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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline, GLP-compliant study. Adequate for assessment.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
The test substance was applied to clipped dorsal skin of groups of pregnant female rats at dose levels of 0, 0.05, 1, 10, 50 or 250 mg/kg bw/d on GD 0-19. Dams were observed daily for clinical signs (including skin reactions: Draize criteria). Maternal body weight was recorded on GD 0, 6, 9, 12, 15, 18 and 20. Food consumption data were recorded regularly (collection period not stated). Dams were sacrificed (carbon dioxide) on GD 20 and subject to gross necropsy, including an examination of the uterus and its contents.

Foetuses were examined for viability and the sex of each and presence of any gross external lesions noted. Approximately one half of the foetuses were processed for soft tissue examination, the remainder for skeletal alterations.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Viscous hydrocarbon liquid
Details on test material:
- Name of test material (as cited in Sponsor documentation): clarified slurry oil, CSO
- Specific gravity = 1.06

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI.
- Age and weight at study initiation: not reported
- Housing (GD 0-19): individually housed in stainless steel, wire mesh-bottomed cages
- Diet (ad libitum): Certified Rodent Chow 5002 (Purina Mills Inc.)
- Water (ad libitum): membrane filtered tap water
- Acclimation period: not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-26 degrees
- Humidity (%): 40-70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: not reported

Administration / exposure

Route of administration:
dermal
Vehicle:
other: applied neat or in acetone
Details on exposure:
The test substance was applied neat to previously clipped intact skin on the dorsum. Animals from the 1-250 mg/Kg bw/d groups along with the sham control group were fitted with “Elizabethan collars” (to minimise ingestion) which were worn continuously from GD 0 to GD 20). Animals from the 0.05 mg/Kg bw/day and acetone control groups were fitted with collars for 6 hr post-treatment.
Analytical verification of doses or concentrations:
no
Details on mating procedure:
Each female was placed with one male (same strain and source) overnight and examined the following day for evidence of mating (e.g. sperm in vaginal smear or presence of copulatory plug taken as Day 1 of pregnancy).
Duration of treatment / exposure:
6 hr / d
Test site cleaned with acetone post-treatment.
Frequency of treatment:
Daily, GD 0-19
Duration of test:
From GD 0 to GD 19
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 0.05, 1, 10, 50, 250 mg/Kg bw/d
Basis:
nominal conc.
No. of animals per sex per dose:
24 females per treatment group
Control animals:
yes, concurrent vehicle
yes, sham-exposed
Details on study design:
Test sample applied open (i.e. test site not covered)

Treatments for the 1, 10, 50 and 250 mg/Kg bw/day groups were applied neat; controls were sham-treated.
Treatments for the 0.05 mg/Kg bw/day group were diluted in acetone and applied at a volume of 1 ml/kg bw/day; controls received acetone.

Examinations

Maternal examinations:
- Clinical observations
- Body weight
- Food consumption
- Gross necropsy
Ovaries and uterine content:
- The number of corpora lutea, number of implantation sites and number of early/late resportions recorded
- Uteri from apparently non-gravid dams were stained and examined while transilluminated and pressed between two glass plates
Fetal examinations:
- Viability, gender, body weight and gross external alterations
- Approx. 50% of foetuses fixed in Bouin's solution and examined for soft tissue alterations (Wilson technique)
- Remainder eviscerated, cleared and stained with alizarin red and examined for skeletal alterations

Foetal alterations were defined by the study authors as either:
(i) malformations: irreversible changes that occur at low incidence in this species and strain; or
(ii) variations: common findings in this species and strain and/or reversible delays in development.
Statistics:
- Maternal body weight and food consumption: Bartletts test followed by ANOVA/Dunnett's test (if variances equal) or Kruskal-Wallis test/Dunn's Summed Rank test (if variances unequal)
- Reproductive / Litter data: ANOVA followed by weight or unweighted GLM analysis

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
- Clinical signs:
There were no maternal deaths, abortions, premature deliveries or any irritation of the test site in any group during the study.

- Body weight / body weight gain:
Maternal feed consumption and maternal body weight were significantly decreased, in a dose-related manner, at 1 mg/kg bw/day and above. Net maternal body weight was significantly decreased in a dose related manner (approx. 5-25%) and accompanied by a marked dose-related reduction (approx. 30-90%) in net maternal body weight gain.

-Necropsy:
Gross necropsy findings were unremarkable.

- Pregnancy
Red vaginal discharge was observed in 20-80% of animals dosed with the test substance at 1 mg/kg bw/day and above (affecting 0, 9, 5, 14 and 19 dams given 0.05, 1, 10, 50 or 250 mg/kg bw/day, respectively). Significant, dose-related reductions in gravid uterine weight (decreased 40-95%) were apparent in dams receiving 1 mg/kg bw/day and above.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
0.05 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
0.05 mg/kg bw/day
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
- Litter parameters:
The number of dead or resorbed foetuses was increased 8-16 fold over that of the controls in the 1, 10 and 50 mg/Kg bw/day groups with total litter resorption in high dose dams.

- Body weight:
Foetal body weight was also significantly decreased (14-25%) at 1 mg/kg bw/day and above.

- Soft tissue findings:
There were no treatment-related gross external malformations, however the incidence delayed development in soft tissues (defined as variations, and covering moderate dilation of the renal pelvis, slight dilation of the lateral ventricles of the brain) was stated to be increased significantly at 1, 10 and 50 mg/Kg bw/day (data not presented in publication). No adverse or statistically significant findings were apparent at 0.05 mg/Kg bw/day group.

- Skeletal findings:
A reduction in the extent of ossification of the caudal vertebrae, metacarpals and phalanges (defined as variations) was apparent in the 1, 10 and 50 mg/Kg bw/day groups. No adverse or statistically significant findings were apparent at 0.05 mg/Kg bw/day group.

Effect levels (fetuses)

Remarks on result:
other: see Details on embyotoxic/teratogenic effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Maternal parameters (mean, [SD]) by dose level

Maternal bw (g), GD 20:
Data presented graphically, no tabulated (quantitative) information available.

Corrected maternal bw (g), GD 20:
294.3 [24.6], NC, NC, 275.2 [19.6]*, 269.7 [18.7]**, 247.3 [25.9]**, 217.2 [20.4]**

Corrected maternal bw gain (g), GD 0 to GD 20:
68.6 [18.7], NC, NC, 49.5 [12.5]**, 46.8 [17.0]**, 20.9 [23.2]**, -7.9 [16.5]**

Gravid uterine weight (g):
83.0 [6.9], NC, NC, 49.9 [20.9]**, 29.8 [24.6]**. 8.8 [11.9]**, 3.5 [2.1]**

Number of dams:
20, 23, 24, 21, 23, 22, 22

Caesarian section and foetal litter data (total or mean and [SD]) by dose level

Live foetuses:
314, 344, 362, 224, 118, 20, 0

Litter size / live foetuses per litter:
14.3 [1.4], 15.0 [2.4], 15.1 [1.7], 9.3 [4.2], 4.9 [4.8], 0.9 [2.2]*, 0 [0]*

Total resorptions per litter:
0.6 [0.7], 1.1 [1.4], 0.8 [1.0], 5.0 [4.7]*, 9.4 [5.3]*, 14.0 [2.8]*, 14.3 [2.1]*

Early resorptions per litter:
0.6 [0.7], 1.1 [1.4], 0.8 [1.0], 4.7 [4.8]*, 9.2 [5.4]*, 13.9 [3.0]*, 14.1 [2.1]*

Dead or resorbed foetuses per litter:
4.1 [4.6], 7.0 [8.2], 4.6 [6.6], 33.8 [30.6]*, 43.6 [24.9]*, 67.6 [14.4]*, -

Foetal body weight – g per litter:
3.52 [0.22], 3.51 [0.41], 3.54 [0.34], 2.94 [0.33]*, 3.02 [0.27]*, 2.62 [0.07]*, -

Number (%) of dams examined:
22 (100%), 23 (92%), 24 (96%), 24 (100%), 24 (100%), 23 (95.8%), 23 (95.8%)

Number of litters with foetuses with any alteration (%) by dose level:
8 (36.4), 7 (30.4), 10 (41.7), 8 (33.3), 7 (46.7), 2 (50.0), -

Number of foetuses with any alteration (%) by dose level:
8 (2.5), 12 (3.5), 16 (4.4), 11 (4.9), 9 (7.6)*, 3 (15.0)*, -

Foetuses evaluated for alterations (N):
314, 344, 362, 224, 118, 20, 0

Litters evaluated for alterations (N):
22, 23, 24, 24, 15, 4, 0


Statistically significant changes in ossification sites (per foetus per litter) by dose level:
Caudal vertebrae:
5.03 [0.50], 5.01 [0.62], 5.00 [0.43], 4.20 [0.79]*, 4.24 [0.41]*, 3.52 [0.67]**, -

Metacarpals, mean sites per paw:
3.69 [0.33], 3.59 [0.32], 3.58 [0.38], 3.46 [0.35]**, 3.46 [0.32], 3.02 [0.05]**, -

Phalanges, mean sites per paw :
5.00 [0.00], 4.97 [0.23], 5.00 [0.00], 3.90 [1.77]*, 4.37 [1.40], 1.58 [1.82]*, -

Dose levels: 0 (sham), 0 (vehicle), 0.05, 1, 10, 50 or 250 mg/kg bw/day
Statistics: * = P<0.05; ** = P<0.01
NC = data not collected
- = not applicable (no live foetuse
s)

Applicant's summary and conclusion

Conclusions:
NOAEL for maternal and developmental toxicity = 0.05 mg/Kg bw/day
Executive summary:

The developmental toxicity of clarified slurry oil (0, 0.05, 1, 10, 50 or 250 mg/Kg bw/day) was investigated in groups of pregnant SD rats after dermal application on GD 0-19. The dams were housed individually and fitted with collars to minimise ingestion during the 6 hr exposure period, and the test site wiped clean (acetone) at the end of the exposure.

There were no maternal deaths, abortions, premature deliveries or any irritation of the test site in any group during the study, however red vaginal discharge (affecting 20-80% of animals) was observed at 1 mg/Kg bw/day and above. Maternal feed consumption and maternal body weight were significantly decreased, in a dose-related manner at 1 mg/Kg bw/day and above. Net maternal body weight and net body weight gain were also significantly decreased in a dose related manner at 1 mg/kg bw/d and above.Significant, dose-related reductions in gravid uterine weight (decreased 40-95%) and an increased incidence of early resorptions (elevated 8-24 fold) were apparent in dams receiving 1 mg/Kg bw/day and above. The number of dead or resorbed foetuses was increased 8-16 fold over that of the controls in the 1, 10 and 50 mg/Kg bw/day groups with total litter resorption in high dose dams. Foetal body weight was also significantly decreased (14-25%) at 1 mg/kg bw/day and above. There were no treatment-related gross external malformations, however the incidence delayed development in soft tissues (variations, including moderate dilation of the renal pelvis, slight dilation of the lateral ventricles of the brain) was stated to be increased significantly (data not presented in publication). A reduction in the extent of ossification of the caudal vertebrae, metacarpals and phalanges (defined as variations) was also apparent in the 1, 10 and 50 mg/Kg bw/day groups. No adverse or statistically significant foetal findings were apparent in 0.05 mg/Kg bw/day group.

The NOAEL for maternal toxicity was 0.05 mg/Kg bw/day (based on decreased net body weight/net body weight gain, a reduction in food intake and the occurrence of vaginal discharge); the NOAEL for developmental toxicity was also 0.05 mg/Kg bw/d (reflecting decreased gravid uterine weight, increased resorptions, decreased live litter size and a reduction in foetal weight).