surviving 7 days × 100)/(number of pups retained at 4 days)
- 14-Day survival index= (Number of pups surviving 14 days × 100)/(number of pups retained at 4 days).
- 21-Day survival index= (Number of pups surviving 21 days × 100)/(number of pups retained at 4 days).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related clinical findings. Areas of alopecia were sporadic and considered incidental.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No deads reported

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Though not statistically significant, except for the final body weight values in female rats (day 93–95), there was a marginal decrease in mean body weights of the 2.0% group compared to the control group. The mild decrease in absolute body weights for this group of rats is due primarily to a depression in body weight gain during the first 2–3 wk of the study. Body weight values were normal for female rats of the 0.2 and 0.7% groups.

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:

effects observed, treatment-related

Description (incidence and severity):

For female rats, statistical significant differences in haematology parameters were few in number and not considered treatment related, due to (1) lack of consistency with time, (2) lack of dose response, or (3) the difference being of small magnitude (clinically insignificant). Decreases in total leukocyte count (14% lower than the control mean), haemoglobin (5%), and haematocrit (4%) were observed in the female rats of the 2.0% group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

For female rats, statistical significant differences in serum chemistry parameters (except serum thyroid hormones) were few in number and not considered treatment related, due to (1) lack of dose response, or (2) the difference being of small magnitude (clinically insignificant). Increases in cholesterol (~30% higher than the control mean), total protein (5%), and albumin (9%) were observed in both the 0.7% and 2.0% groups of female rats.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

For female rats, decreases in mean absolute organ weights included the brain (0.7% and 2.0%), ovaries (0.7% and 2.0%), and heart (2.0%). Mean relative brain (2.0%), kidney (2.0%), and liver (0.7%) weights were increased. Mean relative ovaries weights were decreased (0.7% and 2.0%). Note that the mean body weight was statistically significantly decreased in the 2.0% female rats compared to the control female rats.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no clinically significant or exposure-related gross lesions in any of the study animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no lesions of clinical significance in any treated (2.0% group) or control group animals. Tissues from animals in the 0.7% or 0.2% groups were not examined microscopically, due to the lack of microscopic findings in rats of the control and 2.0% groups.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant and concentration-related increases in T4 and rT3 were observed in female rats after 7 or 14 wk of exposure. T3 levels were decreased in a concentration-related manner in these animals. For TSH, an increase was observed in exposed male rats after 7 wk of exposure, but there was no concentration relationship. After 14 wk of exposure, male and female rats of the 2.0% group had statistically significant increases in TSH levels compared to control rats. A marginal and statistically significant increase in TSH was observed in female rats of the 0.2% group, but this effect was not statistically significant in female rats at 0.7%.

Number of abortions:

no effects observed

Description (incidence and severity):

There were no statistically significant differences between treated and control groups in Gestation index, Mean number of pups per litter and Live birth index). The decrease in mean live birth index (not statistically significant compared to the control group) and unusually high standard deviation for rats of the 2.0% group were due to 1 dam that delivered no live pups.

Pre- and post-implantation loss:

not examined

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no statistically significant differences between treated and control groups in Gestation index, Mean number of pups per litter and Live birth index). The decrease in mean live birth index (not statistically significant compared to the control group) and unusually high standard deviation for rats of the 2.0% group were due to 1 dam that delivered no live pups.

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no statistically significant differences between treated and control groups in all indices and endpoints measured Gestation index, Mean number of pups per litter and Live birth index). The decrease in mean live birth index (not statistically significant compared to the control group) and unusually high standard deviation for rats of the 2.0% group were due to 1 dam that delivered no live pups.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

There were no statistically significant differences between treated and control groups in Mean gestation length.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There were no statistically significant differences between treated and control groups in Mating index, Fecundity index and Fertility index.

Other effects:

not examined

Key result

Dose descriptor:

NOAEC

Effect level:

20 000 ppm

Based on:

test mat.

Basis for effect level:

other: Highest dose tested

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no statistically significant differences between control and exposure groups in mean pup weights.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no statistically significant differences between treated and control groups Mean number of pups per litter and Live birth index. The decrease in mean live birth index (not statistically significant compared to the control group) and unusually high standard deviation for rats of the 2.0% group were due to 1 dam that delivered no live pups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no statistically significant differences between treated and control groups in Pup sex ratio. The decrease in pup sex ratio in the 2.0% group was statistically significant, but it was not considered exposure related. The concurrent control group range in pup sex ratio (male/female), on a per titter basis, was 0.38 to 2.33. For the 2.0% group, the range was 0.22 to 2.00. Further, there was no exposure trend in the means of pup sex ratio

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no statistically significant differences between treated and control groups in Mean number of pups per litter

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

In addition, there were no statistically significant differences between control and exposure groups in mean pup survival indices

External malformations:

no effects observed

Description (incidence and severity):

No abnormalities are reported

Skeletal malformations:

not examined

Visceral malformations:

not examined

Key result

Dose descriptor:

NOAEC

Effect level:

20 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest dose tested

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Analytical verification of chamber atmosphere

The test material was 99.7+%, which remained stable throughout the study period. Analytical chamber concentrations matched the target exposure concentrations of 0 (control), 0.2, 0.7, and 2.0%.

The nominal mean concentrations (standard deviation) were 2.08% (0.09), 0.72% (0.03), and 0.24% (0.015) for the target concentrations of 2.0%, 0.70%, and 0.20%. The ratio between analytical/nominal concentrations as calculated from the rotometer settings for the air and test substance were 0.97, 0.98 and 0.83 for the 2%, 0.7%, and 0.2% target concentrations, respectively.

Chamber temperature and relative humidity means for all exposure groups ranged from 73.5 to 74.2°F and 43.4 to 54.8%, respectively. The oxygen concentration of the 2.0% chamber was approximately 19%. The deviation in this study from the accepted 10–14 air changes per hour during animal exposure to 5 air changes per hour did not have an impact on exposure temperature, relative humidity, oxygen concentration, or substance concentration.

Conversion of NOAEC

2% test substance is corresponding to 20.000 ppm or 16024.4 mg/m3 (based on a molecular weight of 195.91)

Conclusions:

Exposure of 2.0% test substance vapor for approximately 14 weeks produced minimal maternal toxicity and no developmental toxicity in Sprague-Dawley rats. The NOAEC for maternal and developmental toxicity is set at 2.0%, which is the highest dose tested

Executive summary:

A combined repeated dose/ reproductive screening study was performed. The purpose of this study was to determine and evaluate the potential of the substance to produce reproductive toxicity and to provide additional information on the effect of test substance exposure on the thyroid. Groups of 16 male and 16 female rats were exposed (6hr/day) to substance vapor at concentrations of 0 (control), 0.2, 0.7, and 2.0% using whole body inhalation chambers. Prior to mating, rats were exposed for 4 weeks (5 days/wk). Exposures were 7 days/wk during the periods of mating (2 wk), gestation (3 wk), and lactation (3 wk). First generation pups were not exposed to substance vapor. In maternal animals, there were no clinical signs of toxicity except for a minimal decrease in mean body weight in female rats at 2.0%. At necropsy, gross findings, mean serum chemistry levels, mean haematology values mean bone marrow micronuclei scores (detailled in section 7.6.2), and mean organ weights were similar for all exposure groups, including the air control group. Statistically significant differences were considered incidental. There were no treatment-related histopathologic tissue findings, including the thyroid organ. Analysis of reproductive indices and developmental parameters indicate that the substance is not a reproductive toxicant. Results of serum thyroid hormone levels (e.g., T3, T4, rT3, and TSH), indicated concentration-related increases in TSH, T4, and rT3. T3 levels were decreased. First generation pup survival and mean body weights were similar in all exposure groups, including the control. Exposure of 2.0% vapor for approximately 14 weeks produced minimal general toxicity and no reproductive toxicity in Sprague-Dawley rats. On the basis of serum TSH concentrations, the no-observable-effect-level (NOEL) is 0.7%. The NOAEC for systemic toxicity and reproductive effects was set at 2%.

Cross-reference 3

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Dec 1996 to Jan 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

An erythrocyte micronucleus assay was performed as part of a combined repeated dose and reproductive toxicity screening study was performed in Sprague-Dawley Crl:CD(SD)BR rats. In total the study had 16 male and 16 female rats/group to yield at least 12 pregnant females at term. The animals were assigned to one of four groups (0, 0.2, 0.7, and 2.0%) and exposed to substance vapor for 4 wk (6 h/day, 5 days/wk) prior to mating. An additional six male and three female rats were assigned to a fifth group only to serve as positive controls in the micronuclei assay. Animals were exposed 6 h/day, 7 days/wk, during the mating, gestation, and lactation phases of the study. However, dams were not exposed from gestation day 21 through lactation day 4 to allow for parturition and early lactation. The remaining 8 male rats/group and 16 female rats/group were terminated over a 5-day period subsequent to when the last female rat on study reached lactation day 21. Animals were killed by CO2 inhalation overdose followed by exsanguination. Bone marrow cells were collected from the femur and smears were prepared from all rats. Positive control rats were administered a single dose of cyclophosphamide (7.5 mg/kg) intraperitoneally 24 hrs prior to termination. Slides were stained by the Giemsa/May-Greenwald method and observed microscopically at 100x.

GLP compliance:

no

Type of assay:

mammalian erythrocyte micronucleus test

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD (SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, MA.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 11 weeks at the start of exposure
- Weight at study initiation: around 225 g at SD 0. Body weights of males are not reported.
- Housing: The rats were housed in wire-bottom cages during exposure (one per cage except during the mating period) and in plastic shoebox cages with bedding (two males per cage, one female per cage, except during the mating period) during nonexposure periods. Exposure cages were rotated in a clockwise manner within the chamber each exposure day.
- Diet: certified rodent diet (PMI Feeds, Inc., St. Louis, MO) was available ad libitum during nonexposure periods.
- Water: water was available ad libitum during nonexposure periods.
- Acclimatization period: 2 to 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): between 21 and 26
- Humidity (%): between 35 and 65
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Whole-body inhalation exposures were performed in 690-L chambers made of stainless steel and glass.
- Source and rate of air: The substance and air for dilution were controlled through flow meters
- Temperature, humidity, oxygen and ammonia in air chamber: Relative humidity and temperature of the exposure atmosphere were constantly monitored and recorded. Oxygen concentration of the high concentration chamber was analyzed on occasion using a Hudson O2 sensor model 82T (MDA, Lincolnshire, IL). Ammonia levels during exposure were estimated not to be of biological importance
- Air flow and air change rate: To conserve the use of test material and minimize cost, total chamber air flow was reduced to 60 L/min (approximately 5 air changes/h).

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

- 6 hours/day, 5 days/wk prior to mating
- 6 hours/day, 7 days/ wk during the mating, gestation, and lactation phases of the study.
- Dams were not exposed from gestation day 21 through lactation day 4 to allow for parturition and early lactation.
- Pups were not exposed

Dose / conc.:

2 000 ppm

Remarks:

equivalent to 0.2%

Dose / conc.:

7 000 ppm

Remarks:

equivalent to 0.7%

Dose / conc.:

20 000 ppm

Remarks:

equivalent to 2.0%

No. of animals per sex per dose:

16 (to yield at least 12 pregnant females); positive control: 6 male and 3 female animals

Control animals:

yes, concurrent vehicle

Positive control(s):

Single dose of cyclophosphamide (7.5 mg/kg) intraperitoneally 24 hrs prior to termination.

Tissues and cell types examined:

Bone marrow erythrocytes

Details of tissue and slide preparation:

Bone marrow cells were collected from the femur and smears were prepared from all rats. Positive control rats were administered a single dose of cyclophosphamide (7.5 mg/kg) intraperitoneally 24 hrs prior to termination. Slides were stained by the Giemsa/May-Greenwald method and observed microscopically at 100x.

Evaluation criteria:

The frequency of micronucleated cells were evaluated by random observation of 1000 polychromatic erythrocytes (PCE) per sample. The ratio between PCE and normochromatic erythrocytes (NCE) was determined by scoring approximately 1000 erythrocytes as an indicator oftoxicity ofthe test agent.

Statistics:

For micronuclei data the Wilcoxon Rank Sum test was used to increase the statistical sensitivity when comparing control group values to treated group values.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Micronuclei scores for male and female rats exposed to CF3I for 7 or 14 wk were similar to micronuclei scores in the control animals. Positive control animals had mean micronuceli scores that were two- to five-fold higher than negative control values. The weak response in the positive control animals was due to the administration of a small dose of cyclophosphamide to induce micronuclei formation. The ratio ofPCE/NCE (an indicator of bone marrow cell toxicity) was similar in all study groups, control or CF3I exposure.

Conclusions:

Bone marrow micronucleus assay did not no show any dose related mutagenic alterations in male or female Sprague Dawley rats at the high exposure dose of 2.0 %.

Executive summary:

An erythrocyte micronucleus assay was performed as part of a combined repeated dose and reproductive toxicity screening study was performed in Sprague-Dawley Crl:CD(SD)BR rats (detailed description in Section 7.8.1). In total the study had 16 male and 16 female rats/group to yield at least 12 pregnant females at term. The animals were assigned to one of four groups (0, 0.2, 0.7, and 2.0%) and exposed to substance vapor for 4 wk (6 h/day, 5 days/wk) prior to mating. An additional six male and three female rats were assigned to a fifth group only to serve as positive controls in the micronuclei assay (Single dose of cyclophosphamide (7.5 mg/kg) intraperitoneally 24 hrs prior to termination.). Animals were exposed 6 h/day, 7 days/wk, during the mating, gestation, and lactation phases of the study. However, dams were not exposed from gestation day 21 through lactation day 4 to allow for parturition and early lactation. The remaining 8 male rats/group and 16 female rats/group were terminated over a 5-day period subsequent to when the last female rat on study reached lactation day 21. Animals were killed by CO2 inhalation overdose followed by exsanguination. Bone marrow cells were collected from the femur and smears were prepared from all rats. Positive control rats were administered a single dose of cyclophosphamide (7.5 mg/kg) intraperitoneally 24 hrs prior to termination. Slides were stained by the Giemsa/May-Greenwald method and observed microscopically at 100x. The frequency of micronucleated cells was evaluated by random observation of 1000 polychromatic erythrocytes (PCE) per sample. The ratio between PCE and normochromatic erythrocytes (NCE) was determined by scoring approximately 1000 erythrocytes as an indicator of toxicity of the test agent. Micronuclei scores for male and female rats exposed to CF3I for 7 or 14 wk were similar to micronuclei scores in the control animals.

In the current investigation, CF3I was negative in the in vivo micronucleus test since there was no increase in micronuclei frequency in the bone marrow erythrocytes of rats exposed to the test item.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

REPRODUCTIVE TOXICITY SCREEN OF TRIFLUOROIODOMETHANE (CF3I) IN SPRAGUE-DAWLEY RATS

Author:

Dodd DE, Leahy HF, Feldmann ML, English JH, Vinegar A

Year:

1999

Bibliographic source:

Inhalation Toxicology, 11:1041–1055, 1999

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

A combined repeated dose and reproductive toxicity screening study was performed in Sprague-Dawley Crl:CD(SD)BR rats. The study had 16 male and 16 female rats/group to yield at least 12 pregnant females at term. The animals were assigned to one of four groups (0, 0.2, 0.7, and 2.0%) and exposed to substance vapor for 4 wk (6 h/day, 5 days/wk) prior to mating. An additional six male and three female rats were assigned to a fifth group only to serve as positive controls in the micronuclei assay. Animals were exposed 6 h/day, 7 days/wk, during the mating, gestation, and lactation phases of the study. However, dams were not exposed from gestation day 21 through lactation day 4 to allow for parturition and early lactation. Pups were not exposed to and remained in their home cages under observation. Following the 14-day mating period, eight male rats/group were terminated (study wk 7). The remaining 8 male rats/group and 16 female rats/group were terminated over a 5-day period subsequent to when the last female rat on study reached lactation day 21. Animals were killed by CO2 inhalation overdose followed by exsanguination.
Examinations included clinical observations (twice daily), bodyweight, haematology, clinical chemistry, serum thyroid hormone levels and bone marrow erythrocyte micronucleus test (summarized in section 7.6.2). At termination a gross examination was performed, organ weights were determined and selected tissues from male and female rats of the control and high-dose groups were subjected to histopathologic examination. Reproductive indices and parameters were assessed for all dose groups.
All pups were examined as soon as possible after birth to determine number of viable and stillborn members of each litter. Survival indices were calculated at 0, 4, 7, 14, and 21 days after birth. Live pups were counted, sexed, and examined grossly at birth (postnatal day 0) and weighed individually on days 1, 4, 7, 14, and 21 after birth. Standardization of litter sizes, 4 per sex selected randomly when possible, occurred on postnatal day 4. Pups were terminated at weaning and subjected to a gross pathologic examination.

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Trifluoroiodomethane

EC Number:

219-014-5

EC Name:

Trifluoroiodomethane

Cas Number:

2314-97-8

Molecular formula:

CF3I

IUPAC Name:

trifluoroiodomethane

Specific details on test material used for the study:

Supplier: Ajay North America, LLC, Powder Springs, GA
Purity > 99.78%
Appearance: Colourless gas

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, MA.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 11 weeks at the start of exposure
- Weight at study initiation: around 225 g at SD 0. Body weights of males are not reported.
- Housing: The rats were housed in wire-bottom cages during exposure (one per cage except during the mating period) and in plastic shoebox cages with bedding (two males per cage, one female per cage, except during the mating period) during nonexposure periods. Exposure cages were rotated in a clockwise manner within the chamber each exposure day.
- Diet: certified rodent diet (PMI Feeds, Inc., St. Louis, MO) was available ad libitum during nonexposure periods.
- Water: water was available ad libitum during nonexposure periods.
- Acclimatization period: 2 to 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): between 21 and 26
- Humidity (%): between 35 and 65
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Whole-body inhalation exposures were performed in 690-L chambers made of stainless steel and glass.
- Source and rate of air: The substance and air for dilution were controlled through flow meters
- Temperature, humidity, oxygen and ammonia in air chamber: Relative humidity and temperature of the exposure atmosphere were constantly monitored and recorded. Oxygen concentration of the high concentration chamber was analyzed on occasion using a Hudson O2 sensor model 82T (MDA, Lincolnshire, IL). Ammonia levels during exposure were estimated not to be of biological importance
- Air flow and air change rate: To conserve the use of test material and minimize cost, total chamber air flow was reduced to 60 L/min (approximately 5 air changes/h).

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: a maximum of 14 days
- Proof of pregnancy: The observation of vaginal or dropped copulatory plugs and/or vaginal sperm was considered evidence of successful mating (reffered to as GD 0)
- There was no replacement of males if mating did not occur. For any female that did not show evidence of successful mating after 14 days of cohabitation, the last scheduled mating day was considered gd 0 for that female and the animal was treated similarly to the other dams for subsequent events.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Continuous analysis of the chamber air for test substance was performed using infrared absorption spectrometers. Instrumental calibration was performed using known concentrations of freshly prepared test substance in air contained in Tedlar sample bags. Calibration checks were performed at appropriate intervals.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

- 6 hours/day, 5 days/wk prior to mating
- 6 hours/day, 7 days/ wk during the mating, gestation, and lactation phases of the study.
- Dams were not exposed from gestation day 21 through lactation day 4 to allow for parturition and early lactation.
- Pups were not exposed

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 000 ppm

Remarks:

equivalent to 0.2%

Doses / concentrations 2

Dose / conc.:

7 000 ppm

Remarks:

equivalent to 0.7%

Doses / concentrations 3

Dose / conc.:

20 000 ppm

Remarks:

equivalent to 2.0%

No. of animals per sex per dose:

16 (to yield at least 12 pregnant females). An additional six male and three female rats were assigned to a fifth group only to serve as positive controls in the micronuclei assay (section 7.6.2).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: 2 and 13 wk repeated dose toxicity studies are available. Exposure concentrations selected for this study extended beyond those of the 13-wk study (Dodd 1997)

Positive control:

Single dose of cyclophosphamide (7.5 mg/kg) for the micronucleus assay (please refer to section 7.6.2).

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS
The animals were observed twice daily (morning and afternoon), including inhalation exposure periods. Signs of toxicity were recorded. Females were observed twice daily beginning on gd 20 for evidence of littering. The dams were allowed to rear their young to day 21 postpartum.

BODY WEIGHT
The body weight of the male rats were determined prior to the first exposure and weekly thereafter. The body weights of female rats were determined and recorded in the same manner until confirmation of mating. During gestation, female rats were weighed on gestational days 0, 7, 14, and 20. Dams producing litters were weighed on days 0, 4, 7, 12, and 21 postpartum.

HAEMATOLOGY
Routine haematology evaluations were conducted on blood samples taken immediately prior to termination from all animals. The blood was sampled via the posterior vena cava. Haematologic parameters were determined according to established procedures, utilizing a Cell-Dyn 3500 (Abbott Diagnostics, Chicago). Assessed parameters included blood cell count, haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, hematocrit, total and differential leukocyte count and platelet count.

CLINICAL CHEMISTRY
Routine clinical chemistry evaluations were conducted on blood samples taken immediately prior to termination from all animals. The blood was sampled via the posterior vena cava. Serum chemistry parameters were determined according to established procedures, utilizing a Vitros 250XR (Johnson and Johnson, Rochester, NY). Assessed parameters included total protein, albumin, globulin, alanine aminotransaminase (ALT), aspartate aminotransaminase (AST), alkaline phosphatase, urea nitrogen, creatinine, calcium, glucose, potassium, phosphorus, sodium, magnesium, trigylcerides and cholesterol.

SERUM THYROID LEVELS
The following serum thyroid hormone levels were determined in control and exposed rats: thyroxine (T4), triiodothyronine (T3), reverse T3 (rT3), and thyroid-stimulating hormone (TSH).
Assays for T4, T3, rT3, and TSH were performed using radioimmunoassay (RIA) kits and were carried out according to manufacturer’s instructions. For all thyroid hormone or TSH measurements, assay kits were prepared for each animal termination period (study wk 7 or 14) with the same batch number and the same expiration date. Tracer (125I) radioactivity was measured with a Packard gamma counter (Packard Instrument Co., Meriden, CT). For T3, the RIA assay kit was purchased from Diagnostic Product Corp. (Los Angeles, CA), and canine T3 antibody-coated tubes were used. For T4, the RIA assay kit was purchased from Diagnostic Product Corp. (Los Angeles, CA), and T4 antibody coated tubes were used. For rT3, the RIA assay kit was purchased from Wein Laboratories (Succasunna, NJ), and rT3 antiserum raised in the rabbit was used. For TSH, the RIA assay kit was purchased from Amersham Corp. (Arlington Heights, IL), and both lyophilized rabbit anti-rat TSH serum and Amerlex-M second antibody (donkey anti-rabbit serum coated onto magnetized polymer particles containing sodium azide) were used.

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Testes and epididymides weights were determined and histopathology was performed.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 4 pups/ sex /litter

PARAMETERS EXAMINED
All pups were examined as soon as possible after birth to determine number of viable and stillborn members of each litter. Survival indices were calculated at 0, 4, 7, 14, and 21 days after birth. Live pups were counted, sexed, and examined grossly at birth (postnatal day 0) and weighed individually on days 1, 4, 7, 14, and 21 after birth. Pups were terminated at weaning and subjected to a gross pathologic examination.

Postmortem examinations (parental animals):

SACRIFICE
Following the 14-day mating period, eight male rats/group were terminated (study wk 7). The remaining 8 male rats/group and 16 female rats/group were terminated over a 5-day period subsequent to when the last female rat on study reached lactation day 21. Animals were not fasted prior to termination, since fasting might affect thyroid hormone levels.

GROSS NECROPSY
A gross examination was performed at necropsy.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organs weighed included adrenal glands, heart, lungs, liver, kidneys, ovaries, testes, epididymides, brain, spleen, and thymus. Thyroid glands (including parathyroid glands) were removed with the trachea, fixed in buffered formalin, then dissected free of the trachea, and weighed. A single technician performed the dissection of the thyroid glands. These organs and the pituitary gland, nasal cavity, trachea, bronchi, bone marrow (sternal and femoral sections and smear), pancreas, urinary bladder, stomach, duodenum, ileum, colon, prostate, scrotum, seminal vesicles, vagina, uterus, and other tissues with gross lesions were removed from all animals and preserved in 10% neutral-buffered Formalin for possible histopathological examination. Select tissues (lungs, ovaries, testes, epididymides, Thyroid glands (including parathyroid glands), nasal cavity, trachea, bronchi, bone marrow (sternal and femoral sections and smear), prostate, scrotum, seminal vesicles, vagina, uterus, and other tissues with gross lesions from male and female rats of the control and high-dose groups were subjected to histopathologic examination. Testes and epididymides were fixed in Bouin’s fixative; sections were stained with periodic acid and Schiff’s (PAS) and counterstained with hematoxylin.

For animals dying during the study, a complete gross necropsy and histopathologic examination were conducted to determine the possible cause of death.

Postmortem examinations (offspring):

Pups were terminated at weaning and subjected to a gross pathologic examination.

Statistics:

For body weight data, a repeated-measures analysis of variance (ANOVA) was conducted. An ANOVA with Bonferroni multiple comparisons was conducted on the thyroid hormone data. Fisher’s exact test was used on the reproductive data that are calculated on a group basis (e.g., gestational index). For reproductive data calculated on an individual basis (e.g., live birth index), micronuclei data, haematology and serum chemistry data, and organ weight data, the Wilcoxon rank sum test was used to increase the statistical sensitivity when comparing control group values to treated group values.

Reproductive indices:

- Number of females paired (placed with a male)
- Mating index= (Number of females with plug or sperm positive × 100)/(number of females paired)
- Fecundity index= (Number of females delivering a litter × 100)/(number of females with plug or sperm positive)
- Fertility index= (Number of females delivering a litter × 100)/(number of females paired)
- Mean gestation length
- Gestation index (%)= (Number of females with live litters × 100)/(number of females delivering a litter)
- Mean number of pups per litter
- Pup sex ratio= (Number of male pups per group)/(number of female pups per group)
- Live birth index= (Number of live pups at birth × 100)/(number of pups born)

Offspring viability indices:

- 4-Day survival index= (Number of pups surviving 4 days × 100)/(number of live pups at birth)
- 7-Day survival index= (Number of pups surviving 7 days × 100)/(number of pups retained at 4 days)
- 14-Day survival index= (Number of pups surviving 14 days × 100)/(number of pups retained at 4 days).
- 21-Day survival index= (Number of pups surviving 21 days × 100)/(number of pups retained at 4 days).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related clinical findings. Areas of alopecia were sporadic and considered incidental.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No deads reported

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Though not statistically significant, except for the final body weight values in female rats (day 93–95), there was a marginal decrease in mean body weights of the 2.0% group compared to the control group. The mild decrease in absolute body weights for this group of rats is due primarily to a depression in body weight gain during the first 2–3 wk of the study. Body weight values were normal for male and female rats of the 0.2 and 0.7% groups.

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:

effects observed, treatment-related

Description (incidence and severity):

For male and female rats, statistical significant differences in haematology parameters were few in number and not considered treatment related, due to (1) lack of consistency with time (7-wk compared to 14- wk values for male rats), (2) lack of dose response, or (3) the difference being of small magnitude (clinically insignificant). Decreases in total leukocyte count (14% lower than the control mean), haemoglobin (5%), and haematocrit (4%) were observed in the female rats of the 2.0% group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

For male and female rats, statistical significant differences in serum chemistry parameters (except serum thyroid hormones) were few in number and not considered treatment related, due to (1) lack of consistency with time (7-wk compared to 14- wk values for male rats), (2) lack of dose response, or (3) the difference being of small magnitude (clinically insignificant). Increases in cholesterol (~30% higher than the control mean), total protein (5%), and albumin (9%) were observed in both the 0.7% and 2.0% groups of female rats.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no lesions of clinical significance in any treated (2.0% group) or control group animals. Tissues from animals in the 0.7% or 0.2% groups were not examined microscopically, due to the lack of microscopic findings in rats of the control and 2.0% groups.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant and concentration-related increases in T4 and rT3 were observed in male and female rats after 7 or 14 wk of exposure. T3 levels were decreased in a concentration-related manner in these animals. For TSH, an increase was observed in exposed male rats after 7 wk of exposure, but there was no concentration relationship. After 14 wk of exposure, male and female rats of the 2.0% group had statistically significant increases in TSH levels compared to control rats. A marginal and statistically significant increase in TSH was observed in female rats of the 0.2% group, but this effect was not statistically significant in female rats at 0.7%.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no statistically significant differences between treated and control groups in all indices and endpoints measured. The decrease in mean live birth index (not statistically significant compared to the control group) and unusually high standard deviation for rats of the 2.0% group were due to 1 dam that delivered no live pups.

Effect levels (P0)

1

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

20 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest dose tested

2

Key result

Dose descriptor:

NOAEC

Remarks:

reproductive toxicity

Effect level:

20 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest dose tested

3

Dose descriptor:

NOEC

Remarks:

systemic toxicity

Effect level:

7 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Serum TSH concentrations

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment- related clinical observations in the first-generation pups from birth to postnatal day 21.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no statistically significant differences between control and exposure groups in mean pup survival indices

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no statistically significant differences between control and exposure groups in mean pup weights.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

The decrease in pup sex ratio in the 2.0% group was statistically significant, but it was not considered exposure related. The concurrent control group range in pup sex ratio (male/female), on a per titter basis, was 0.38 to 2.33. For the 2.0% group, the range was 0.22 to 2.00. Further, there was no exposure trend in the means of pup sex ratio

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormalities are reported

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

20 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest dose tested

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Analytical verification of chamber atmosphere

The test material was 99.7+%, which remained stable throughout the study period. Analytical chamber concentrations matched the target exposure concentrations of 0 (control), 0.2, 0.7, and 2.0%.

The nominal mean concentrations (standard deviation) were 2.08% (0.09), 0.72% (0.03), and 0.24% (0.015) for the target concentrations of 2.0%, 0.70%, and 0.20%. The ratio between analytical/nominal concentrations as calculated from the rotometer settings for the air and test substance were 0.97, 0.98 and 0.83 for the 2%, 0.7%, and 0.2% target concentrations, respectively.

Chamber temperature and relative humidity means for all exposure groups ranged from 73.5 to 74.2°F and 43.4 to 54.8%, respectively. The oxygen concentration of the 2.0% chamber was approximately 19%. The deviation in this study from the accepted 10–14 air changes per hour during animal exposure to 5 air changes per hour did not have an impact on exposure temperature, relative humidity, oxygen concentration, or substance concentration.

Conversion of NOAEC

2% test substance is corresponding to 20.000 ppm or 16024.4 mg/m3 (based on a molecular weight of 195.91)

Applicant's summary and conclusion

Conclusions:

Exposure of 2.0% test substance vapor for approximately 14 weeks produced minimal general toxicity and no reproductive toxicity in Sprague-Dawley rats. On the basis of reproductive indices and parameters, the NOAEC for this study is 2.0%.

Executive summary:

A combined repeated dose/ reproductive screening study was performed. The purpose of this study was to determine and evaluate the potential of the substance to produce reproductive toxicity and to provide additional information on the effect of test substance exposure on the thyroid. Groups of 16 male and 16 female rats were exposed (6hr/day) to substance vapor at concentrations of 0 (control), 0.2, 0.7, and 2.0% using whole body inhalation chambers. Prior to mating, rats were exposed for 4 weeks (5 days/wk). Exposures were 7 days/wk during the periods of mating (2 wk), gestation (3 wk), and lactation (3 wk). First generation pups were not exposed to substance vapor. In parental animals, there were no clinical signs of toxicity except for a minimal decrease in mean body weight in female rats at 2.0%. At necropsy, gross findings, mean serum chemistry levels, mean haematology values mean bone marrow micronuclei scores (detailled in section 7.6.2), and mean organ weights were similar for all exposure groups, including the air control group. Statistically significant differences were considered incidental. There were no treatment-related histopathologic tissue findings, including the thyroid organ. Analysis of reproductive indices and developmental parameters indicate that the substance is not a reproductive toxicant. Results of serum thyroid hormone levels (e.g., T3, T4, rT3, and TSH), indicated concentration-related increases in TSH, T4, and rT3. T3 levels were decreased. First generation pup survival and mean body weights were similar in all exposure groups, including the control. Exposure of 2.0% vapor for approximately 14 weeks produced minimal general toxicity and no reproductive toxicity in Sprague-Dawley rats. On the basis of serum TSH concentrations, the no-observable-effect-level (NOEL) is 0.7%. The NOAEC for systemic toxicity and reproductive effects was set at 2%.