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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2002-06-20 to 2002-07-12
Reliability:
other: dose range finding experiment for a pre-natal developmental toxicity study
Rationale for reliability incl. deficiencies:
other: Rating according to Klimisch is not applicable, since this reference is a dose range finding experiment for a pre-natal developmental toxicity study.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(adopted 2001-01-22)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS - Sprague-Dawley [Crl: CD® (SD)IGS BR]
- Source: Charles River Laboratories, Portage, Michigan
- Age at study initiation: approximately 10 weeks old
- Weight at study initiation: 210 - 243 g
- Housing: rats were individually housed in suspended, stainless steel, wire-mesh type cages, except during mating when the females were housed in similar cages with males.
- Diet (ad libitum during non-exposure period): meal Lab Diet® #5002, PMI Nutrition International, Inc., St. Louis, Missouri)
- Water (ad libitum during non-exposure period ): tap water
- Acclimation period: one week

A sample each of diet and water were collected and analysed for total antimony (diet: <0.5 µg/g; water: <0.005 µg/g).

ENVIRONMENTAL CONDITIONS
- Temperature: 16.1 - 23.3 °C
- Relative humidity: 48 - 73%
- Photoperiod (hrs dark / hrs light): approximately 12 hours per day

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure (if applicable):
nose only
Vehicle:
clean air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: the exposures were conducted in a 63 L stainless steel and acrylic nose-only exposure chamber with a stainless steel baffle.
- Method of holding animals in test chamber: for the exposure, each animal was removed from the home cage and placed in a nose-only restraint
tube. Each animal nose protruded from a small opening in the conical end of the tube. The conical end of the tube was inserted into the chamber prior to generation of test atmosphere.

- System of generating particulates/aerosols: the test article was generated into the breathing air of the treated animals. Dust aerosol atmosphere of the test article were generated using a Wright Dust feeder (WDF). Prior to any generation, the test article was packed into a cylindrical holder (cup) with high pressure to provide a compact uniform test article surface. A scraping blade in the WDF rotated over the test article surface in the cup, removing small amounts of test article. The test article that was dislodged by the scraping blade was entrained in the air stream, passed through the WDF, and transported out of the WDF into the elutriation chamber prior to entering the exposure chamber. Dilution air was introduced into the chamber as necessary to increase the chamber airflow and achieve correct chamber concentration.

The control animals were exposed to clean air by the same exposure regimen as the treated groups except that the test article was not introduced into the chamber and a WDF was not used; compressed air was introduced into the chamber from the opening in the top.

- Airflow, temperature, humidity: a chamber airflow of at least 0.6 L per minute per animal supplied by the generation system resulted in at least 10 chamber air changes per hour and an oxygen level at or above 19%. The chamber was maintained to the maximum extent possible at a mean temperature between 21 to 22°C and a mean relative humidity between 4 to 6%. Chamber temperature , percent relative humidity, and airflow rate were monitored continuously and recorded at 30 minutes intervals during the exposure period.

- Method of particle size determination: one particle size distribution was performed at least once during each exposure using the TSI Aerodynamic Particle Sizer (APS), to determine the mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of any aerosol present.
The instrument consisted of the APS, a diluter (model 3302), and a computer. The diluter reduced the particle concentration of high concentration aerosols. If the aerosol concentration was too high, coincidence counting would have occurred. To keep the coincidence error below 5%, the particle concentration was maintained at less than 2850 particles per cubic centimeter for 0.5 µm particles, or 1193 particles per cubic centimeter for 10 µm particles.
The following mean MMAD ± standard deviation /mean GSD ± Standard deviation were determined:
0.39 mg/m³: 1.96 µm ± 0.300 / 1.63 ± 0.147
0.73 mg/m³: 1.89 µm ± 0.309 / 1.60 ± 0.069
1.48 mg/m³: 1.73 µm ± 0.440 / 1.63 ± 0.111
6.07 mg/m³: 1.59 µm ± 0.241 / 1.68 ± 0.152

TEST ATMOSPHERE
- Brief description of analytical method used: chamber atmosphere samples for determination of the test article exposure level were collected. The samples were withdrawn from the exposure chamber through metricel membrane filters mounted on an open-faced filter holder. The gravimetric concentrations were calculated with the use of atomic absorption.
Prior to initiation of animal exposures, samples were taken to show that the test article was evenly distributed from port to port on the nose-only chamber.

The amount of test article delivered by the generation system during the exposure was divided by the total volume of air passing through the chamber to give the nominal concentration.
Mean nominal concentration:
0.39 mg/m³: 9.7 ± 10.50 mg/m³
0.73 mg/m³: 15.2 ± 8.61 mg/m³
1.48 mg/m³: 12.9 ± 8.06 mg/m³
6.07 mg/m³: 25.5 ± 13.81 mg/m³

In an attempt to control the exposures at these very low exposures levels, the test article was generated at a higher more stable level and then diluted down to achieve the targeted exposure level. As a result the nominal exposure levels were higher than the actual measured levels. In addition, this difference also represents the normal losses seen with deposition of the test article on the walls within the chamber and generation system.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Please refer to the field "Details on exposure" above.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 female :1 male
- Proof of pregnancy: vaginal plug and/or sperm referred to as day 0 of gestation
Duration of treatment / exposure:
Day 0 to day 19 of gestation
Frequency of treatment:
approximately six hours/day
Duration of test:
20 days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.39 ± 0.455 mg/m³
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0.73 ± 1.017 mg/m³
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
1.48 ± 1.986 mg/m³
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
6.07 ± 6.479 mg/m³
Basis:
analytical conc.
No. of animals per sex per dose:
5 mated females
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice each day, seven days a week
- Cage side observations checked: morbidity, mortality, and signs of injury.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily from Days 0 through 20 of gestation, each rat was removed from the cage and given a detailed clinical examination. During the treatment period, these examinations were conducted as animals were removed from the exposure chambers. The first group of animals examined was randomized each day of the exposure period.

BODY WEIGHT: Yes
- Time schedule for examinations: individual body weights were recorded on Days 0, 3, 6, 9, 12, 15, 18, and 20 of gestation. Individual body weight change was calculated for the following gestation day intervals: 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18, 18 - 20, and 0 - 20. Adjusted body weight (Day 20 gestation body weight minus the gravid uterine weight) and adjusted body weight change (Days 0 - 20 of gestation) were also calculated.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined: Yes
Food consumption was recorded on the corresponding body weight days and calculated for the following intervals: Days 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18, 18 - 20, and 0- 20 of gestation.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
On day 20 of gestation, each female was euthanized by carbon dioxide inhalation and immediately subjected to a laparohysterectomy.
A complete necropsy was performed on all dams. Special emphasis was placed on structural abnormalities or pathologic changes that may have influenced the pregnancy. Gross lesions and/or target organs were saved in 10% neutral buffered formalin for possible microscopic examination, and the carcasses were discarded.
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
- Beginning at the distal end of the left uterine horn, location of viable and nonviable foetuses was recorded. The position of the cervix was also recorded. The placentae were grossly examined.
Fetal examinations:
Foetuses were individually weighed, measured for crown-rump distance, sexed externally, and examined for external malformations and variations. No external malformations or developmental variations were found, this no statistical analyses were performed for these endpoints.
Statistics:
1) Group pair-wise comparisons (Levene's test, Dunnett's test, and Welch's t-test with a Bonferroni correction)
Endpoints examined: gestation body weights, gestation body weight changes, gestation food consumption, adjusted body weights, adjusted body weight changes (Days 0-20), gravid uterine weights, corpora lutea/dam, total implantations/dam, litter size/dam, viable foetuses/dam, total number resorptions/dam, number early resorptions/dam, number late resorptions/dam, and mean crown-rump distance/litter.

2) Fisher's Exact Test with Bonferroni correction
Endpoints examined: pregnancy index

3) Arcsin-Square-Root Transformation
Endpoints examined: foetal sex ratio (% males/litter), % preimplantation loss/dam, and % postimplantation loss/dam

4) Descriptive statistics (means, standard deviations, and number of animals for each group and time period)
Endpoint examined: nonviable foetuses/dam

5) Covariate Analysis (test of assumptions for Analysis of Covariance, Dunnett's test)
Endpoint examined: mean foetal body weights/litter

The following computer systems were used during the conduct of this study: ARTEMIS II, SAS and Microsoft Office Professional.
Indices:
Pregnancy index
Historical control data:
Historical control data was provided on Crl: CD (SD) BR rats, which was obtained during the period from 1995 - 2000. The data included different parameters relevant to developmental toxicity and was collect by the testing laboratory.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
- Mortality: all animals survived to scheduled euthanasia on Day 20 of gestation.
- Detailed clinical observations: no effect of treatment with antimony trioxide was evident from the detailed clinical examinations. Findings seen in the treated groups occurred at low incidence or with similar frequency as in controls and were considered unrelated to treatment. Several animals in the control and treated groups had findings involving material around the eyes and/or nose. This was attributed to the housing of the animals during the nose-only exposures and their inability to groom during this period.
- Body weights and body weight changes: no effect of treatment with antimony trioxide was evident from gestation body weights and body weight gain. Mean body weights and body weight gain during gestation in the treated groups were comparable to controls. All groups (control and treated) experienced a weight loss over the Gestation Day 6 - 9. This was attributed to many of the animals in each group being weighed post-exposure on Gestation Day 9. These animals had been without feed and water over the 6-hour period and most animals failed to gain or lost weight over this period. For all other intervals during the study animals were weighed in the morning prior to the start of exposures.
- Food consumption: no effect of treatment with antimony trioxide was evident from food consumption during gestation. Generally, food consumption during gestation in the treated groups was comparable to controls. There were a few intervals (Gestation Days 15 - 18 and over the entire study period Gestation Days 0 - 20) when food consumption for the 1.48 mg/m³ group was statistically higher than controls but these differences were considered incidental and unrelated to treatment.
- Macroscopic observation: no macroscopic findings were seen in the treated animals.
- Uterine and ovarian examinations: pregnancy indices were 100% in the control and each treated group providing five litters per group for evaluation on Gestation Day 20.
No effect of treatment was evident from uterine implantation data. The mean number of corpora lutea, implantations, foetuses, resorptions, preimplantation loss, and postimplantation loss in the treated groups was comparable to controls.
Gravid uterine weights and adjusted Day 20 gestation body weights in the treated groups were comparable to controls. Body weight gain over Gestation Days 0 - 20 using the adjusted Gestation Day 20 body weights were comparable to controls in the 0.39, 0.73 and 6.07 mg/m^3. In the 1.48 mg/m³ group the adjusted body weight gain for Gestation Days 0 - 20 was statistically (p<0.05) higher than controls. This was considered to represent an incidental finding and unrelated to treatment.
Please also refer for further information to the field "Any other information on results incl. tables" below.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOEC
Effect level:
6.07 mg/L air (analytical)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEC
Effect level:
> 6.07 mg/L air (analytical)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
- Body weights: foetal body weights at the 6.07 mg/m³ dose level were slightly smaller than controls. Male foetuses in the 6.07 mg/m³ were about 6% lighter than controls, female foetuses were about 9% lighter, and as the sexes combined foetuses were about 8% lighter. These differences were not statistically significant, but considered suggestive of a treatment-related response. In the lower dose groups, foetal body weights were comparable to controls and unaffected by treatment.
- Sex ratio: no effect of treatment was evident from foetal sex ratios. These ratios in the treated groups ranged from 45.1% to 55.8% and were considered comparable to the 46.5% foetal sex ratio in the controls.
- Crown-rump distance: foetal crown-rump distance was shorter than controls in the 6.07 mg/m³ group. Male foetuses were about 3% shorter than controls (not statistically significant), female foetuses were about 5% shorter (p<0.05) and as the sexes combined foetuses were about 4% lower (p<0.05). The shorter size of these foetuses is consistent with the lower foetal body weights seen in this group. No effect of treatment on crown-rump distance was seen at the lower dose levels.
- External examination: no findings (malformations or developmental variations were seen in foetuses from the control or treated groups.
- There were no nonviable foetuses or late resorptions detected at the time of uterine examination.
Please also refer for further information to the field "Any other information on results incl. tables" below.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1: Maternal and foetal observations

Endpoint

0 mg/m³

0.39 mg/m³

0.73 mg/m³

1.48 mg/m³

6.07 mg/m³

No. females on study

5

5

5

5

5

No. females - Mortality

0

0

0

0

0

No. pregnant

5

5

5

5

5

Pregnancy index - Percent

100.0

100.0

100.0

100.0

100.0

No. abortions

0

0

0

0

0

No. early deliveries

0

0

0

0

0

No. females with all resorptions

0

0

0

0

0

No. females with viable foetuses - Day 20 Gestation

5

5

5

5

5

Corpora lutea – No. per animal -

Mean ± SD

14.0 ± 2.00

16.0 ± 1.41

16.4 ± 1.67

15.0 ± 1.41

15.0 ± 1.41

Implantation sites – No. per animal –

Mean ± SD

13.0 ± 1.73

14.2 ± 1.30

14.6 ± 1.52

14.0 ± 1.00

14.0 ± 1.00

Preimplantation loss - % per animal –

Mean ± SD

6.98 ± 4.578

11.24 ± 2.737

10.88 ± 4.773

6.46 ± 4.209

6.46 ± 4.209

Postimplantation loss - % implants per animal

Mean ± SD

1.43 ± 3.194

4.62 ± 6.880

2.86 ± 6.389

5.83 ± 8.122

5.62 ± 9.297

Viable foetuses

No. per animal -

Mean ± SD

12.8 ± 1.64

13.6 ± 2.07

14.2 ± 1.92

13.6 ± 2.88

13.2 ± 1.48

Nonviable foetuses – No. per animal –

Mean ± SD

0.0 ± 0.00

0.0 ± 0.00

0.0 ± 0.00

0.0 ± 0.00

0.0 ± 0.00

Foetal sex ratio

% males per animal –

Mean ± SD

46.5 ± 4.09

47.3 ± 9.05

55.8 ± 13.46

51.3 ± 28.33

45.1 ± 16.93

Resorptions: early + late – No. per animal –

Mean ± SD

0.2 ± 0.45

0.6 ± 0.89

0.4 ± 0.89

0.8 ± 1.10

0.8 ± 1.30

Resorptions: early – No. per animal –

Mean ± SD

0.2 ± 0.45

0.6 ± 0.89

0.4 ± 0.89

0.8 ± 1.00

0.8 ± 1.30

Resorptions: late – No. per animal –

Mean ± SD

0.0 ± 0.00

0.0 ± 0.00

0.0 ± 0.00

0.0 ± 0.00

0.0 ± 0.00

Foetal weight [g] – Males

Mean ± SD

3.43 ± 0.137

3.37 ± 0.384

3.44 ± 0.060

3.49 ± 0.166

3.23 ± 0.287

Foetal weight [g] – Females

Mean ± SD

3.26 ± 0.072

3.19 ± 0.305

3.16 ± 0.076

3.39 ± 0.066

2.96 ± 0.355

Foetal weight [g] – Males + Females

Mean ± SD

3.34 ± 0.103

3.28 ± 0.338

3.32 ± 0.037

3.46 ± 0.102

3.09 ± 0.296

SD = Standard deviation

 

Applicant's summary and conclusion

Conclusions:
In this inhalation rat range-finding developmental toxicity study with antimony trioxide, the No-Observed-Effect-Concentration (NOEC) for maternal toxicity was 6.07 mg/m³, the highest dose level evaluated. The NOEC for developmental toxicity was greater than 6.07 mg/m³.