Butanedioic acid, sulfo-, 4-[2-[(2-hydroxyethyl)amino]ethyl] ester, N-C18-unsatd. acyl derivs., disodium salts

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2013

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 for classification.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Butanedioic acid, 2 (or 3) - sulfo-, 4 - [2- [(1-oxo (C12 - C18 (even numbered) and C18unsaturated) alkyl)) amino] ethyl] esters, disodium salts

IUPAC Name:

Butanedioic acid, 2 (or 3) - sulfo-, 4 - [2- [(1-oxo (C12 - C18 (even numbered) and C18unsaturated) alkyl)) amino] ethyl] esters, disodium salts

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)

Sex:

male/female

Details on test animals or test system 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.

Details on mating procedure:

- 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
- After approx. 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: yes. This mating-procedure was repeated until at least 8 pregnant dams were available for each group.
- After successful mating each pregnant female was caged (how): 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. On the other side of the animal room than the males with each dose group separated by an empty row.

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).

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

Details on study schedule:

- Age at mating of the mated animals in the study:10 weeks

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.

Examinations

Parental animals: Observations and 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
Number of (viable) pups day0/4


REPRODUCTIVE INDICES:
Gestation Index
Fertility Index
Birth Index
Live Birth Index
Viability Index
Pre-implantation loss [%]
Post-implantation loss [%]

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight, epididymis weight
At the time of sacrifice or death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
The following organs or parts of organs of all male adult animals were fixed in 7% formalin; testes and epididymides were fixed in Bouin' s fixative:
Epididymis (2), Gross lesions, Prostate, Seminal vesicle, Testicle (2).
Detailed histopathologic examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure) of all adult males of groups 1 to 4 following H-E and PAS staining.

Litter observations:

STANDARDISATION OF LITTERS
- screening study: Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter, were carefully examined externally for gross abnormalities.

PARAMETERS EXAMINED
Number of pups absolute (total/live)
Number of pups per dam (total/live)
Number of male and female pups (total/live)
Number of stillbirths
Mean pup weight

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals: The male animals are sacrificed after a minimum total dosing period of 28 days if no longer needed for further mating.
- Maternal animals: All surviving animals: Dams with offspring are sacrificed on day 4 postpartum, or shortly thereafter. Females showing no evidence of copulation are sacrificed 24 days after the last day of the mating period.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Microscopic examination and organ weights of all adult male animals identified as left or right: Epididymis (2) ,Testicle (2)
The following organs or parts of organs of all adult animals are fixed in 7% formalin; testes and epididymides will be fixed in Bouin' s fixative:
Epididymis (2), Gross lesions, Mammary gland, Ovary (2), Prostate, Seminal vesicle, Testicle (2), Uterus (incl. cervix and oviducts), Vagina.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations macroscopic examination) as follows:
Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter, were carefully examined externally for gross abnormalities.
The animals were sacrificed under ether anaesthesia by cutting the aorta abdominalis, exsanguinated, weighed, dissected and inspected macroscopically. All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition of the thoracic viscera were noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined.
The lungs were removed and all pleural surfaces examined under suitable illumination.
The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

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 ≤0.01)

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

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.

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: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

slightly increased salivation in one male rat dosed at 1000 mg/kg bw/day

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

slight reduction iin male and female rats dosed at 1000 mg/kg bw/day

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

slight reduction iin male and female rats dosed at 1000 mg/kg bw/day

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
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.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
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.

A slightly statistically significant (p ≤ 0.01) increase in relative food consumption by 10.3% was noted in the high dose males during the 2nd test week. This was caused by the reduced body weight of the rats of the high dose group.
No influence on food consumption was noted in any treatment group in the females.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Pre-coital time: No test item-related influence was noted.
Gestation length: No test item-related influence was noted.
Evaluation of reproduction parameters of the dams:
No test item-related differences were noted in the number of corpora lutea, the number of implantation sites and the number of pups between the dams from the control group and those from the treatment groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No test item-related influence was noted.
For other organs: see Section 7.5.1.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No effects on the reproductive organs.
For other organs: see Section 7.5.1.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No effects on the reproductive organs.
For other organs: see Section 7.5.1.

Effect levels (P0)

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
No test item-related influence was noted on the survival rate of the pups.

BODY WEIGHT (OFFSPRING)
No test item related influence was noted on the mean and the total body weight of the pups on lactation day 1 and 4.

GROSS PATHOLOGY (OFFSPRING)
No visible gross abnormalities were noted between the control and the treatment groups

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1. Fertility and Reproductive parameters Parental generation

Parameter	Group 1 Control	Group 2 100 mg/kg	Group 3 300 mg/kg	Group 4 1000 mg/kg
No. of females evaluated for pre-coital time	10	10	10	10
Mean precoital interval (days)	4.5	3.4	3.9	2.6
No. of females evaluated for fertility	10	10	10	10
Number of pregnant dams	8	9	9	10
Fertility index (%)	80	90	90	100
No. of females evaluated for gestation length	8	9	9	10
Gestation length (days)	22.0	22.2	22.2	22.1
Number of dams with live pups	8	9	9	10
Gestation Index (%)	100	100	100	100
Corpora lutea(total)	110	137	129	142
Corpora lutea(mean)	13.8	15.2	14.3	14.2
Implantation sites (total)	110	131	128	139
Implantation sites (mean)	13.8	14.6	14.2	13.9
Number of pups at birth (total)	96	125	123	127
Number of pups at birth (mean)	12.0	13.9	13.7	12.7
Birth Index (mean %)	90.4	95.5	95.9	90.1
Birth Index (total^# %)	87	95^1	96^1	90.1
Number of stillbirths	0	0	1	0
No. of dams with stillborn pups	0	0	1	0
Number of live born pups (total)	96	125	123	126
Number of live born pups (mean)	12.0	13.9	13.7	12.6
Live birth index (mean %)	100.0	100.0	100.0	97.5
Live birth index (total^#1 %)	100	100	100	99
Pre-implantation loss (mean %)	0.0	4.4	0.7	1.9
Pre-implantation loss (total^#2 %)	0.0	4.4^2	0.8	2.1
Post-implantation loss (mean %)	9.6	4.5	4.1	11.2
Post-implantation loss (total^#3 %)	12.7	4.6^2	3.9^2	9.4
Number of runts	0	0	0	0
Number of malformed pups	0	0	0	0

^# based on the total No. of implantation sites and total No. of pups at birth (alive and dead)

^#1 based on the total No. live born pups and total No. of pups at birth (alive and dead)

^#2 based on the total No. corpora lutea and total No. of implantation sites

^#3 based on the total No. implantation sites and toal number of live born pups

¹ p≤0.05 Chi²-test

² p<0.05 Chi²-test

Applicant's summary and conclusion

Conclusions:

NOAEL (no-observed-adverse-effect level) for reproductive toxicity: >= 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 test item/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 test item/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 test item/kg bw/day). A slight reduction in body weight was noted for the male and female rats of the high dose group (1000 mg test item/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.

Effects on reproduction parameters and organs

No test item-related influence was noted on the reproduction parameters in any treatment group (100, 300 and1000 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 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): >=1000 mg/kg bw/day, p.o.