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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 27. 1990 to February 20. 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP conform study following modern procedures.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Naphthalene, NAP
- Source: Aldrich Chemical Co., Milwaukee, WI;
- Substance type: organic, aromatic hydrocarbon
- Physical state: solid
- Analytical purity: > 99% by HPLC analysis
- Lot/batch No.: M082890/03

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc., Raleigh, NC
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 209-271 g
- Housing: individually housed in solid-bottom polycarbonate cages with stainless steel wire lids
- Diet: Ground Purina Certified Rodent Chow (#5002) ad libitum
- Water: Deionized/filtered water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72°F (range 69-75°F) [22°C (range 20.5-24°C)]
- Humidity (%): 55% (range 49-60%) and 48% (range 44-63%).
- Photoperiod (hrs dark / hrs light): lights on from 06:00h to 18:00h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Mazola
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For both study replicates, each dose of NAP was formulated independently in a sufficient quantity to last the entire dosing period. The chemical/vehicle mixtures had to be refrigerated to insure stability. To avoid repeated heating and cooling of the NAP solutions, each dose formulation was divided into aliquots, such that only one aliquot of each formulation was needed for any particular day of dosing. Pre- and post-dosing analysis performed by RTI of the NAP dose formulations for each replicate were within 96%-108% of their theoretical concentrations. Thus, the NAP/vehicle formulations were considered to be stable throughout the period of use for each study replicate.

VEHICLE
- Concentration in vehicle: The actual dose delivered to each animal was adjusted daily, according to a weight taken prior to dosing.
- Amount of vehicle (if gavage): 5 ml/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Pre- and post-dosing analysis performed by RTI of the NAP dose formulations for each replicate were within 96%-108% of their theoretical concentrations.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: 1 night
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
On gestational days 6 through 15
Frequency of treatment:
Daily
Duration of test:
20 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 150 and 450 mg/kg body weight/day
Basis:
analytical conc.
No. of animals per sex per dose:
28
Control animals:
yes, concurrent vehicle
yes, historical
Details on study design:
- Dose selection rationale:

The dose range selected for this study was based on preliminary data furnished by EHRT (NTP, 1990). EHRT studied the effects of naphthalene (0, 100, 400, 500, 600 or 800 mg/kg/day administered by gavage on gestational days 6 through 15) on a limited number of maternal and fetal endpoints. Significant maternal and fetal toxicity was observed at 600 and 800 mg/kg/day. For example, 67% of the dams died during treatment in the 800 mg/kg/day dose group and of those dams that survived the treatment, 33% were found to have totally resorbed their litters. Further, slight maternal and fetal toxicity was observed for dams in the 400 and 500 mg/kg/day groups, indicating that the maternal LD50 for naphthalene was between 400 and 500 mg/kg/day. Since it was desirable for the highest dose used in the current study to cause less than 10% maternal death and because the maternal LD10 for naphthalene could not be determined from the EHRT study, 450 mg/kg, a dose midway between the 400-500 mg/kg doses was chosen. The lower doses were selected so that a no effect level could be established for both maternal and fetal toxicity.

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 3, 6-15, 18, 20

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: On days 0, 3, 6, 9, 12, 15, 18, and 20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20.
- Organs examined: The maternal body, liver, and intact uterus were weighed and corpora lutea were counted. Uterine contents were examined to determine the number of implantation sites, resorptions, dead fetuses and live fetuses. Uteri which had no visible implantation sites were stained with ammonium sulphide (10%) to detect very early resorptions.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
Statistics:
General Linear Models (GLM) procedures, were applied for the analyses of variance (ANOVA) of maternal and fetal parameters. Prior to GLM analysis, an arcsine-square root transformation was performed on all litter-derived percentage data and Bartlett's test for homogeneity of variance was performed on all data to be analyzed by ANOVA. GLM analysis determined the significance of dose-response relationships and the significance of dose effects, replicate effects and dose x replicate interactions. When ANOVA revealed a significant (p<0.05) dose effect, Williams' and Dunnett's Multiple Comparison Test compared NAP-exposed to control groups. One-tailed tests were used for all pairwise comparisons except maternal body and organ weights, maternal food and water consumption, fetal body weight and percent males per litter. Nonsignificant (p>0.05) dose x replicate effects on selected fetal parametric measures were considered justification for pooling data across replicates for nonparametric analysis on related measures. When a significant (p<0.05) dose x replicate interaction occurred, the data for that endpoint and for any related nominal scale data were analyzed separately for dose effects within each replicate in the study, as well as for all replicates combined. Nominal scale measures were analyzed by a X2 test for independence and by a test for linear trend on proportions. When a x2 test showed significant group differences, a one-tailed Fisher's exact probability test was used for pairwise comparisons of NAP and control groups.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: Clinical signs of toxicity, reduced food/water consumption, reduced body weight

Details on maternal toxic effects:
Three dams had to be removed from the study. One dam in the 150 mg/kg/day-group was removed due to urinary calculi, a condition which probably existed prior to the initiation of dosing. In the 450 mg/kg/day group, one dam was removed because it delivered on day 20 and another dam in that group was removed because it had microphthalmia, a condition which existed prior to the start of dosing. Two dams died during dosing and both were in the 50 mg/kg/day group. Necropsy data indicated that the deaths were unrelated to dosing errors.

NAP-treated animals exhibited clinical signs of toxicity which were observed in all treatment groups on the first day of dosing. Lethargy, slow breathing, and prone body posture were the most common clinical signs during the first five days of dosing, with the incidence of these effects showing a marked dose-dependence. For example, on gd 6, the first day of dosing, 81% of the dams in the 50 mg/kg/day group exhibited one or more of the above clinical signs as compared to 96% for both, the 150 and 450 mg/kg/day groups. By the third day of dosing, the clinical signs of toxicity had essentially disappeared in the 50 mg/kg/day group, indicating that the dams had acquired tolerance to these effects of NAP. A similar trend was noted for 150 mg/kg/day NAP, but tolerance was not noted until the sixth day of dosing (gd 11). In the 450 mg/kg/day group the incidence of NAP-induced lethargy and slow respiration also declined during the dosing period, but it never fell below 15%. As the rats became tolerant to the depressant effects of NAP, the incidence of rooting behavior increased; rooting behavior is commonly observed in rodents following gavage administration of compounds which have a noxious odor or which act as a local irritant. The occurrence of rooting behavior, which was observed in less than 10% of the dams during the first few days of dosing, gradually increased as dosing progressed. By gd 15, 24% of the dams in the 150 mg/kg/day group and 92% of the dams in the 450 mg/kg/day group exhibited rooting behavior, as compared to 0% for both, the control and 50 mg/kg/day NAP groups.

NAP also reduced maternal food and water consumption in the two highest dose groups. On gd 6 to 9, relative food intake was suppressed 25% by 150 mg/kg NAP and 37% by the higher dose. However, by gd 9 to 12, food consumption in the two groups had returned to control levels. A significant elevation in food consumption was noted on gd 18 to 20 in the 450 mg/kg/day group. Relative water intake in both groups displayed a pattern similar to food consumption. From gd 6 to 9, a 19% decrease was observed in the 150 mg/kg/day group (significant) and a 13% reduction in the 450 mg/kg/day group (nonsignificant); water consumption in both groups showed significant elevations of 15%-25% on gd 9 to 12 through gd 15 to 18, be-fore returning to control levels by gd 18 to 20. Absolute food and water consumption were similarly affected.

NAP also caused a significant dose-dependent decrease in maternal body weight and body weight gain; the effect was confined to the 150 mg/kg and 450 mg/kg groups, the same groups which exhibited suppressed food and water consumption. In these two groups, dams actually lost weight during the first three days of dosing, coincident with the time when nutrient intake displayed its greatest decreases in those animals. After gd 9, when food and water consumption were at or above control levels, the rats in the 150 mg/kg/day and 450 mg/kg/day groups gained weight at nearly the same rate as controls. Nevertheless, weight gain from gd 6 to 15 was decreased 31% and 53% relative to controls in the 150 and 450 mg/kg/day groups respectively. This indicated that the rats were not able to offset the initial NAP-induced weight deficits. Weight gain from gd 0 to 20 was also significantly less than controls (13-20%) in these two groups. Thus, even after the dosing period had ended, the dams were not able to fully recover from the earlier effects of NAP. As a result of the effects on weight gain, maternal body weights in the two groups remained consistently below control values (6-12%) throughout dosing and this trend persisted until sacrifice on gd 20. The adverse effects of 150 mg/kg/day and 450 mg/kg/day NAP on maternal weight and weight gain were not secondary to decreased uterine weight because deficits were observed for both corrected and absolute weight gain from gd 0 to 20.

Maternal liver weights (absolute or relative) were comparable across groups. There was a trend toward decreased gravid uterine weight with increasing dose of NAP. Gravid uterine weights of the 50, 150, and 450 mg/kg/day groups were 105%, 95% and 89% of control values respectively, but
there was no significant difference among groups by ANOVA.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEL
Remarks:
(highest dose tested)
Effect level:
450 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEL
Remarks:
(lowest dose tested)
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Results from the uterine examination revealed that the number of corpora lutea per dam and the number of implantation sites per litter in the NAP-treated dams were within 95-102% of control values and the number of live fetuses per litter was likewise unaffected. There were also no statistically significant effects of treatment upon the incidence of resorptions or late fetal deaths. However, there was a significant trend test for the percent adversely affected implants per litter. The incidence of conceptuses adversely affected (ie. non-live or malformed) was 8%, 7%, 13% and 20% in the control through high-dose groups, respectively. Average fetal body weight per litter also exhibited a significant trend test. Fetal body weights in the 50, 150, and 450 mg/kg/day groups were 105%, 99% and 95% of control values respectively. However, ANOVA did not detect a significant overall effect of dose for either the percent adversely affected fetuses or the average fetal body weight per litter.

Analysis of anatomical defects indicated that NAP treatment tended to increase the incidence of malformations. Both the percent fetuses malformed per litter (4%, 4%, 7% and 10%) and the percent litters with malformed fetuses (23%, 27%, 33% and 50%) showed a significant trend test. The largest difference was seen in the 450 mg/kg/day group where the percent fetuses malformed per litter were 2.5 times greater than controls (ie. 10% vs.4%). Nevertheless, ANOVA did not detect a significant effect of dose for the percent malformed fetuses per litter, nor was there a significant difference among groups for the percent litters with malformations by the Chi-Square Test.

A review of the individual malformations suggested an effect of treatment on the incidence of enlarged ventricles of the brain. Indeed, separate statistical analysis of this malformation indicated that there was a significant dose-dependent increase in the percent fetuses per litter and the percent litters with enlarged ventricles (significant trend test for both). For example, the percent fetuses with enlarged ventricles in the 50, 150, and 450 mg/kg/day groups was 1.1-, 1.6-, and 2.8-fold higher than control values respectively. However, the main affect for dose in the ANOVA was not significant and a significant effect of replicate was detected for this measure. In the first replicate, there was no effect of treatment on the incidence of enlarged lateral ventricles, with 9% of the fetuses per litter affected in the 450 mg/kg/day group versus 6% in the controls. In contrast, the percentage of fetuses per litter in the second replicate displaying this malformation was only 0.6% in the controls, as compared to 9% in the 450 mg/kg/day group (significant). Thus, the overall effect of treatment on this particular malformation (and malformations in general) is confounded by a 10-fold change in the incidence of this malformation between replicates within the control group.

In contrast to the effect of NAP on malformations, the overall incidence of variations showed a decreasing trend. The percent fetuses with any variation per litter was 45% lower than controls in both the 150 and 450 mg/kg/day groups, but the main effect for dose in the ANOVA was
nonsignificant.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1. Maternal Toxicity in Sprague-Dawley Rats exposed to Naphthalene on Gestational Days 6 through 15

 Naphthalene (mg/kg/day)

0

50

150

450

Subjects (dams)

 

 

 

 

    Total Treated

28

28

28

28

    No. Removed or dead

0

2

1

2

    No. (%) pregnant at sacrifice

26 (93%)

26 (93%)

25 (93%)

26 (100%)

Maternal Body Weight Gain (g)(±SEM)

 

 

 

 

    Treatment period (gd 6 – 15)

55.1 ± 2.0§

53.2 ± 3.1

37.8 ± 2.4*

26.0 ± 3.1*

    Gestation period (gd 0 – 20)

161.0 ± 3.7§

161.3 ± 4.2

140.5 ± 4.8*

129.3 ± 5.6*

    Corrected gestation bw gain

(-gravid uterus)

              

77.0 ± 3.4§

73.1 ± 3.3

60.4 ± 3.0*

54.5 ± 2.8*

    Gravid uterine weight

84.0 ± 2.4§

88.2 ± 3.8

80.2 ± 3.7

74.8 ± 5.2*

Maternal liver weight

 

 

 

 

    Absolute (g)

17.4 ± 0.3

16.8 ± 0.3

16.4 ± 0.4

16.6 ± 0.4

    Relative (% body weight)

4.36 ± 0.05

4.23 ± 0.06

4.34 ± 0.07

4.54 ± 0.09

* Dunnett’s or William’ test, p<0.05

§Test for linear Trend, p<0.05

 


Table 2. Developmental Toxicity in Sprague-Dawley Rats Following Maternal Exposure

to Naphthalene on Gestational Days 6 through 15

Naphthalene (mg/kg/day)

All data on per litter basis (±SEM)

0

50

150

450

All litters (No.)

26

26

25

26

    No. corpora lutea per pregnant dam

15.2 ± 0.4

15.5 ± 0.6

14.5 ± 0.7

15.2 ± 0.5

    No. Implantation sites

14.9 ± 0.4

15.1 ± 0.7

14.4 ± 0.6

14.3 ± 0.7

    % Resorptions

3.9 ± 0.9

2.6 ± 0.9

5.8 ± 4.0

11.3 ± 5.5

    % Litters with one or more resorption

50

31

32

27

    % Late fetal deaths

0.2 ± 0.2

0.0 ± 0

0.3 ± 0.3

0.0 ± 0

    % Litters with one or late fetal death

4

0

4

0

    % Nonlive implants per litter

4.2 ± 0.9

26 ± 0.9

6.1 ± 4.0

11.3 ± 5.5

% Litters with one or more nonlive implants

54

31

36

27

    % Litters with 100% nonlive implants

0

0

4

8

    % Adversely affected implants

8.2 ± 1.9 §

6.8 ± 2.3

13.1 ± 4.4

20.2 ± 5.6

% litters with one or more adversely affected implants

65

50

56

65

Live Litters

26

26

24

24

    No. Live fetuses

14.3 ± 0.5

14.7 ± 0.7

14.5 ± 0.4

14.3 ± 0.7

    Average fetal body wt (g)

 

 

 

 

         Male fetuses

3.8 ± 0.05 §

4.00 ± 0.12

3.74 ± 0.06

3.61 ± 0.06

         Female fetuses

3.62 ± 0.04 §

3.71 ± 0.06

3.63 ± 0.07

3.45 ± 0.06

    % Male fetuses

56 ± 3

55 ± 3

51 ± 2

50 ± 3

Malformations

 

 

 

 

    % Fetuses malformed

4.1 ± 1.9 §

4.3 ± 2.2

7.5 ± 2.6

10.0 ± 3.0

         Male fetuses

4.7 ± 2.1

3.7 ± 1.5

6.7 ± 3.0

10.4 ± 3.6

         Female fetuses

3.8 ± 2.1 §

4.1 ± 3.1

7.4 ± 2.9

9.7 ± 3.0

    % Litters with malformed fetuses

23

27

33

50

    % Fetuses with enlarged ventricles (brain)

 

 

 

 

         Replicate I

5.9 ± 3.4

7.1 ± 4.2

7.6 ± 3.7

8.8 ± 4.3

         Replicate II

0.6 ± 0.6 §

0.0 ± 0

2.1 ± 2.1

8.9 ± 4.4 *

Variations

 

 

 

 

    % Fetuses with variations

18.9 ± 4.2 §

19.9 ± 4.0

10.4 ± 3.5

10.3 ± 2.8

    % litters with one or more variations

81

69

42 **

58

* Dunnett’s or William’ test, p<0.05

** Fisher’s Exact Test,p<0.05

§Test for linear Trend, p<0.05

Applicant's summary and conclusion

Conclusions:
In conclusion, naphthalene was not fetotoxic or teratogenic. There is some evidence that minimal developmental toxicity with an increasing trend of visceral malformation occurs at clearly maternally toxic doses.

Executive summary:

In this well conducted study, groups of 28 female Sprague-Dawley rats were treated by gavage with 0, 50, 150 or 450 mg/kg/day naphthalene on days 6-15 of gestation. Caesarean sections were performed on day 20. The results from this study indicate that naphthalene administered orally had minimal effects on fetal development in the presence of significant maternal toxicity.

Developmental toxicity was expressed only as significant trends toward decreased fetal weight and increased incidence of visceral malformations in the naphthalene-treated groups. The dose of naphthalene which caused the largest developmental changes (450 mg/kg/day) also significantly reduced maternal corrected weight gain and food consumption.

There was an increase in the incidence of one visceral malformation (enlarged lateral ventricles of the brain), but only in one replicate in the 450 mg/kg/day group. It is equivocal whether this represents a direct effect of naphthalene on fetal development, because there was an order of magnitude difference in the incidence of these malformations between replicates in the control group. Additionally, the incidence of enlarged lateral ventricles of the brain in the background control data from RTI laboratory has been reported to be highly variable in recent years. In six NTP-sponsored rat developmental teratology studies conducted at RTI from January 1989 through December, 1990, the occurrence of this particular malformation has ranged from 0% to 27%.

Therefore, apparent developmental toxicity of naphthalene cannot be based solely on an increase in the incidence of enlarged lateral ventricles in one replicate of the high dose group. Therefore, it can be concluded a developmental NOAEL for orally administered naphthalene is closer to 450 mg/kg/day and the developmental LOAEL is probably higher than 450 mg/kg/day naphthalene. In the absence of other data, 150 mg/kg bw/d has been adopted as unequivocal NOAEL.

Naphthalene caused significant maternal toxicity. Two dams died during dosing in the 50 mg/kg/day group, but the absence of any deaths in the higher dose groups does not suggest an association between naphthalene-treatment and maternal mortality.

More consistent were the significant reductions in maternal body weight and weight gain in the 150 mg/kg/day and 450 mg/kg/day naphthalene groups. The effects of these two doses on maternal weight parameters may be secondary to naphthalene-induced suppression of food and water consumption on gd 6 to 9. The fact that maternal body weight began to rise in these two groups at essentially the same rate as controls after food and water consumption returned to normal, supports this view. Furthermore, when maternal nutritional status was not affected by treatment (50 mg/kg/day naphthalene), no effect on maternal weight or maternal weight gain was observed. The threshold for effects of naphthalene on respiration and motor activity was lower than that for weight reduction since 50 mg/kg/day naphthalene was comparable to 450 mg/kg/day naphthalene in producing shallow breathing and lethargy during the early phases of dosing.

The LOAEL for naphthalene-related maternal toxicity is 50 mg/kg/day.