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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:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Accepted, well conducted study. Exposure period from GD 6-15
Cross-reference
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Accepted, well conducted study. Exposure period from GD 6-15
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Exposure on GD 6-15
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
CD-1
Details on test animals and environmental conditions:
Source - Charles River Labs, Portage, MI
Age at receipt - 8 weeks
Acclimation period - 2 or 3 weeks
Feed - Purina certified Rodent chow #5002 (available except during exposure)
Water - Ad libitum
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Details on exposure:
All animals were exposed in 16 cubic meter glass and stainless steel chambers. Chamber ventilation was provided by an HVAC system separate from the general laboratory air handling system. This air was particulate-filtered and controlled for temperature and humidity. Chamber air flow rate, temperature, and relative humidity were monitored every half-hour during exposure periods.

To generate test atmospheres, nitrogen was heated to 200 deg C by passage through a 1-liter stainless steel cylinder fitted with a 1500-watt band heater and was then introduced at the bottom of a glass column 7.6 cm in diameter and 30 cm long, packed with glass beads. The liquid test material was delivered by a fluid metering pump from a stainless steel safety can, through Teflon tubing, to the bottom quarter of the column. The test sample was vaporized as it flowed up the column with the nitrogen. The vapors were passed to the chamber inlet where dilution with chamber ventilation air reduced the concentration to the desired exposure levels.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Exposure levels were monitored with a gas-phase infrared spectrometer. Measurements were made on an hourly basis throughout the study. Accuracy of this method was confirmed by the use of vapor standards. Additionalyy, the composition (on a weight percentage basis) of the test material within each exposure chamber was determined by gas chromatography during the first exposure week.
Details on mating procedure:
Female mice were paired 1:1 with male mice for mating. Female mice were confirmed to have been mated by visual presence of a vaginal plug.
Duration of treatment / exposure:
Gestation day 6-15
Frequency of treatment:
6 hrs/day
Duration of test:
Gestation day 0-18
No. of animals per sex per dose:
30 mice/group
Control animals:
yes, sham-exposed
Details on study design:
Mated female mice were randomly assigned to one of four treatment groups, each containing 30 animals. Contrl group was exposed to room air only.
Maternal examinations:
Mice were examined twice daily for viability and overt changes in appearance and behavior. Presence and duration of clinical signs were recorded daily from GD6-GD15 and also on GD18. Maternal body weight was measured on GD0 and daily from GD6 to GD18.
Ovaries and uterine content:
Surviving females were sacrificed on GD18. Uterine contents were examined for the number and location of viable and non-viable fetuses, early and late resorptions. The number of total implantations and corpora lutea were recorded. Uteri were excised and weighed.
Fetal examinations:
Fetuses were individually weighed, sexed, tagged and examined for external malformations and variations. Approximately half of the fetuses were dissected, internally sexed and examined for visceral malformations and variations.
Hearts were dissected by a modification of the method described by Staples (1974). Heads were fixed in Bouin's solution for soft tissue examination by the razor blade sectioning technique. Remaining fetuses were fixed in alcohol, macerated with potassium hydroxide, stained with Alizarin Red S and cleared with glycerin. Features were subjected to skeletal examination.
Statistics:
Fertility indices and male/female sex ratios were compared by the Chi-square test criterion. Proportions of litters with malformations were compared by the Fisher's exact probability test to determine the significance of difference. Proportions of resorbed and dead fetuses, pre- and post-implantation losses and pup survival indices were compared by the Mann-Whitney U-test to determine the significance of difference.

Numbers of corpora lutea, total implantations and live fetuses, mean fetal body weights, parental body weights, maternal body weight changes, organ weights and hematological parameters were compared by one-way ANOVA, Bartlett's test for homogeneity of variance and the appropriate t-test for equal and unequal variance using Dunnett's multiple comparison tables to determine the level of significance.

Mean numbers of liveborn pups per litter and mean pup weight were also compared by ANOVA and the appropriate t-test. All statistical analyses compared the treatment groups to control with the levels of significance at p < 0.05 and p < 0.01. All means were accompanied by standard deviations.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
The highest concentration (1500 ppm; nominal concentration) produced severe maternal toxicity. Mortality in this group was 44% (12 mice died between GD 8-16 and 2 on GD 6). Mean maternal body weight gain in the 1500 ppm group was significantly reduced during exposure periods (GD 6-15) and from GD0-18. clinical observations made in this dose group included abnormal gait, labored breathing, hunched posture, weakness, inadequate grooming, circling and ataxia.

In the 500 ppm group, 2 mice died during the exposure period with one due to treatment-unrelated injury and the other undetermined. Maternal body weight gain in the 500 ppm group showed a statistically significant reduction compared to chamber controls. there were no statistically significant reductions in body weight gain in the 100 ppm group and clinical signs of toxicity were absent in both the 100 and 500 ppm groups.
There were no statistically significant differences in maternal organ weights in any exposure group. Hematological evaluations revealed significant decreases in percent hematocrit and mean corpuscular volume values in the 1500 ppm group compared to controls. Leukocyte count was reduced in the 500 ppm group but was not considered toxicologically relevant since no such effect was noted in the 1500 ppm group.
Key result
Dose descriptor:
NOAEC
Effect level:
ca. 100 ppm (nominal)
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEC
Effect level:
ca. 100 ppm (nominal)
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Number of live fetuses per litter and the mean fetal body weight were significantly reduced in the 1500 ppm group. Post-implantation loss was significantly elevated, ossification was delayed particularly in the skull and sternebrae and number of fetuses with cleft palate was substantially increased.
Fetal body weight was significantly reduced in the 500 ppm group with no other evidence for developmental toxicity, malformation or developmental delay. Number of live fetuses/litter was significantly reduced in the 100 ppm group but was considered toxicologically irrelevant as there was no such effect in the 500 ppm group.
Key result
Dose descriptor:
NOAEC
Effect level:
100 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental Toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
No selective developmental effects were noted. C9 aromatic naphtha only induced developmental effects at maternally toxic exposures.
Executive summary:

Inhalation developmental studies have been conducted on the following category members. In a study with C9 aromatic naphtha (a C9 aromatic solvent), mice were exposed to 0, 100, 500, 1500 ppm from GD 6 -15. In this study, conducted in mice exposed to C9 aromatic naphtha, anomalies (delayed sternebral and cranial ossification) and definitive birth defects (increased incidence of cleft palate) were noted in the presence of severe maternal toxicity (44% mortality) at the highest concentration tested (1500 ppm).  The increased incidence of cleft palate in the pups has been shown to be related to stress in the dams at toxic dose levels (1500 ppm – 44% mortality). Reduction in fetal body weights in the 500 ppm group also occured in the presence of statistically significant reductions in maternal body weight at the same dose level. Overall, exposure resulted in some fetal effects in the presence of maternal toxicity, but no selective developmental effects. The fetal NOAEC values range from 100 to 300 ppm and maternal NOAECs range from 100 to 300 ppm (500 - 1500 mg/m3).

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Exposure on GD 6-15
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
deTest material specially prepared to meet specific compositional requirements as specified in the USEPA TSCA test rule (Fed. Reg. 50: 20662-20667, May 17, 1985).
Test material - Defined as a high-flash aromatic naphtha type 1 (ASTM method D-3734)
Results of gas chromatography analysis for composition -

Compound Weight percent (%)
o-xylene 3.20
Cumene 2.74
n-propylbenzene 3.97
4-ethyltoluene 7.05
3-ethyltoluene 15.1
2-ethyltoluene 5.44
1,3,5-trimethylbenzene 8.37
1,2,4-trimethylbenzene 40.5
1,2,3-trimethylbenzene 6.18
≥ C10’s 6.19
Total 98.74

Test animals

Species:
mouse
Strain:
CD-1
Details on test animals and environmental conditions:
Source - Charles River Labs, Portage, MI
Age at receipt - 8 weeks
Acclimation period - 2 or 3 weeks
Feed - Purina certified Rodent chow #5002 (available except during exposure)
Water - Ad libitum

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Details on exposure:
All animals were exposed in 16 cubic meter glass and stainless steel chambers. Chamber ventilation was provided by an HVAC system separate from the general laboratory air handling system. This air was particulate-filtered and controlled for temperature and humidity. Chamber air flow rate, temperature, and relative humidity were monitored every half-hour during exposure periods.

To generate test atmospheres, nitrogen was heated to 200 deg C by passage through a 1-liter stainless steel cylinder fitted with a 1500-watt band heater and was then introduced at the bottom of a glass column 7.6 cm in diameter and 30 cm long, packed with glass beads. The liquid test material was delivered by a fluid metering pump from a stainless steel safety can, through Teflon tubing, to the bottom quarter of the column. The test sample was vaporized as it flowed up the column with the nitrogen. The vapors were passed to the chamber inlet where dilution with chamber ventilation air reduced the concentration to the desired exposure levels.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Exposure levels were monitored with a gas-phase infrared spectrometer. Measurements were made on an hourly basis throughout the study. Accuracy of this method was confirmed by the use of vapor standards. Additionalyy, the composition (on a weight percentage basis) of the test material within each exposure chamber was determined by gas chromatography during the first exposure week.
Details on mating procedure:
Female mice were paired 1:1 with male mice for mating. Female mice were confirmed to have been mated by visual presence of a vaginal plug.
Duration of treatment / exposure:
Gestation day 6-15
Frequency of treatment:
6 hrs/day
Duration of test:
Gestation day 0-18
No. of animals per sex per dose:
30 mice/group
Control animals:
yes, sham-exposed
Details on study design:
Mated female mice were randomly assigned to one of four treatment groups, each containing 30 animals. Contrl group was exposed to room air only.

Examinations

Maternal examinations:
Mice were examined twice daily for viability and overt changes in appearance and behavior. Presence and duration of clinical signs were recorded daily from GD6-GD15 and also on GD18. Maternal body weight was measured on GD0 and daily from GD6 to GD18.
Ovaries and uterine content:
Surviving females were sacrificed on GD18. Uterine contents were examined for the number and location of viable and non-viable fetuses, early and late resorptions. The number of total implantations and corpora lutea were recorded. Uteri were excised and weighed.
Fetal examinations:
Fetuses were individually weighed, sexed, tagged and examined for external malformations and variations. Approximately half of the fetuses were dissected, internally sexed and examined for visceral malformations and variations.
Hearts were dissected by a modification of the method described by Staples (1974). Heads were fixed in Bouin's solution for soft tissue examination by the razor blade sectioning technique. Remaining fetuses were fixed in alcohol, macerated with potassium hydroxide, stained with Alizarin Red S and cleared with glycerin. Features were subjected to skeletal examination.
Statistics:
Fertility indices and male/female sex ratios were compared by the Chi-square test criterion. Proportions of litters with malformations were compared by the Fisher's exact probability test to determine the significance of difference. Proportions of resorbed and dead fetuses, pre- and post-implantation losses and pup survival indices were compared by the Mann-Whitney U-test to determine the significance of difference.

Numbers of corpora lutea, total implantations and live fetuses, mean fetal body weights, parental body weights, maternal body weight changes, organ weights and hematological parameters were compared by one-way ANOVA, Bartlett's test for homogeneity of variance and the appropriate t-test for equal and unequal variance using Dunnett's multiple comparison tables to determine the level of significance.

Mean numbers of liveborn pups per litter and mean pup weight were also compared by ANOVA and the appropriate t-test. All statistical analyses compared the treatment groups to control with the levels of significance at p < 0.05 and p < 0.01. All means were accompanied by standard deviations.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
The highest concentration (1500 ppm; nominal concentration) produced severe maternal toxicity. Mortality in this group was 44% (12 mice died between GD 8-16 and 2 on GD 6). Mean maternal body weight gain in the 1500 ppm group was significantly reduced during exposure periods (GD 6-15) and from GD0-18. clinical observations made in this dose group included abnormal gait, labored breathing, hunched posture, weakness, inadequate grooming, circling and ataxia.

In the 500 ppm group, 2 mice died during the exposure period with one due to treatment-unrelated injury and the other undetermined. Maternal body weight gain in the 500 ppm group showed a statistically significant reduction compared to chamber controls. there were no statistically significant reductions in body weight gain in the 100 ppm group and clinical signs of toxicity were absent in both the 100 and 500 ppm groups.
There were no statistically significant differences in maternal organ weights in any exposure group. Hematological evaluations revealed significant decreases in percent hematocrit and mean corpuscular volume values in the 1500 ppm group compared to controls. Leukocyte count was reduced in the 500 ppm group but was not considered toxicologically relevant since no such effect was noted in the 1500 ppm group.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEC
Effect level:
ca. 100 ppm (nominal)
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEC
Effect level:
ca. 100 ppm (nominal)
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
Number of live fetuses per litter and the mean fetal body weight were significantly reduced in the 1500 ppm group. Post-implantation loss was significantly elevated, ossification was delayed particularly in the skull and sternebrae and number of fetuses with cleft palate was substantially increased.
Fetal body weight was significantly reduced in the 500 ppm group with no other evidence for developmental toxicity, malformation or developmental delay. Number of live fetuses/litter was significantly reduced in the 100 ppm group but was considered toxicologically irrelevant as there was no such effect in the 500 ppm group.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEC
Effect level:
100 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental Toxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
No selective developmental effects were noted. C9 aromatic naphtha only induced developmental effects at maternally toxic exposures.
Executive summary:

Inhalation developmental studies have been conducted on the following category members. In a study with C9 aromatic naphtha (a C9 aromatic solvent), mice were exposed to 0, 100, 500, 1500 ppm from GD 6 -15. In this study, conducted in mice exposed to C9 aromatic naphtha, anomalies (delayed sternebral and cranial ossification) and definitive birth defects (increased incidence of cleft palate) were noted in the presence of severe maternal toxicity (44% mortality) at the highest concentration tested (1500 ppm).  The increased incidence of cleft palate in the pups has been shown to be related to stress in the dams at toxic dose levels (1500 ppm – 44% mortality). Reduction in fetal body weights in the 500 ppm group also occured in the presence of statistically significant reductions in maternal body weight at the same dose level. Overall, exposure resulted in some fetal effects in the presence of maternal toxicity, but no selective developmental effects. The fetal NOAEC values range from 100 to 300 ppm and maternal NOAECs range from 100 to 300 ppm (500 - 1500 mg/m3).