Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from August 1993 to 11 September 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant study conducted according to OECD guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
See confidential details on test material section

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan-Winkelmann, Borchen
- Age at study initiation: not documented
- Weight at study initiation: 187 to 236 g
- Fasting period before study: no
- Housing: During the adaptation period, the females were kept in groups in Type III Makrolon cages, and starting from gestation day 0 they were iindividually accomodated in Type II Makrolon cages on low-dust wood shavings supplied by ssniff GmbH in Soest.
- Diet (e.g. ad libitum): standard diet (Altromin 1324) given ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 7 days before mating.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30-70%
- Air changes (per hr): at least 10 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial lighting from 6 a.m. to 6 p.m.

IN-LIFE DATES: From: 24 Aug 1993 To: 27 Oct 1993

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Administration formulations (solutions) were prepared with demineralised water. The stability (over 8 days) of the active ingredient in the formulations were analytically verified before initiation of the study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A content check of the formulations in all dose levels was carried out. The results revealed no significant devioations of the active ingredient content from the nominal value in the formulations.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 2 females with one male
- Length of cohabitation: one night
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
from day 6 to day 15 p.c.
Frequency of treatment:
daily
Duration of test:
see above
No. of animals per sex per dose:
27 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected according to a preceding range-finding study in 5 rats per group with doses of 200, 300, 400 and 500 mg/kg bw. From the dose of 300 mg/kg and above breathing sounds, decreased feed intakes and body weight gains were observed (300 mg/kg marginally). One dam of the 400 mg/kg group died and all dam of the 500 mg/kg group revealed bloody snouts and decreased motility. The resorption rate and the external examination of the fetuses revealed no embryotoxic potential up to and including 500 mg/kg.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: from day 0 to 20 p.c. all animals were inspected twice daily on the basis of their appearance and behavior and not was given to any alterations in the excretory products.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule:see above

BODY WEIGHT: Yes
- Time schedule for examinations: on day 0 p.c., daily from day 6 to 15 p.c and on day 20 pc.

FOOD CONSUMPTION: Yes
The quantity of food consumed for each female was recorded for the following intervals: days 0-6, 6-11, 11-16 and 16-20 p.c.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: gross pathological evaluation (no further details)
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes / No / No data
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
- Other:
Fetal examinations:
- External examinations: Yes, all fetuses
- Soft tissue examinations: Yes, evaluation of about half of the fetuses.
- Skeletal examinations: Yes, evaluation of about half of the fetuses.
- Head examinations: No data


- External examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data
- Soft tissue examinations: Yes: [all per litter / / #? per litter ] / No / No data
- Skeletal examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data
- Head examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data
Statistics:
Animals that had no implantation sites were not used for statistical evaluation. For calculation of body weight gain, feed intakes and fetal parameters only dams with living fetuses were used.

Statistical significance was tested using the following methods:

a. Fisher's exact significance test at significance levels of alpha = 5% and alpha = 1% (two-tailed) was used for the:
- fertility rate
-gestation rate

b. The F-test, and t-test or Welch t-test were used for the:
- feed intakes
- Body weight gains
- Corrected body weight gains
- Number of corpora lutea per dam
- Number of implantations per dam
- Number of live fetuses per dam
- Percentage of live fetuses in proportion to the number of implantations per dam
- Number of male or female fetuses per dam
- Fetal weights per group and per dam
- Placental weights per group and per dam

c. The Yates-corrected Chi2 test was used for the:
- Number of fetuses or litters exhibiting malformations
- Pre-implantation loss per dam
- Number of male or female fetuses per group
- Number of resorptions per dam

d. 2 by N CHI2-test; in case of signficant differences Fisher's exact test with Bonferonni correction for:
- Number of fetuses or litters with skeletal findings.

Indices:
see Statistics section
Historical control data:
Historical control data were provided to allow comparison with concurrent controls. These included data for clinical signs, pathological and anatomical findings, food consumption during pregnancy, placenta findings, fertility and pregnancy index, caesarian section data, summary of reproduction data, number of malformations, spontaneous malformations, external and visceral deviations of fetuses, skeletal retardations and 14th rib, skeletal findings of fetuses/litters.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: Reduced body weight gain and feed intake at 60 and 360 mg/kg

Details on maternal toxic effects:
The following parameters of maternal toxicity were impaired: The body weight gain in the 60 and 360 mg/kg group dams and the feed intake in the 360 mg/kg group (60 mg/kg marginally) were reduced during the treatment period (details in Tables 1 and 2). Decreased water consumption and reduced feces excretion were observed in some dams of the 360 mg/kg group.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: based on maternal toxicity
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: based on developmental toxicity
Remarks on result:
other: observed with maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: Increased numbers of fetuses with skeletal variations and retardations were observed at 360 mg/kg

Details on embryotoxic / teratogenic effects:
The following parameters of intrauterine development were affected: In the 360 mg/kg group increased numbers of placentas with necrotic border and slightly reduced placental weights were observed (details below and in Table 4). In the 360 mg/kg group increased numbers of fetuses with skeletal variations and retardations were observed (details below and in Table 5).
At the dose of 360 mg/kg b.w. increased incidences of vertebral and multiple malformations that may occur spontaneously were evident, while the overall incidence of malformations was comparable between control and 360 mg/kg group. A treatment related effect for this slightly different distribution cannot be excluded.
However, primary developmental toxicity was not evident, since all embryotoxic findings observed at the dose of 360 mg/kg appeared in a maternally toxic dose range.

Effect levels (fetuses)

Remarks on result:
other: not specified

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Appearance, behavior and mortality:

Appearance, behavior and mortality of the dams were unaffected up to and including the dose of 360 mg/kg b.w.

Gross pathological findings in dams:

No treatment related findings were evident at necropsy up to and including the dose of 360 mg/kg b.w.

General Reproduction data:

The number of corpora lutea, preimplantation losses and implantations was comparable in all experimental groups, with exception of the significantly lower preimplantation losses in the 360 mg/kg group (details in Table 3).

Gestation rate:

The gestation rate was unaffected by the treatment with doses up to and including 360 mg/kg.

Weight and appearance of placentas:

The placental weight was statistically significantly decreased at the dose of 360 mg/kg when calculated on litter basis and from 60 mg/kg and above when calculated in individual basis.Since the toxicologically more relevant calculation on litter basis was not affected at the dose of 60 mg/kg and as the values even of the 360 mg/kg group lay within the range of historical control data, the statistical significance at 60 mg/kg is not regarded as toxicologically relevant and even the values obtained at the dose of 360 mg/kg as equivoqual. Nevertheless, together with the increase number of placentas with necrotic placental borders at 360 mg/kg, this dose is considered to be the effect level for placental weight and appearance (details in Table 4).

Resorption rate, number of fetuses:

The resorption rate and the mean number of fetuses were not affected by the treatment up to and including the dose of 360 mg/kg.

Sex and fetuses:

The number of male and female fetuses did not diffe from those in the controls to any extent at levels up to and including 360 mg/kg.

Fetal weight:

The fetal weight was unaffected by the treatment up to and including 360 mg/kg.

Skeletal system deviations (variations, retardations):

At the highest dose of 360 mg/kg, slightly reduced ossification of the parietal and interparietal bones and the 7th cervical vertebra were observed as well as an increase in the incidence of fetuses with wavy ribs. These findings are considered to be treatment related.

Malformations (details in Table 5):

Neither the type nor the incidence of malformations were affected by treatment with doses up to and including 60 mg/kg.

The number of abnormal fetuses at 360 mg/kg was comparable to the control group. All findings in the control and the 360 mg/kg were also observed in previous controls. Nevertheless, there was a slight difference concerning the distribution of these findings. Microphthalmia mainly observed in the control group of this study occured in comparable incidences in previous controls, while the incidence of vertebral and multiple malformations mainly obtained in the 360 mg/kg group was rather low in previous controls. Therefore a treatment related effect on the incidence of the verteral and multiple malformations cannot be excluded. However, incresed incidences of spontaneoulsy occuring malformations are not regarded as indication of a specific teratogenic potential.

The remaing individual fetal findings (deviations) observed at visceral examinations revealed no evidence of tes substance-related effects in any of the dose troups.

Applicant's summary and conclusion

Conclusions:
The test item, Reaction product of naphthalene, butanol, sulfonated and neutralized by caustic soda was administered by gavage, once daily from days 6 to 15 p.c. to inseminated Wistar rats at doses of 0, 10, 60, or 360 mg/kg/day in a GLP-compliant study in accordance with OECD Guideline 414.
On the basis of the results obtained in this study:
- The No Observed Adverse Effect Levels (NOAEL) for maternal toxicity was 10 mg/kg b.w./day.
- The NOAEL for developmental toxicity was 60 mg/kg b.w./day.

At the dose of 360 mg/kg b.w. increased incidences of vertebral and multiple malformations that may occur spontaneously were evident, while the overall incidence of malformations was comparable between control and 360 mg/kg group. A treatment related effect for this slightly different distribution cannot be excluded. However, primary developmental toxicity was not evident, since all embryotoxic findings observed at the dose of 360 mg/kg appeared in a maternally toxic dose range.

Therefore, in the absence of evident direct embryotoxic effect, the registered substance is not classified for reprotoxicity according to the classification criteria of the Regulation (EC) 1272/2008 (CLP) and of the Directive 67/548/EEC.
Executive summary:

Groups of 27 inseminated Wistar rats each were treated daily orally by gavage with Reaction product of naphthalene, butanol, sulfonated and neutralized by caustic soda formulated in demineralized water from day 6 to day 15 p.c. in doses of 0, 10, 60, or 360 mg/kg body weight, respectively. The fetuses were delivered by cesarian section on the 20th day of gestation. Investigations were performed to determine the general tolerance of the test substance by the dams, as well as its effect on intrauterine development.

Mortality, behavior and appearance of the dams were not affected by the treatment up to and including 360 mg/kg. The body weight gain in the 60 and 360 mg/kg group dams and the feed intake in the 360 mg/kg group (60 mg/kg marginally) were reduced during the treatment period. Decreased water consumption and reduced feces excretion were observed in some dams of the 360 mg/kg group. There were no test substance-related findings at necropsy in any of the experimental groups.

With respect to intrauterine development, the gestation rate, the resorption rate and accordingly the number of fetuses, and the fetal sex as well as fetal weight were unaffected by the treatment up to and incuding 360 mg/kg.

In the 360 mg/kg group increased numbers of placentas with necrotic border and slightly reduced placental weights were observed.

External, visceral and skeletal examination of the fetuses revealed no treatment related effects on intrauterine development up to and including the dose of 60 mg/kg. At the dose of 360 mg/kg b.w. increased incidences of vertebral and multiple malformations that may occur spontaneously were evident, while the overall incidence of malformations was comparable between control and 360 mg/kg group. A treatment related effect for this slightly different distribution cannot be excluded.

However, primary developmental toxicity was not evident, since all embryotoxic findings observed at the dose of 360 mg/kg appeared in a maternally toxic dose range.

The No Observed Adverse Effect Levels (NOAEL) were thus:

Maternal toxicity : 10 mg/kg b.w./day

Developmental toxicity : 60 mg/kg b.w./day

Therefore, in the absence of evident direct embryotoxic effect, the registered substance is not classified for reprotoxicity according to the classification criteria of the Regulation (EC) 1272/2008 (CLP) and of the Directive 67/548/EEC.