Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 2016 - March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Januari 2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Identification: Sodium alkylnaphthalene sulfonate
Chemical name: Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated sodium salts
CAS Number: 1258274-08-6
Appearance: Tan powder
Batch: SR123887B
Purity/Composition: 96.37 %
Test item storage: At room temperature container flushed with nitrogen
Stable under storage conditions until: 21 February 2018 (expiry date)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: SR123887B
- Expiration date of the batch: 21 February 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature container flushed with nitrogen
- Stability under test conditions: yes
- Solubility and stability of the test substance in the vehicle: Stability for at least 6 hours at room temperature under normal laboratory light conditions and under nitrogen is confirmed over the concentration range 20 - 200 mg/mL

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: approximately 10-14 weeks
- Weight at study initiation: 169 to 237 grams
- Housing: Females were individually housed in Macrolon plastic cages; sterilized sawdust as bedding material and paper as cage-enrichment/nesting material were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water.
- Acclimation period: At least 5 days prior to treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5 – 20.3
- Humidity (%): 45.3 – 48.8
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23 February 2016 To: 17 March 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test
item, vehicle, and/or formulation. No correction was made for the purity/composition of the test item. Formulations were stored at room temperature flushed with nitrogen.
Dose volume was 5 mL/kg bw. Actual dose volumes were calculated according to the latest body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Details on mating procedure:
Untreated females were mated at the Supplier, and were at Day 0 or 1 post-coitum on arrival at the Test Facility (Day 0 post-coitum was the day of successful mating; confirmed by vaginal plug).
Duration of treatment / exposure:
From Days 6 to 20 post-coitum, inclusive.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Duration of test:
until Day 21 post-coitum
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were selected by the sponsor based on the results of a combined 28-day/repro
screening study, in which dose levels of 0, 100, 300 and 1000 mg/kg bw/day were tested. At 1000 mg/kg bw/day, mortality was observed in 2/10 males after 4 to 5 days and in 4/10 females after 20 to 39 days. This was attributed to tracheal/pulmonary lesions associated with aspiration of test article and acute inflammation/ulceration in the stomach. Moreover, hemorrhage in the mesenteric lymph nodes was noted at 1000 mg/kg bw/day. At 300 mg/kg bw/day, only minimally increased incidence and severity in hemorrhage was observed which was considered not adverse. No reproduction or developmental toxicity was observed by treatment up to 1000 mg/kg bw/day.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from Day 2 post-coitum onwards up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum

FOOD CONSUMPTION: Yes
Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative assessment was introduced as no treatment related effect was suspected.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 21 post-coitum
- Organs examined: according to guidelines

OTHER: the stomach of all animals was dissected and examined
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: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
Pre-implantation loss (%) = ((number of corpora lutea - number of implantation sites) / number of corpora lutea) x 100
Post-implantation loss (%) = ((number of implantation sites – number if live fetuses) / number of implantation sites) x 100
Viable fetuses affected/litter (%) = ((number of viable fetuses affected/litter) /( number of viable fetuses/litter)) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant clinical signs were noted by treatment up to and including 600 mg/kg bw/day. Rales was noted for three females at 100 mg/kg bw/day and five females at 600 mg/kg bw/day for 1-3 consecutive days. Piloerection was observed for one control female, one female at 300 mg/kg bw/day and nine females at 600 mg/kg bw/day for 1-3 consecutive days. As these findings were not consisted over time, they are considered not toxicologically relevant.
Salivation was noted for two females at 100 mg/kg bw/day, eight females at 300 mg/kg bw/day and almost all females at 600 mg/kg bw/day. This is considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test item.
Alopecia was noted for one control female, two females at 100 mg/kg bw/day and two females at 300 mg/kg bw/day. As this occurred within the range of background findings observed in rats of this age and strain under the conditions in this study and no dose response relationship is observed, this is not considered to be treatment related.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Weight gain corrected for uterus weight was significantly lower at 300 and 600 mg/kg bw/day (23.9 g and 23.1 g, respectively), compared to controls (30.7 g). As no dose response relationship is noted and the control value is relatively high compared to our historical control data (mean corrected body weight gain: 27 g), this was considered not toxicologically relevant.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
One female at 600 mg/kg bw/day showed an irregular surface of the glandular stomach, which was probably related to treatment with the test item, but there was no microscopic correlate in this part of the stomach. However, an erosion/ulceration with a lymphogranulocytic inflammation was recorded in the forestomach for this animal.
Alopecia noted for some females confirmed the clinical sign observed in the in-life phase of the animals and was considered not to be treatment related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the stomach at 600 mg/kg bw/day. A slightly increased incidence of minimal inflammation of the glandular stomach was recorded at 600 mg/kg bw/day (5/10 females), compared to 1/10 females of the control group. In one female at 600 mg/kg bw/day a moderate erosion/ulceration with a moderate lymphogranulocytic inflammation of the forestomach was recorded, which was considered to be related to the treatment with the test item.
The remainder of the recorded microscopic findings including the minimal inflammation in the forestomach in single animals were within the normal range of background pathology encountered in rats of this age and strain.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
A relatively high mean value of pre-implantation loss (14.0% per litter) was noted in the 100 mg/kg bw/day group, compared to the other groups. This was not considered to be treatment related as treatment started from implantation onwards (i.e. Day 6 post-coitum). As this value was within normal limits and no dose response relationship was observed, this was considered not treatment related.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
One female at 100 mg/kg bw/day and one female at 300 mg/kg bw/day were not pregnant. This observation is considered not treatment-related.
Other effects:
no effects observed
Details on maternal toxic effects:
Test item-related morphological alterations were present in the stomach at 600 mg/kg bw/day. A slightly increased incidence of minimal inflammation of the glandular stomach was recorded at 600 mg/kg bw/day (5/10 females), compared to 1/10 females of the control group. In one female at this dose a moderate erosion/ulceration with a moderate lymphogranulocytic inflammation of the forestomach was recorded, which was considered to be related to the treatment with the test item.
No maternal toxicity was observed in the 100 and 300 mg/kg bw/day groups.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
>= 600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no systemic effects observed up to and including the highest dose tested.
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
no systemic effects observed
Key result
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: morphological alteration in stomach: slightly increased incidence in minimal inflammation of the glandular stomach; one high dose female with moderate erosion/ulceration with moderate lymphogranulocytic inflammation of the forestomach

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically relevant effects on fetal body weights (per sex and combined for both sexes) noted by treatment up to and including 600 mg/kg bw/day.
Female fetal weights were significantly lower at 600 mg/kg bw/day (4.9 g) when compared to the control group (5.2 g). As this decrease was minimal, the value was only just outside the historical control range (5.0 g - 5.2 g) and fetal skeletal development was unaffected, this is considered to be a result of a slightly high control value (mean historical control value: 5.1 g) and not toxicologically relevant. Mean combined (male and female) fetal body weights were 5.3, 5.3, 5.2 and 5.1 gram for the
control, 100, 300 and 600 mg/kg bw/day groups, respectively.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There were no toxicologically relevant effects on fetal body weights (per sex and combined for both sexes) noted by treatment up to and including 600 mg/kg bw/day.
Female fetal weights were significantly lower at 600 mg/kg bw/day (4.9 g) when compared to the control group (5.2 g). As this decrease was minimal, the value was only just outside the historical control range (5.0 g - 5.2 g) and fetal skeletal development was unaffected, this is considered to be a result of a slightly high control value (mean historical control value: 5.1 g) and not toxicologically relevant. Mean combined (male and female) fetal body weights were 5.3, 5.3, 5.2 and 5.1 gram for the
control, 100, 300 and 600 mg/kg bw/day groups, respectively.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Mean litter sizes were 9.6, 9.0, 10.5 and 10.0 fetuses/litter for the control, 100, 300 and 600 mg/kg bw/day groups, respectively.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment up to and including 600 mg/kg bw/day.
Mean sex ratios (males:females) were 48:52, 52:48, 49:51 and 48:52 for the control, 100, 300 and 600 mg/kg bw/day groups, respectively.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on external morphology following treatment up to and including 600 mg/kg bw/day. Malformations were noted in two fetuses at the high dose level and in one control fetus. At the high dose, these were general anasarca and small lower jaw, astomia (absent mouth) and absent eye bulges (all together in one fetus), and in the control group a combination of exencephaly and open eyelids was observed in one fetus. The occurrence of two dissimilar malformed fetuses at the high dose does not indicate a relation to treatment.
External variations were not seen in any group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on skeletal morphology following treatment up to and including 600 mg/kg bw/day.
Of the malformations, bent limb bones (scapulas and/or humeri) were most frequently observed. These occurred in one control fetus, two fetuses from dose group 100 mg/kg bw/day and three fetuses from dose group 600 mg/kg bw/day. The mean litter incidence at the high dose level was above the maximum historical control value (1.8% versus 0.9% per litter). However, because no dose response was observed for this malformation and it is the most frequently observed skeletal abnormality among historical control fetuses, the finding was not considered to be treatment related. The other skeletal malformations that were noted (rib anomaly in one high dose fetus and fused skull bones in one low dose fetus) occurred singly and therefore were considered chance findings.
Skeletal variations occurred at an incidence of 81.0%, 75.3%, 83.2% and 93.3% per litter in dose groups 0, 100, 300 and 600 mg/kg bw/day, respectively. All the ones noted, were not considered treatment related as they occurred in the absence of a dose-dependent relationship, infrequently, in control fetuses only and/or at frequencies that were within the range of available historical control data.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on visceral morphology following treatment up to and including 600 mg/kg bw/day.
Three fetuses in the high dose group had visceral malformations. The one with multiple malformations observed externally also appeared to have a narrow pulmonary trunk and atrial septum defect and two other fetuses had a small eye observed at soft tissue cephalic examination. The latter finding was also observed in one low dose fetus. The externally noted absent eye bulges in the high dose fetus should be mentioned here as well, but these occurred in a fetus with multiple external and visceral
malformations. As such, this grossly abnormal fetus was considered to have fortuitously occurred. The two remaining small eyes at the high dose level were not considered treatment related as these pertains two cases only without presence of a dose relationship. Moreover, this malformation was previously noted in historical control fetuses.
Besides the occurrence of situs inversus in one control fetus, there were no other viscerally malformed fetuses in this study.
The variations that were noted in this study were small supernumerary lobe(s) and appendix of the liver, convoluted and dilated ureter(s), small renal papilla and partially undescended thymus horn(s). These variations occurred at low incidences and/or in the absence of a doserelated incidence trend and therefore were not considered to be treatment related.
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
No developmental toxicity was observed in the 100, 300 and 600 mg/kg bw/day groups.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of adverse effects up to and including the highest dose tested

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

The concentrations analysed in the formulations of 100, 300 and 600 mg/kg bw/day groups were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%), with mean accuracies of 103%, 96% and 103% in these groups, respectively

The formulations of 100 mg/kg bw/day and 600 mg/kg bw/day were homogeneous (i.e. coefficient of variation ≤ 10%), with a homogeneity (coefficient of varaition) of 1.6 and 3.8% in these groups, respectively.

Table 1: overview developmental toxicity data

Dose level (mg/kg bw/day)

0

100

300

600

Pregnant/total dams

22/22

21/22

21/22

22/22

-early resorptions

-late resorptions

(% per litter)

8.7

0.4

7.7

0.0

4.7

0.0

6.6

0.3

Dams with abortion, early deliveries, stillbirths, resorptions only and/or dead fetuses

0

0

0

0

Pre-implantation loss (number and percent)

16 (7.4%)

30 (14%)

9 (3.5%)

15 (5.8%)

Post-implantation loss (number and percent)

22 (9%)

11 (7.7%)

11 (4.7%)

17 (6.9%)

Body weight on day 21

327

314

322

315

Body weight gain day 6-21 (%)

44

41

43

41

Gravid uterine weight (g)

69.1

63.8

72.5

68.3

Mean live offspring (number)

9.6

9.0

10.5

10.0

Live offspring (percent)

91.0

92.3

95.3

93.1

Mean fetal/pup body weight males (g)

5.4

5.5

5.3

5.2

Mean fetal body weight females

5.2

5.1

5.1

4.9**

Mean fetal body weight (sexes combined)

5.3

5.3

5.2

5.1*

Malformations (including runts) number and percent of fetuses per litter

 

 

3 (2.3%)

 

 

4 (7.6%)

 

 

0 (0%)

 

 

8 (6.6%)

Variations (% per litter)

-external

-soft tissue

-skeletal

 

0

7.7

81.0

 

0

18.3

75.3

 

0

11.0

83.2

 

0

8.6

93.3

Table 2: Historical Control Data Rat: Crl:WI(Han) (outbred, SPF-Quality)

Gestation Day 21

Study Date Range: 2014 – 2015

Mean of Study Means

Endpoint 

Total

Mean

SD

Median

Min

Max

P5

P95

 No of Studies 

13

 Total No. of Animals in the Control Group 

304

 No. of Animals that Died 

0

 No. of Animals that were Euthanized 

0

 No. of Animals that Aborted or Delivered 

3

 Percent Pregnant 

98.8

2.73

100.0

90.9

100.0

97.1

100.0

 No. of Animals Examined at Laparohysterectomy 

301

 No. Nongravid 

4

 No. Gravid 

297

 No. with Only Resorptions 

2

 No. of Dams with Live Fetuses 

295

 Mean No. Viable Fetuses/Dam 

10.7

0.71

10.6

9.1

11.6

10.3

11.2

 Total No. Viable Fetuses 

3194

 Viable Fetuses (%/Litter) 

95.2

2.63

95.9

88.9

98.4

93.6

96.8

 Mean No. Postimplantation Loss/Dam 

0.5

0.15

0.4

0.2

0.7

0.4

0.6

 Total No. Postimplantation Losses 

134

 Postimplantation Loss (%/Litter) 

4.8

2.63

4.1

1.6

11.1

3.2

6.4

 Dead Fetuses (%/Litter) 

0.0

0.11

0.0

0.0

0.4

0.0

0.1

 Early Resorptions (%/Litter) 

4.7

2.62

4.1

1.6

11.1

3.2

6.3

 Late Resorptions (%/Litter) 

0.0

0.11

0.0

0.0

0.4

0.0

0.1

 Mean No. Implantations/Dam 

11.2

0.69

11.1

9.6

12.0

10.8

11.6

 Mean No. Corpora Lutea/Dam 

11.9

0.71

11.7

10.9

13.2

11.5

12.3

 Mean No. Preimplantation Loss/Dam 

0.7

0.32

0.6

0.2

1.3

0.5

0.9

 Total No. Preimplantation Losses 

207

 Preimplantation Loss (%/Litter) 

6.2

3.43

5.8

2.0

14.5

4.2

8.3

 Total No. Male Fetuses 

1617

 Total No. Female Fetuses 

1577

 % Males/Litter 

50.8

2.12

50.7

46.6

53.7

49.5

52.0

 % Female/Litter 

49.2

2.12

49.3

46.3

53.4

48.0

50.5

 Mean Fetal Body Weight (g) 

5.2

0.08

5.2

5.1

5.3

5.1

5.2

 Mean Male Body Weight (g) 

5.4

0.10

5.4

5.2

5.5

5.3

5.4

 Mean Female Body Weight (g) 

5.1

0.07

5.1

5.0

5.2

5.0

5.1

 Mean Male Placenta Weight (g) 1 

0.46

0.01

0.47

0.44

0.47

0.4

0.5

 Mean Female Placenta Weight (g) 1 

0.44

0.01

0.44

0.42

0.45

0.4

0.5

Historical Control Data Rat: Crl:WI(Han) (outbred, SPF-Quality) 

Gestation Day 21 

Study Date Range: 2014 -2015 

 

No. of Studies             13      

 Total No. Fetuses/Litters Examined Externally  3194 /295 

 Total No. Fetuses/Litters Examined Viscerally  2061 /295 

 Total No. Fetuses/Litters Examined Skeletally  2059 /295

Mean of Study Means                            Summary Incidence

(%Per Litter Basis)                                (Total No. Affected)

MALFORMATIONS 

Mean

SD

Median

Min

Max

P5

P95

Fetuses

Litters

 Total External Malformations 

1

1

 Total Visceral Malformations 

7

7

 Total Skeletal Malformations 

15

15

 Total Malformations 

22

22

 EXTERNAL 

 Exencephaly 

0.0

0.14

0.0

0.0

0.5

0.0

0.1

1

1

 Eye(s)-Open 

0.0

0.14

0.0

0.0

0.5

0.0

0.1

1

1

 VISCERAL 

 Diaphragmatic Hernia 

0.0

0.08

0.0

0.0

0.3

0.0

0.1

1

1

 Eye(s)-Absent and/or Small 

0.1

0.26

0.0

0.0

0.9

0.0

0.2

3

3

 Hydrocephaly-External 

0.0

0.12

0.0

0.0

0.5

0.0

0.1

1

1

 Situs Inversus 

0.2

0.34

0.0

0.0

1.0

0.0

0.4

3

3

 SKELETAL 

 Jaw-Upper Jaw Small 

0.1

0.22

0.0

0.0

0.8

0.0

0.2

1

1

 Jaw-Lower Jaw Absent or Small 

0.1

0.22

0.0

0.0

0.8

0.0

0.2

1

1

 Limb Bone(s)-Bent 

0.3

0.44

0.0

0.0

1.1

0.0

0.5

4

4

 Rib Anomaly 

0.1

0.31

0.0

0.0

1.1

0.0

0.3

1

1

 Skull Anomaly 

0.1

0.34

0.0

0.0

1.2

0.0

0.3

2

2

 Sternebra(e)-Fused 

0.1

0.29

0.0

0.0

1.0

0.0

0.3

2

2

 Sternebra(e) Malaligned (Severe) 

0.0

0.08

0.0

0.0

0.3

0.0

0.1

1

1

 Sternoschisis 

0.1

0.22

0.0

0.0

0.8

0.0

0.2

1

1

 Vertebral Anomaly With or Without Associated Rib Anomaly 

0.2

0.53

0.0

0.0

1.9

0.0

0.5

3

3

 Vertebral Centra Anomaly 

0.1

0.22

0.0

0.0

0.8

0.0

0.2

1

1

Mean of Study Means                         Summary incidence

(%Per Litter Basis)                             (Total No. Affected)

VARIATIONS 

Mean

SD

Median

Min

Max

P5

P95

Fetuses

Litters

 EXTERNAL 

 None Observed 

 VISCERAL 

 Kidney(s)-Renal Papilla(e) Absent and/or Small 

0.1

0.25

0.0

0.0

0.9

0.0

0.2

2

2

 Liver-Appendix 

1.2

0.56

1.3

0.3

2.3

0.9

1.6

23

21

 Liver-Discolored 

0.1

0.30

0.0

0.0

1.0

0.0

0.3

3

3

 Liver-Small Supernumerary Lobe(s) 

4.0

1.96

4.0

1.3

7.7

2.8

5.2

69

58

 Spleen-Supernumerary 

0.0

0.14

0.0

0.0

0.5

0.0

0.1

1

1

 Thymus-Partially Undescended Horn(s) 

1.3

1.55

0.8

0.0

4.3

0.3

2.2

34

23

 Thyroid-Discolored 

0.1

0.36

0.0

0.0

1.3

0.0

0.3

1

1

 Ureter(s)-Convoluted 

1.0

2.39

0.0

0.0

8.7

0.0

2.5

43

28

 Ureter(s)-Dilated 

0.9

2.33

0.0

0.0

8.5

0.0

2.3

44

19

 SKELETAL 

 7th Cervical Rudimentary Rib(s) 

1.7

1.34

1.2

0.0

4.4

0.9

2.5

30

26

 7th Cervical Full Rib(s) 

0.1

0.36

0.0

0.0

1.1

0.0

0.4

2

2

 14th Full Rib(s) 

5.7

4.65

5.2

0.0

13.1

2.9

8.5

88

64

 14th Rudimentary Rib(s) 

44.1

19.84

54.4

19.0

72.0

32.1

56.1

798

250

 Metacarpal(s) and/or Metatarsal(s) Unossified 

2.2

1.97

1.0

0.0

6.3

1.0

3.4

41

24

 Pelvic Girdle-Caudal Shift 

6.6

3.77

7.1

1.7

12.8

4.3

8.9

127

71

 Rib(s)-Bent 

10.6

7.78

10.2

0.8

22.3

5.9

15.3

162

85

 Rib(s)-Short 

0.0

0.06

0.0

0.0

0.2

0.0

0.0

1

1

 Skull-Reduced Ossification 

2.7

2.55

1.8

0.0

7.0

1.2

4.3

81

46

 Skull-Supernumerary Site 

0.0

0.14

0.0

0.0

0.5

0.0

0.1

1

1

 Sternebra(e) #1, #2, #3 and/or #4 Unossified 

0.2

0.31

0.0

0.0

0.8

0.0

0.3

3

3

 Sternebra(e) #5 and/or #6 Unossified 

0.9

1.33

0.0

0.0

4.1

0.1

1.7

37

23

 Sternebrae-Malaligned (Slight or Moderate) 

11.1

5.72

8.9

4.4

21.3

7.6

14.5

188

131

 Sternum-Supernumerary Ossification Site 

0.1

0.31

0.0

0.0

1.1

0.0

0.3

1

1

 Vertebral Centra-Reduced Ossification 

0.6

0.88

0.4

0.0

3.0

0.1

1.2

12

12

Applicant's summary and conclusion

Conclusions:
Based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for Sodium alkylnaphthalene sulfonate was established as being 300 mg/kg bw/day for local toxicity based on morphological alterations of the stomach. The maternal NOAEL for systemic toxicity was at least 600 mg/kg bw/day.
The developmental NOAEL was established as being at least 600 mg/kg bw/day.
Executive summary:

Developmental toxicity/teratogenicity for Sodium alkylnaphthalene sulfonate was studied in rats according to OECD 414 guideline. Eighty-eight mated female Wistar Han rats were assigned to four dose groups. Sodium alkylnaphthalene sulfonate was administered once daily by oral gavage from Days 6 to 20 post-coitum at doses of 100, 300 and 600 mg/kg bw/day. The rats of the control group received the vehicle, water, alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. Formulations prepared on one day during treatment were analyzed for accuracy and homogeneity.

All animals surviving to Day 21 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. The stomach was collected and fixed from all animals at necropsy. Histopathological examination was performed on the stomach from 10 selected females per group. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placenta and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative; these fetuses were dissected and examined for visceral anomalies. The other one-half of the fetuses were processed and stained with Alizarin Red S for skeletal examinations.

Accuracy and homogeneity of formulations were demonstrated by analyses.

Test item-related morphological alterations were present in the stomach at 600 mg/kg bw/day. A slightly increased incidence of minimal inflammation of the glandular stomach was recorded at 600 mg/kg bw/day (5/10 females), compared to 1/10 females of the control group. In one female at this dose a moderate erosion/ulceration with a moderate lymphogranulocytic inflammation of the forestomach was recorded, which was considered to be related to the treatment with the test item. No maternal toxicity was observed in the 100 and 300 mg/kg bw/day groups. No developmental toxicity was observed in the 100, 300 and 600 mg/kg bw/day groups.

In conclusion, based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for Sodium alkylnaphthalene sulfonate was established as being 300 mg/kg bw/day for local toxicity based on morphological alterations of the stomach. The maternal NOAEL for systemic toxicity was at least 600 mg/kg bw/day. The developmental NOAEL was established as being at least 600 mg/kg bw/day.