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Developmental toxicity / teratogenicity

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Endpoint:
developmental toxicity
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to GLP and valid methods and is considered relevant and reliable to study effects on offspring. However as it only examines external changes of pups, it is not adequate for classification.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 422
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Butanedioic acid, 2(or 3)-sulfo, 4-[2-[(1-oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts; Disodium Cocamido MEA-Sulfosuccinate
- Physical state: Watery solution
- Analytical purity: 41.5% (active ingredient) ; correction factor 2.41
- Impurities (identity and concentrations): See confidential details
- Composition of test material, percentage of components: See confidential details
- Purity test date: June 1, 2012
- Lot/batch No.: ST025966859
- Expiration date of the lot/batch: May 2013
- Stability under test conditions: Freshly prepared formuations; actual concentrations of the test item in the test item vehicle mixtures were between 99.99% and 102.96% of the nominal concentrations, which were stable at room temperature for at least 24 hours.
- Storage condition of test material: At +2° to +8°C
- Other: Manufacturer/supplier: Evonik Industries AG

Test animals

Species:
rat
Strain:
other: CD® / Crl:CD(SD)
Details on test animals and environmental conditions:
- Source: Charles River Laboratories Research, Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at start of dosing : Males 55 days; Females: 48 days
- Weight range at start of dosing: Males: 281.0 to 308.0 g; Females: 68.9 to 195.7 g
- Fasting period before study: Ad libitum with exception of the night before the day of blood withdrawal for laboratory examination.
- Housing: With exception of the mating period, the animals were kept singly in MAKROLON
cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt/ Arkeburg, Germany) is used as bedding material in these cages. The cages were cleaned and changed once a week.
- Diet (e.g. ad libitum): ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany, ad libitum with exception of the night before the day of blood withdrawal for laboratory examination.
- Water (e.g. ad libitum): Tap water is offered daily ad libitum.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C (maximum range)
- Humidity (%): 55% ± 15% (maximum range)
- Air changes (per hr): Not provided
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
Males From: August 27, 2012 To: October 2, 2012
Females From: August 27, 2012 To: October 19, 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: tap water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Application volume: 5 mL/kg bw/day. The test item was dissolved in the vehicle tap water to concentrations of 20, 60 and 200 mg test item /mL tap water and was administered orally at a constant volume once daily. The amount of the test item was adjusted to the animal's current body weight daily. The test item-vehicle mixture was freshly prepared every day.



Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the test item-vehicle mixtures samples of approx. 2 x 5 mL were taken at the following time points and stored at ≤ -20°C until analysis at LPT.
*Start of treatment period: Analysis of stability and concentration: Immediately after preparation of the test item-vehicle mixtures as well as 8 and 24 hours after storage of the test item preparations at room temperature: 3 samples/dose level group = 9 samples
*End of treatment period: Concentration: During treatment with the test item always before administration to the last animal/dose level group: 3 samples
The samples were labelled with the study number, test item, test species, type of sample, aliquot number, concentration, test day, sampling time and date.
The validation of the analytical method is part of LPT study No. 28344 (14-day dose-range-finding).

The measured actual concentrations of the test item in the test item vehicle mixtures were between 99.99% and 102.96% of the nominal concentrations (table 26).
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1 (1 male and 1 female animal were placed in one cage during the dark period)
- Length of cohabitation: The female was placed with the same male until pregnancy occurred or 2 weeks had elapsed.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:
Males: 2 weeks prior to mating, during the mating period and approx. 2 weeks post mating at least until the minimum total dosing period of 28 days has been completed (up to and including the day before sacrifice).
Females: 2 weeks prior to mating and continuing up to, and including, day 3 post-partum or the day before sacrifice.
Frequency of treatment:
daily
Duration of test:
Males: 2 weeks prior to mating, during the mating period and approx. 2 weeks post mating at least until the minimum total dosing period of 28 days has been completed (up to and including the day before sacrifice).
Females: 2 weeks prior to mating and continuing up to, and including, day 3 post-partum or the day before sacrifice.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels have been selected in agreement with the Sponsor based on the results of a 14-day dose-range-finding study in rats dosed at 100, 300 and 1000 mg act. ingr./kg bw by oral gavage (LPT Study No. 28344). None of the animals died prematurely. None of the male and female rats treated orally with 100 or 300 mg act. ingr./kg bw/day revealed any changes in behaviour, external appearance or faeces. Salivation was noted for 2 of 5 male animals treated at 1000 mg/kg bw/day on 1 or 3 test days starting on test day 9 and increased faeces was noted for 3 of 5 male and 2 of 5 female high dosed animals on 6 or 9 test days starting on test day 5. No test item-related changes on body weight and body weight gain were noted for the male and female rats up to 1000 mg act. ingr./kg bw/day. No test item-related changes on food consumption were noted for the male and female rats treated orally with 100 or 300 mg act. ingr./kg bw/day. The food consumption of
the male and female animals treated with 1000 mg act. ingr./kg bw/day was slightly increased by 9% for the males and by 10% for the females in test week 2 (statistically significant at p ≤ 0.01 for both sexes). No test item-related influence was noted for the drinking water consumption at any of the tested dose levels. None of the male and female rats treated orally with 100, 300 or 1000 mg act. ingr ./kg bw/day revealed changes at macroscopic inspection at necropsy or organ weights.
- Rationale for animal assignment (if not random):
The animals are randomly allocated to the test groups.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the test period, each animal was observed for clinical signs at least once daily. Mortality was recorded twice daily. In addition, animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals were checked regularly from 7:00 a.m. to 11 :00 a.m. with a final check performed at approximately 3:30 p.m.
- Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Additionally, once before the first exposure (to allow within-subject comparisons) and once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena and at the same time, each time preferably by observers unaware of the treatment. Signs noted included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes. Body weights were recorded individually for each adult animal.
- Time schedule for examinations:
Males and females were weighed on the first day of dosing, weekly thereafter and at termination. During gestation, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum) and day 4 post-partum.

FOOD CONSUMPTION: Yes
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period with the execution of the mating period. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment week.

WATER CONSUMPTION:
- Time schedule for examinations: Water consumption was monitored daily by visual appraisal throughout the study.

HAEMATOLOGY: see Section 7.5.1

CLINICAL CHEMISTRY: See Section 7.5.1

NEUROLOGICAL OBSERVATIONS: see Section 7.5.1

REPRODUCTIVE PARAMETERS:
Number of pregnant females
Pre-coital time
Gestation length calculated from day 0 of pregnancy
Corpora lutea
lmplantation sites
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes . Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI.
Examinations included:
- Gravid uterus weight: No
- 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. Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter, were carefully examined externally for gross abnormalities.
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: No
- Head examinations: No
Statistics:
Toxicology and Pathology data were captured, whenever possible, using the departmental computerized systems (Provantis®8 Integrated preclinical software, Instem LSS Ltd.). Raw data not fully compatible with the computerized systems were maintained on paper according to appropriate SOPs.
The test item-treated groups (2 - 4) were compared with the control group (1 ).
The following statistical methods were used:
STUDENT' s t-test All numerical functional tests
(p ≤0.05 and ≤0.01)


Multiple t-test based on Body weight I Food consumption I
DUNNETT, C. W. Haematology I Clinical chemistry I
New tables for multiple Absolute and relative organ weights
Comparisons with a control (p ≤ 0.05 and ≤0.01)
Biometrics,
482-491 (Sept 1 964)

For all numerical values (e.g. body weight, food consumption and organ weight data) homogeneity of variances was tested by using the BARTLETT chi2-test. lf the variances are homogeneous, the DUNNETT test (p ≤ 0.01) was used to compare the experimental groups with the control group.
In case of heterogeneity of variances, the STUDENT's t-test was carried out; limit of significance is p ≤ 0.01.
Exact test of R. A. FISHER Histopathology, if applicable
(p≤0.05)
For the comparison of classification measurements (for example the fertility index) the FISHER's exact test, n < 100 or chi2-test with Yates' correction for continuity, n ≥ 100 (p ≤0.05 and p ≤0.01) were employed.
These statistical procedures were used for all data. Significantly different data were indicated in the tables of the report.
The mean values and standard deviations were calculated to the highest possible degree of accuracy and then rounded to the reported number of decimal places. Hence, deviations to the last decimal place of up to ± 1 may occur caused by rounding.
Indices:
Reproductive indices:
Gestation Index
Fertility Index
Pre-implantation loss [%]
Post-implantation loss [%]

Offspring viability indices:
Birth Index
Live Birth Index
Viability Index

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: Slight reduction in body weight in male and female rats dosed at 1000 mg/kg bw/day; other changes were noted as well for clinical biochemistry, macroscopic and microscopic examination

Details on maternal toxic effects:
A slight reduction in body weight was noted for female rats of the high dose group (1000 mg test item/kg bw/day). For the female rats the reduction in body weight was noted from gestation day 0 (7.6%) until lactation day 4 (9.5%). The body weight at autopsy was reduced accordingly.

The laboratory examinations revealed an increased ALAT activity for female rats of the high dose group (1000 mg test item/kg bw/day).

Microscopic examination revealed the occurrence of squamous cell hyperplasia in the non-glandular mucosa of the forestomach from female rats of the high dose group (1000 mg test item/kg bw/day).

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects. Remark: No test item related influence was noted on the survival rate and the mean and total body weights of the pups.

Details on embryotoxic / teratogenic effects:
No test item related influence was noted on the survival rate and the mean and total body weights of the pups.
Effects on the development of the F1 offsprings (pups) NOAEL (no-observed-adverse-effect level): above 1000 mg/kg bw/day, p.o.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1.Developmental parameters

Parameter

Group 1

Control

Group 2

100 mg/kg

Group 3

300 mg/kg

Group 4

1000 mg/kg

Number of pregnant dams

8/10

9/10

9/10

10/10

Number of pups at birth (total)

96

125

123

127

Number of pups at birth (mean)

12.0

13.9

13.7

12.7

No. of live pups at birth (total)

96

125

123

126

No. of live pups at birth (mean)

12.0

13.9

13.7

12.6

No. of live pups on day 4 (total)

96

125

123

123

No. of live pups on day 4 (mean)

12.0

13.9

13.7

13.7

Viability index day 1-4 (mean %)

100.0

100.0

100.0

90.0

Viability index day 1-4 (total# %)

100.0

100.0

100.0

97.6

Mean body weight day 0/1 male and female pups (g)

6.23

6.34

6.29

6.11

Mean body weight day 4 male and female pups (g)

8.51

9.07

9.11

8.73

Mean total litter weight day 0/1, male and female pups (g)

75.00

87.38

85.91

77.09

Mean total litter weight day 4, male and female pups (g)

102.54

124.08

123.28

118.94

External examinations of pups (No. of pups/litter size)

- no externally visible
 abnormalities     

- stomach without milk        

 

 

 

8/8

0/8

 

 

 

9/9

0/9

 

 

 

9/9

0/9

 

 

 

10/10

1/10 (3/3)*

#: total number of live pups on the respective lactation day

* 3 live born pups from dam no. 77 were found dead without milk on lactation day 2.

Applicant's summary and conclusion

Conclusions:
NOAEL (no-observed-adverse-effect level) of the F1 offspring: above 1000 mg/kg bw/day, p.o.
Executive summary:

The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development according to OECD guideline 422. The test item was administered orally by gavage to rats at dose levels of 100, 300 and 1000 mg active ingredient/kg bw/day. The application started two weeks before mating on test day one and ended on the day or one day before sacrifice. Day of sacrifice was on test day 37 for the male rats and between lactation day 4 and 7 for the female rats.

Effects on the parental generation (general toxicity)
No test item-related premature death was noted in any treatment group (100, 300 and 1000 mg/kg bw/day).

No signs of clinical toxicity were noted for the male and female rats of the low and intermediate dose groups (100 and 300 mg/kg bw/day).

A slightly increased salivation was noted in one male rat, no further signs of clinical toxicity were noted for the male and female rats of the high dose group (1000 mg/kg bw/day).

No test item related influence was noted for the male and female rats of all treatment groups (100, 300 and 1000 mg/kg bw/day) during the observational and functional (grip strength and spontaneous motility) neurological screenings.

A slight reduction in body weight was noted for the male and female rats of the high dose group (1000 mg/kg bw/day). For the male rats the reduction in body weight was noted from test day 8 (4.4%) until test day 36 (5.5%) and for the female rats from gestation day 0 (7.6%) until lactation day 4 (9.5%). The body weight at autopsy was reduced accordingly.

The laboratory examinations revealed an increased ALAT activity for the male and female rats of the high dose group (1000 mg/kg bw/day) and a decrease in the albumin concentration for the male rats of the high dose group.

No test item-related changes were noted during the macroscopic inspection at autopsy with the exception of changes in the stomach from 2 male animals from the high dose group (1000 mg/kg bw/day).

Microscopic examination revealed the occurrence of squamous cell hyperplasia in the non-glandular mucosa of the forestomach from the male and female rats of the high dose group (1000 mg/kg bw/day). Further microscopic findings occurred in form of pulmonary congestion in the male rats from the high dose group.

Effects on reproduction parameters and organs

No test item-related influence was noted on the reproduction parameters in any treatment group (100, 300 and 1000 mg/kg bw/day).

Microscopic examination revealed no changes in the reproductive organs from the male and female rats of the high dose group (1000 mg/kg bw/day).

Effects on the development of the F1offsprings (pups)

No test item related influence was noted on the survival rate and the mean and total body weights of the pups.

External examination of the pups revealed no visible changes related to the test item.

The following no-observed-effectlevelswere established:

Effects on the F0-generation

NOAEL (no-observed-adverse-effect level): 300 mg/kg bw/day, p.o.

Effects on reproductive toxicity

NOAEL (no-observed-adverse-effect level): above 1000 mg/kg bw/day, p.o.

Reproductive indices:

Effects on the development of the F1offsprings (pups)

NOAEL (no-observed-adverse-effect level): above 1000 mg/kg bw/day, p.o.