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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:
weight of evidence
Study period:
04 Aug - 05 Dec 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 22 January 2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted August 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSa No 8147, 24 November 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium N-lauroylsarcosinate
EC Number:
205-281-5
EC Name:
Sodium N-lauroylsarcosinate
Cas Number:
137-16-6
Molecular formula:
C15H29NO3.Na
IUPAC Name:
sodium [dodecanoyl(methyl)amino]acetate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD® (SD)IGS BR
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Margate, UK
- Age at study initiation: not specified in report, animals purchased time-mated
- Weight at study initiation: 197-268 g (females on arrival, prior to GD3)
- Housing: individual in solid-floor propylene cages with stainless steel lids in a single air-conditioned room, bedding with softfood flakes
- Diet: pellet diet (Rodent 2018C Teklad Global Certified Diet, Harlan; Oxon; UK) ad libitum, environmental enrichment provided in the form of wooden chew blocks and cardboard fun tunnels
- Water: ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 04 Aug 2013 (first day of treatment) To: 22 Aug 2013 (final day of necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
the test item was prepared at the appropriate concentrations as a solution in distilled water.

VEHICLE
- Concentration in vehicle: 6, 20 and 50 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined by high performance liquid chromatography (HPLC/UV) using an external standard technique.The formulations investigated during the study were found to comprise test item in the range of 101% to 104% and, thus, the required content limit ± 10% with reference to the nominal content was met. The test item was found to be stable in the formulations when kept for twenty one days in the refrigerator due to results which met the variation limit of 10% from the time-zero mean. Thus, the results indicate the accurate use of the test item and distilled water as vehicle during this study. The formulations were found to be homogeneously prepared, and sufficient formulation stability under storage conditions was approved.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
Gestation Day 5-19
Frequency of treatment:
daily, 7 days/week
Duration of test:
until Gestation Day 20 (terminal sacrifice)
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 P females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on previous toxicity data.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked included: following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioural changes once daily during the gestation period. An additional observation was also performed five hours after dosing during the normal working week. All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: individual body weights were recorded for each surviving individual animal at Day 3, 5, 6, 7, 8, 11, 14, 17 and 20 (termination kill) of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined: Yes, at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily by visual inspection of water bottles
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: ovaries and uteri of pregnant females, full external and internal examination; any macroscopic abnormalities were recorded; the stomach was retained from all females
Ovaries and uterine content:
The ovaries and uterine content were 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

- Other: fetal sex, placental weight, position and type of intrauterine implantation
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: No
Statistics:
Group mean values were calculated to include data from all females with live fetuses on Day 20 of gestation. As the litter was the standard unit of assessment, values were first calculated within the litter, and group mean values represent the mean of these individual litter values.
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:
Female body weight change, food consumption and gravid uterus weight: Bartlett´s test for homogeneity of variance and one way analysis of variance, followed by Dunnett´s multiple comparison test or, if unequal variances were observed, an alternative multiple comparison test.
All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.
Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis non-parametric analysis of variance and Mann-Whitney ‘U’ test.
Probability values (p) demonstrating different levels of significance were chosen as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p>=0.05 (not significant)
Indices:
Pre implantation loss: [(number of corpora lutea-number of implantations) / number of corpora lutea] x 100;
Post-implantation loss: [(number of implantations–number of live foetuses)/number of implantations] x 100;
Sex ratio: % male fetuses (sex ratio)= [Number of male foetuses / Total number of foetuses] x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
250 mg/kg bw/day: the female found dead on Day 10 did not show any clinical signs prior to Day 10. The female found dead on Day 18 had noisy respiration on Days 10 and 17 and increased salivation and pilo-erection on Day 17.
5/22 females showed incidences of increased salivation between Days 10 and 19 and 3/22 females also showed noisy respiration on either Day 6 or 7.

100 mg/kg bw/day: 1/24 females showed increased salivation on Day 18 and 1/24 females had noisy respiration on Day 13.

Observations of this nature are commonly observed following the oral administration of an unpalatable or irritant test item formulation and are considered not to represent true systemic toxicity.

Control: 1/24 females had fur loss on Day 18. In the absence of treatment this was considered of no toxicological importance.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
250 mg/kg bw/d: One female treated was found dead on Day 10 and another female was found dead on Day 18.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
250 mg/kg bw/day: body weight gain of the females was lower throughout the treatment period and cumulative body weight gain to Day 14 (where body weight is not greatly influenced by the weight of the gravid uterus) was 24% lower than the corresponding control value. By termination on Day 20, cumulative body weight gain was 15% lower than the corresponding control values. When final body weight was adjusted for the gravid uterus, an overall body weight gain reduction was evident during gestation.

100 mg/kg bw/day: females showed a slight reduction in cumulative body weight gain from Day 7 onwards. When final body weight was adjusted for the gravid uterus, an overall body weight gain reduction was evident during gestation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
250 mg/kg bw/day: females showed a reduction in food consumption throughout the treatment period. Statistically significant reductions were evident between Days 8 and 11 (p < 0.001) and Days 14-17 (p< 0.05) compared with the control group.
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
Description (incidence and severity):
The female treated with 250 mg/kg bw/day that was found dead on Day 10 had gaseous distention in the stomach and gastro-intestinal tract. The other interim death female had sloughing on the non-glandular region of the stomach, seven dead foetuses in the right uterine horn and five dead foetuses in the left uterine horn. The remaining females treated with 250 mg/kg bw/day that were terminated on Day 20 all had sloughing on the non-glandular region of the stomach.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
not examined

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
Females from all treatment groups showed a statistically significant reduction (p<0.05 - p<0.01) in the number of corpora lutea. The intergroup differences did not show a true dose related response and were considered to be incidental and unrelated to treatment due to ovulation and mating occurring prior to the administration of the test item. As a consequence of incidentally lower number of corpora lutea in treated females litter size was statistically significantly reduced (p<0.05) in all treated females. The intergroup differences did not show a true dose related response and in the absence of any effect on post-implantation loss and mean fetal weights it supports the assumption that the intergroup differences in litter size was considered to be incidental and of no toxicological importance.
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There was no obvious adverse effect of maternal treatment on litter data as assessed by the mean number of implantations, early and late embryonic/fetal deaths and live fetuses or sex ratio, as assessed by percentage male.
For all dose groups, there were no significant treatment-related trends in the proportion of fetuses (or litters) with evidence of visceral or skeletal anomalies. The types of external, visceral and skeletal anomalies were those commonly observed for this type of study. There were no findings that were considered to represent any known malformations.

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No adverse effect on developmental toxicity observed at the highest tested dose level.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

All treated females showed statistically significant reduction of corpora lutea and in consequence reduced litter sizes. The intergroup differences did not show a true dose related response and were considered to be incidental and unrelated due to ovulation and mating occurring prior to the administration of the test item starting on GD5. The absence of any effect on post-implantation loss and mean fetal weights supports the assumption that these intergroup differences in litter size were considered to be incidental and of no toxicological importance. Foetuses of the 250 mg/kg bw/day group showed a statistically significant increase in the percent of foetuses showing one or more ossified forepaw phalanges. Due to the isolated occurrence of this observation it is not considered to be a true developmental effect and it is therefore of no biological importance.

Table A: Group Mean Litter Data Values (Mean ± SD)

Dose Level (mg/kg bw/day)

Number of Litters

Number of Corpora Lutea

Number of Implants

Number of Embryonic/Fetal Deaths

Number of Live Implants

Early

Late

Total

Male

Female

Total

0 (control)

23

15.2 ± 1.8

13.9 ± 1.6

0.1 ± 0.3

0.1 ± 0.6

0.3 ± 0.7

6.9 ± 1.8

6.8 ± 1.9

13.7 ± 2.1

30

21

14.3* ± 1.8

13.1 ± 2.0

1.0 ± 2.4

0.1 ± 0.4

1.0 ± 2.5

6.0 ± 2.7

6.1 ± 2.4

12.1* ± 3.4

100

24

13.7* ± 1.9

13.0 ± 1.8

0.6 ± 1.3

0.2 ± 0.7

0.8 ± 1.4

6.5 ± 2.1

5.6 ± 1.7

12.1* ± 2.5

250

22

13.9** ± 1.2

13.2 ± 1.1

0.5 ± 1.4

0.2 ± 0.9

0.7 ± 1.6

6.5 ± 2.0

6.0 ± 1.9

12.5* ± 2.2

p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 not significant

Table A: Group Mean Litter Data Values (continued) (Mean ± SD)

Dose Level (mg/kg bw/day)

Implantation Loss (%)

Male foetuses (%)

Mean Male Fetal Weight (g)

Mean Female Fetal Weight (g)

Mean Fetal Weight (g)

Mean Placental Weight (g)

Litter Weight (g)

Total Placental Weight (g)

Pre

Post

0 (control)

8.4 ± 7.4

2.3 ± 7.2

50.4 ± 11.3

4.117 ± 0.260

3.935 ± 0.264

4.026 ± 0.257

0.564 ± 0.047

54.883 ± 8.672

7.667 ± 1.141

30

8.0 ± 10.0

8.4 ± 19.4

48.6 ± 15.2

4.152 ± 0.405

3.881 ± 0.372

4.005 ± 0.365

0.578 ± 0.155

48.345 ± 13.761

6.729 ± 1.942

100

5.0 ± 5.2

6.9 ± 12.2

53.7 ± 12.8

4.166 ± 0.339

3.886 ± 0.269

4.036 ± 0.289

0.542 ± 0.060

49.142 ± 11.318

6.540 ± 1.503

250

4.8 ± 5.3

5.5 ± 12.5

52.2 ± 13.7

4.221 ± 0.347

4.033 ± 0.327

4.126 ± 0.320

0.540 ± 0.057

51.237 ± 8.200

6.744 ± 1.369

 

Table B: Group Mean Cumulative Body Weight Change Values (Mean ± SD)

Dose Level (mg/kg bw/day)

Number of Animals

Cumulative Body Weight Change (g) from GD5

GD6

GD7

GD8

GD11

GD14

GD17

GD20

0 (control)

23

4.0 ± 3.4

7.6 ± 3.7

13.0 ± 5.1

33.6 ± 6.6

51.6 ± 8.1

82.3 ± 10.4

127.8 ± 14.9

30

21

5.1 ± 5.4

7.1 ± 8.2

12.9 ± 10.2

34.0 ± 12.9

53.5 ± 15.5

82.2 ± 19.2

127.0 ± 23.4

100

24

2.3 ± 5.3

6.1 ± 5.5

8.8 ± 7.5

27.6 ± 9.9

44.8 ± 13.8

74.2* ± 12.4

114.3* ± 17.2

250

24/22+

0.5 ± 9.0

3.6 ± 10.1

7.3 ± 11.2

21.7** ± 14.6

39.3* ± 17.8

68.2** ± 20.0

109.1** ± 24.7

+ = n=23 on GD10, n=22 on GD18
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 not significant

 

Table C: Group Mean Gravid Uterus Weight , Adjusted Body Weight and Body Weight Change Values (Mean ± SD)

Dose Level (mg/kg bw/day)

Number of Animals

Body Weight (g) on Days of Gestation

Body Weight Change (g) during GD5-20

Gravid Uterus Weight (g)

Adjusted Body Weight (g) GD20

Adjusted Body Weight (g) Change GD5-20

GD5

GD20

0 (control)

23

262.6 ± 20.6

390.4 ± 29.8

127.8 ± 14.9

84.663 ± 12.502

305.8 ± 23.4

43.2 ± 8.2

30

21

261.7 ± 12.9

288.7 ± 27.3

127.0 ± 23.4

75.544 ± 18.733

313.2 ± 28.6

51.5 ± 22.7

100

24

260.6 ± 14.4

374.9 ± 24.8

114.3** ± 17.2

75.893 ± 15.615

299.0 ± 20.7

38.4* ± 14.6

250

24/22+

257.6 ± 15.5

368.1 ± 32.4

109.1** ± 24.7

77.535 ± 11.935

290.6 ± 25.0

31.6** ± 18.7

+ = n=23 on GD10, n=22 on GD18
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 not significant

 

Table D: Group Mean Food Consumption Values (Mean ± SD)

Dose Level (mg/kg bw/day)

Number of Animals

Food Consumption (g/rat/day) between Days of Gestation

3-5

5-8

8-11

11-14

14-17

17-20

0 (control)

23

23.0 ± 3.1

25.1 ± 2.5

26.5 ± 2.6

26.1 ± 2.8

25.9 ± 3.0

28.0 ± 2.4

30

21

23.6 ± 3.7

24.5 ± 3.9

27.1 ± 3.7

26.5 ± 3.7

26.0 ± 4.2

28.7 ± 3.8

100

24

22.8 ± 2.9

23.9 ± 2.8

24.9 ± 3.3

24.6 ± 3.7

24.5 ± 3.4

27.5 ± 2.9

250

24/22+#

22.3 ± 3.4

22.4 ± 5.3

21.5*** ± 4.7

23.3 ± 4.3

22.6* ± 4.2

25.4 ± 3.8

+ = n=23 on GD10, n=22 on GD18
# = Food consumption not performed on GD 8 and 11 for female Nos. 85 to 90 due to technician error
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 not significant

In conclusion the oral administration of the test substance to pregnant rats by oral gavage during gestation at dose levels of 30, 100 and 250 mg/kg bw/day resulted in treatment related effects detected in females treated with 250 and 100 mg/kg bw/day. The NOAEL was therefore considered to be 30 mg/kg bw/day.

No toxicological significant changes were detected in the offspring parameters measured. The NOEL for developmental toxicity was therefore considered to be 250 mg/kg bw/day. 

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study the test substance did not induce any treatment-related biologically relevant malformations in the developing unborn organism in the presence of maternal toxicity. Therefore, the test substance does not meet the criteria for classification for prenatal developmental toxicity according to Regulation (EC) No 1272/2008. The available data is thus conclusive but not sufficient for classification.

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