Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Based on the generated data from the available OECD 422 study on Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1) in rats, the NOAEL of 675.50 mg a.i./kg bw/day was identified for the reproduction/developmental toxicity. The study is performed according to OECD 422 guideline and under GLP and has reliability rating 1.

 

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of a maximum of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed. Animals of an additional control group were handled identically as the dose groups but received sterile water, the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats.

 

The following doses were evaluated:

Control (C):                0           mg/kg body weight/day

Low Dose (LD):          67.55    mg/kg body weight/day

Medium Dose (MD):   202.65  mg/kg body weight/day

High Dose (HD):         675.50  mg/kg body weight/day

 

The dose levels referred to active ingredient Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1). In order to correct for the purity of 40.8 % of active ingredient, the test material was weighed under consideration of a correction factor of 2.451. Dose volumes were adjusted individually based on the body weight most recently measured. The administration volume was 5 mL/kg body weight.

 

On the basis of this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1) in male and female Wistar rats with dose levels of 67.55, 202.65 and 675.50 mg/kg body weight/day the following conclusions can be made:

 

The percentage of pre- and post- implantation losses were higher in HD group (10.20 % and 24.36, respectively) compared to corresponding control (3.77 % and 19.33, respectively). This is based on an incidental finding, single female no. 79 whose number of implantation sites was lower and who did not litter. In absence of associated findings is assumed to be without relation to the test item.

 

The delivery index was lower in HD group i.e., 83 % compared to 100 % of control. The lower delivery index in HD group is based on single female no. 79 who did not litter. In absence of associated findings is assumed to be without relation to the test item. Based on the generated data the high dose (HD) level 675.50 mg/kg body weight/day is considered to be the NOAEL for the systemic toxicity for male and female rats. The high dose (HD) level 675.50 mg/ kg body weight/day is considered to be the NOAEL for the reproduction/ developmental toxicity.

 

Available endpoint data within the substance group covers the smallest (shortest alky, lowest number amine and carboxymethylated groups, CAS no2098351-38-1) as well as the biggest structure (longest alkyl-unsaturated, highest number amine and carboxymethylated groups, CAS no 2060541-49-1) within the amphoteric, glycinate group. The NOAEL values from these two studies are comparable, but the study on Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1) is identified to have the lowest NOAEL value.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-09-12 to 2018-03-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Software update

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: approx. 14-15 weeks old
Body weight at the allocation of the animals to the experimental groups:
males: 332 - 390 g (mean: 356.95 g, ± 20 % = 285.56 – 428.34 g)
females: 198 - 240 g (mean: 221.13 g, ± 20 % = 176.90 – 265.35 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes. 
This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities. Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.

Housing and Feeding Conditions
-- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Animals were housed in groups of 5 animals / sex / cage in type IV polysulphone cages during the premating period for both males and females and during post-mating period for males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages and males were returned to their original cage. In each cage Altromin saw fibre was used as bedding.
- Nesting material was provided on GD 18 for all mated females
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (6 days) under laboratory conditions

Preparation of the Animals
Prior to the start of the treatment period, a detailed clinical observation outside the home cage was made. None of the animals showed adverse clinical symptoms. Before initiation of dosing, all females were screened for two weeks for regular estrous cyclicity and females (10 females/ group) with regular estrous cycle (4-5 day cycle) were used in the study.
Before the first administration, all animals to be used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively. Randomisation was performed with validated IDBS Workbook 9.4.0 software.
Each animal was marked with its identification number by individual ear tattoo.

Route of administration:

oral: gavage

Vehicle:

other: aqua ad iniectabilia

Remarks:

Manufacturer: AlleMan Phama; Batch No.: 601101; Physical State: liquid; Storage Conditions: at room temperature; Expiry Date: December 2018

Details on exposure:

The dose levels were referred to active ingredient (CAS no. 97659-51-3). In order to correct for the purity of 40.8 % of active ingredient, the test material was weighed under consideration of a correction factor of 2.451.
The test item was weighed into a tared plastic vial on a precision balance. The test item was dissolved in aqua ad iniectabilia.
The dose formulations were prepared by adding the required volume of aqua ad iniectabilia and further vortexing it for 2-3 minutes.
The vehicle was selected as suggested by the sponsor based on the test item’s characteristics and testing guideline. The test item and control formulations were prepared once in 10 days and stored at room temperature. This was decided based on the available stability data (Eurofins Munich study no. 162865).
The vehicle was also used as control item.

According to the results of the dose range finding study (BSL Munich study no. 163484) and in consultation with the sponsor the following doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and one control group (C).

Control: 0 mg/kg/d
Low Dose: 67.55 mg/kg/d
Medium Dose: 202.65 mg/kg/d
High Dose: 675.50 mg/kg/d


The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and a NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume as used for the dose groups.

The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 5 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Details on mating procedure:

Mating was performed in a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the mating. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation.
The cages were arranged in such a way that possible effects due to cage placement were minimized.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During the study, samples were collected from control, LD, MD and HD groups for the investigation of substance concentration. The samples were collected in study week 1 (pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) from all groups (16 samples).
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 5 mL). The A-samples were analysed at Eurofins Munich. After the receipt at the analytics department samples were measured within 10 days based on available stability data (Eurofins Munich study no. 162865). The procedures followed for the study sample analysis were mentioned in a phase plan (study no. 162866) that was amended to the study plan. The B-samples will be retained at -15 to -35 °C at BSL Munich (test facility) and discarded after completion of the final study report. The results are reported in the appendix of the final report.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females. Afterwards, in females treatment was continued during the gestation period and up to post-natal day 12. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Frequency of treatment:

daily

Details on study schedule:

Arrival of the Test Item: 18 April 2016
Study Initiation Date: 12 September 2016
Amendment to Study Plan: 20 September 2016
2nd Amendment to Study Plan: 26 September 2016
3rd Amendment to Study Plan: 26 June 2017
Delivery of Animals: 08 September 2016
Acclimatisation Period: 08 September 2016 to 12 September 2016
Experimental Starting Date: 13 September 2016
Treatment Period: 28 September 2016 to 29 November 2016
Necropsies: 03 October 2016, 24 October 2016, 26 October 2016 to 28 October 2016, 02 November 2016, 07 November 2016, 09 November 2016,
29 November 2016, 16 November 2016 to 21 November 2016
Experimental Completion Date: 29 November 2016
Completion Date of Delegated
Phase (Histopathology): 18 July 2017
Completion Date of Delegated
Phase (Formulation Analysis): 12 February 2018
Study Completion Date: date of the study director’s signature

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

67.55 mg/kg bw/day

Dose / conc.:

202.65 mg/kg bw/day

Dose / conc.:

675.5 mg/kg bw/day

No. of animals per sex per dose:

100 animals (40 males and 60 females) were included in the study. All females were screened for regular estrous cycles for 14 days before the treatment initiation and only 40 females (10 females/group) showing regular estrous cycles were continued in the study.

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

Clinical Observations
General clinical observations were made once a day, approximately same time after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Once before the first exposure, and weekly thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

Functional Observations
Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and during the last week of lactation in 5 randomly selected females (only lactating females were evaluated) of each group outside the home cage using a functional observational battery of tests:
Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

Body Weight and Food Consumption
The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4 and PND 13 along with the pups. All animals were weighed directly before termination.
Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

Haematology
Haematological parameters were examined in 5 randomly selected males and females (only lactating females were evaluated) from each group at the end of the treatment prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in EDTA-coated tubes. The following haematological parameters were examined: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso) and large unstained cells (Luc).

Blood Coagulation
Coagulation parameters from 5 randomly selected males and all females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in citrate tubes. The following coagulation parameters were examined: prothrombin time (PT) and activated partial thromboplastin time (aPTT).

Clinical Biochemistry
Parameters of clinical biochemistry from 5 randomly selected males and all females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in serum separator tubes. The following parameters of clinical biochemistry were examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na) and potassium (K).
From 2 female pups/litter on day 4 after birth, from all dams and 2 pups/litter (1 male and 1 female) at termination on day 13 and from all adult males at termination. Blood samples were collected by cardiac puncture in pups and from abdominal aorta in adult animals. All blood samples were stored under appropriate conditions. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4). Pup blood was pooled by litter for thyroid hormone analysis.

Urinalysis
A urinalysis was performed with samples from 5 randomly selected males and females (only lactating females were evaluated). Additionally, urine colour/ appearance were recorded. The following parameters were measured using qualitative indicators (Heiland Urine Stripes URI 10SL): specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood and leukocytes.

Oestrous cyclicity (parental animals):

Estrous cycles of all females were monitored before the start of treatment to select the females with regular cyclicity (using vaginal smears). Further on, vaginal smears were also examined daily from the beginning of the treatment period until the evidence of mating.

Sperm parameters (parental animals):

For the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides. Please refer to histopathology section.

Litter observations:

The duration of gestation was recorded and calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after the delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of littering (PND 0) and on PND 4 and PND 13. Live pups were identified by tattoo mark on paw. In addition to the observations of the parent animals, any abnormal behaviour of the offspring was recorded.
The anogenital distance (AGD) of each pup was measured on PND 0. Pup body weight measured on PND 0 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD / Cube root of pup weight). The number of nipples / areolae in male pups was counted on PND 12.

Postmortem examinations (parental animals):

Pathology
All surviving male animals were sacrificed after the completion of the mating period (after a total dosing period of 28-29 days) on study day 29 and 30, while female animals were sacrificed on the respective PND 13 by using anaesthesia (ketamine/xylazine, 2:1). Pups used for blood collection on PND 4 were sacrificed by bleeding after cardiac puncture under anesthesia. All surviving pups were killed by cervical dislocation/decapitation on PND 13.
Dead pups and all surviving pups on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.
Non-pregnant females were sacrificed on day 26 from the day of sperm positive vaginal smear or form the last day of mating period.
All adult animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), the thyroid/parathyroid glands and all organs showing macroscopic lesions of all adult animals were preserved in 4 % neutral-buffered formaldehyde, except for testes and epididymides which were preserved in modified Davidson’s Solution for 24 hours and then transferred to 70 % ethanol.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Organ Weights
The wet weight of the organs [testes (paired weight), levator ani + bulbocavernosus, muscle complex (complete weight), epididymides (paired weight), glans penis, prostate, seminal vesicles and coagulating glands (complete weight), uterus with cervix, Cowper’s gland (paired weight), ovaries (paired weight), thyroid/parathyroid glands (from 1 pup/sex/litter/group and from all adult males and females) – weighed after fixation (complete weight), thymus, liver, spleen, kidneys (paired weight), brain, adrenal (paired weight), heart and pituitary gland] of 5 randomly selected male and female animals (only lactating females were evaluated) from each group were recorded as soon as possible. Paired organs were weighed together. Organ weights of animals found dead or euthanised for animal welfare reasons were not recorded.
Reproductive organs were weighed from all animals.
Thyroid/parathyroid glands from 1 pup/sex/litter/group (sacrificed on PND 13) and from all adult males and females were preserved. Weight of thyroid/parathyroid glands were measured after fixation.
Adrenal glands, all gross lesions, aorta, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides, eyes with optic nerve and Harderian gland, femur with knee joint, heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs, lymph nodes (mandibular), lymph nodes (mesenteric and axillary), mammary gland area (male and female), oesophagus, ovaries, oviducts, pancreas, pituitary, prostate and seminal vesicles with coagulating glands as a whole, rectum, salivary glands (sublingual, submandibular), sciatic nerve, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar segments),
spleen, sternum (with bone marrow), stomach, testes, thymus, thyroid/parathyroid glands, tongue, trachea, ureters, urinary bladder, uterus with cervix and vagina of the same selected animals from each group were preserved in 4% neutral-buffered formaldehyde except for testes and epididymides that were fixed in Modified Davidson’s Fixative for approximately 24 hours before they were transferred to 70% ethanol. All animals found dead and/or intercurrently euthanised for animal welfare reasons were subjected to a gross necropsy and the organs preserved for a histopathological examination.
Thyroid/parathyroid glands from 1 pup/sex/litter/group sacrificed on PND 13 and non-selected adult animals were preserved for potential histopathological examination.

Histopathology
A full histopathology was carried out on the preserved organs and tissues of the selected animals of the control and high dose groups which were sacrificed at the end of the treatment period.
A full histopathology was carried out on the preserved organs and tissues of all animals which died during the study.
Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and eosin and examined in control and HD animals and in non-pregnant female animals of the LD and MD animals. Testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland) were also examined in the mating partners of the non-pregnant female LD and MD animals.
Gross lesion macroscopically identified was examined microscopically in all animals.
For the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland (test site for histopathology). The study phases from test site 1 and 2 were performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1] (Status as of 01 December 2012). Blocking, embedding, cutting, H&E staining and scientific slide evaluation was performed according to the corresponding SOP’s of the test sites.
The principal investigator for histopathological tissue processing sent all specimen and raw data (including blocks, slides, remaining wet tissues, paper raw data, statement of compliance and quality assurance statement) to the study director.
The principal investigator for histopathological evaluation provided the histopathology results to the study director by e-mail and sent a pathology phase report to the study director upon the completion of the study.

Postmortem examinations (offspring):

Dead pups and all surviving pups on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.

Statistics:

A statistical assessment of the results of body weight, food consumption, parameters of haematology, blood coagulation, clinical biochemistry and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights were statistically analyzed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.1.3 software (p<0.05 was considered as statistically significant).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results P0

Mortality:

mortality observed, non-treatment-related

Description (incidence):

see Details on results P0

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results P0

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results P0

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results P0

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results P0

Urinalysis findings:

no effects observed

Description (incidence and severity):

see Details on results P0

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

see Details on results P0

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results P0

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

see Details on results P0

Reproductive function: sperm measures:

effects observed, non-treatment-related

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results P0: Thyroid Hormone (T4) Analysis, Precoital Interval and Duration of Gestation, Pre- and Postnatal Data, Reproductive Indices

Clinical Observations
In males, there were no clinical symptoms in LD group. Alopecia (2/10), slight piloerection (1/10) and crust (1/10) were observed in a few male rats of MD group and additionally, piloerection was seen transiently in male no. 25. Therefore, these findings in MD group males were not related to test item treatment. In HD group, abnormal breathing and chromodacryorrhea were seen on single occasion in male no. 38 and diarrhoea was observed on single occasion in male no. 39. These findings were transient and incidental in nature. One male (no. 28) of MD group had accidentally swallowed a piece of gavaging cannula but did not show adverse findings until the terminal sacrifice.
In females, crust (2/10), erythema (1/10), hunched posture (1/10) and piloerection (1/10) were seen in control group. In LD group, moving the bedding (1/10) and salivation (1/10) was observed on one occasion during gestation. These findings in C and LD groups were not of toxicological relevance. In MD group, abnormal breathing, slightly reduced spontaneous activity and piloerection were in female no. 68 and salivation in female no. 66 on a single occasion. In the HD group, abnormal breathing, alopecia, diarrhoea and regurgitation were observed on single occasion in single isolated females. These findings in the MD and HD groups were considered incidental in nature.
Moving the bedding and salivation were seen immediately after the dose administration in mostly all male and female rats of MD and HD groups. These finding were considered to be related to local effect of test item treatment by gavage.
Clinical observation of decedents before found dead:
Male no. 21 (MD group). No clinical findings
Female no. 68 (MD group) PMD 12: abnormal breathing; PMD 12, 14: slightly reduced spontaneous activity; PMD 14: slight piloerection; MD 1: moving the bedding; GD 1-5: moving the bedding
Female no. 71 (HD group) PMD 6-14: moving the bedding; MD 1: moving the bedding; GD 0-15, 17-19: moving the bedding
Female no. 75 (HD group) PMD 5-6: moving he bedding
Female no. 77 (HD group) PMD 5-6: moving he bedding
Female no. 80 (HD group) PMD 3-14: moving the bedding; PMD 14: slightly increased salivation; MD 1-2: moving the bedding,
GD 0-10: moving the bedding; GD 9: moderately increased salivation
PMD= premating day, GD= gestation day, MD= mating/post mating day

Mortality
There was mortality in males and females during the treatment. In MD group, male no. 21 was found dead on day 17 and female no. 68 was found dead on GD 6. In HD group females 71, 75, 77 and 80 were found dead on GD 20, premating day 6, GD 16 and GD 10, respectively. Microscopically, the cause of death was deemed to be related to test item aspiration.

Body Weight Development
In males, the mean body weight was statistically significantly lower in HD (-7% to -6%) group from day 14 onwards. The body weight gain was lower during the entire treatment period (premating day 1 to terminal sacrifice) in MD (24.33 g) and HD (12.60 g) groups compared to control (33.10 g) attaining statistical significance in HD group. The lower weight gain in HD group was due to transiently observed lower weight gain during the 2nd week of premating. The terminal body weight of males in HD group was statistically significantly lower compared to the control group. This finding in the absence of other adverse changes and also considering the absolute body weight being slightly lower (≤7%) compared to control, the statistically significantly lower weight gain was of little toxicological relevance.
In females, the body weight and body weight gain were not affected in test item groups compared to the control group. Although, there was statistically significantly higher weight gain on premating days 7-14 in HD group and GD 7-14 in MD and HD groups, they were not considered to be of toxicological relevance.

Food Consumption
The food consumption was not statistically significantly different in female test item groups compared to the corresponding control group. However, the food consumption was slightly lower in male MD group and moderate to markedly lower in HD group compared to the control. The lower food consumption in males corroborated lower weight gain. In the absence of other adverse findings, this was of little toxicological relevance.

Haematology and Coagulation
In males and females, the mean values of measured hematological parameters were not statistically significantly different in test item groups compared to the corresponding control group, with the exception of the mean haemoglobin in male HD group which was statistically significantly higher in the HD group (16.33 g/dL) compared to the control group (15.00 g/dL). As the mean and individual haemoglobin values were within the range of historical control data (14.2 to 19.0 g/dL), the finding was not considered adverse.
The mean PT and aPTT values in males and females were not statistically significantly different in test item groups compared to the corresponding control group.


Clinical Biochemistry
In males and females, the mean of clinical biochemistry parameters were not statistically significantly different from the corresponding control group, except the mean TBA value in male HD group which was statistically significantly higher (16.91 µmol/L) compared to the control group (8.43 µmol/L). The TBA value was also markedly higher in the LD group (15.55 µmol/L) and was not statistically significantly different compared to the control group. Thus, there was no dose-response relationship. The mean and individual TBA values were also within the range of historical control data (9.98 to 59.19 µmol/L). Therefore, the finding was not considered adverse.

Urinanalysis
In males and females, the urinalysis indicated no significant differences between the test item and the corresponding control group.

Functional Observations
The functional neurological assessment revealed no significant differences between the control and test item group animals. There were no ophthalmoscopic findings in any of the animals of this study.

Organ Weights
In males, the relative spleen weight was statistically significantly lower in the MD group and epididymides weight was statistically significantly lower in the HD group when compared to the control group. Histopathological examination in HD animals showed no lesions in spleen and epididymides of test item groups. Further, the absolute and relative pituitary gland weights were lower in the LD (-15 to -17 %) and the HD (-16 to -22%) groups attaining statistical significance for absolute pituitary weight in the HD group. There was no dose-response relationship and no macroscopic findings associated with the pituitary gland. Thus, the finding was not considered an adverse effect of test item treatment.
In females, the absolute and relative thymus weight was statistically significantly lower in the HD group; the relative liver weight was statistically significantly higher in the HD group compared to the control group. Histopathologically, there were no lesions in thymus and liver of females and therefore, the organ weight findings were not considered to be adverse.
The thyroid weight of male pups measured on PND 13 was not affected in test item groups compared to control group. The thyroid weight was higher in female pups of the MD and HD group, however not statistically significantly different compared to the control group. Thus, the finding was not considered to be adverse.

Pathology
At necropsy macroscopic findings revealed the following:
Male no. 21 (MD group) Lung: abnormal color, dark, red
Male no. 28 (MD group) Adrenal gland (left sided): foci (2-6 no.s)
Female no. 68 (MD group) -
Female no. 71 (HD group) Lung: abnormal surface, red and lung failed to collapse
Female no. 75 (HD group) Lung: abnormal color, dark
Female no. 77 (HD group) Lung: abnormal color, dark and filled with test item fluid
Female no. 80 (HD group) Lung: failure to collapse
Microscopic examination of these gross lesions could not be attributed to treatment with the test item.

Histopathology
The following animals were found dead during the study:
Male no. 21 (MD group), Day 17:
Organs Related to Moribidity/Death: Lung: abnormal color, dark, red; Foreign material, alveolar, multifocal, grade 1, alveolar hemorrhage, focal, grade 1; interstitial inflammation, multifocal, subacute, grade 2; alveolar macrophages, focal/multifocal, grade 2. Adrenal glands: diffuse hypertrophy, bilateral, grade 2
Cause of Morbidty/Death: Possible aspiration of test item and stress-related findings (adrenal gland)
Female no. 68 (MD group), Day 22; Organs Related to Moribidity/Death: - ; Cause of Morbidty/Death: -
Female no. 71 (HD group), Day 35:
Organs Related to Moribidity/Death: Lung: abnormal surface, red; microemboli, multifocal, grade 4; Stomach: ulceration, forestomach, foal, grade 3; Liver: hepatocellular hypertrophy, centrilobular, grade 3; Necrosis, hepatocellular, multifocal, centrilobular, grade 2; Thymus: lymphoid atrophy, grade 3
Cause of Morbidty/Death: Possible aspiration of test item and shock symptomatic (micro-emboli), stress-related findings (stomach, thymus), Incidental findings in liver
Female no. 75 (HD group), Day 6:
Organs Related to Moribidity/Death: Lung: abnormal color, dark; alveolar hemorrhage, focal, grade 1; alveolar macrophages, focal/multifocal, grade 2; interstitial inflammation, multifocal, subacute, grade 1
Cause of Morbidty/Death: Possible aspiration of test item
Female no. 80 (HD group), Day 27:
Organs Related to Moribidity/Death: Lung: failure to collapse; alveolar edema, grade 3
Cause of Morbidty/Death: Possible aspiration of test item
In addition, female no. 77 (HD group, Day 31) was considered to be an accidental death. The animal died immediately after the dosing. Test item (fluid) release from the lungs was noted.
There was no morphological reason for non-mating of male 8 and 11 with females 48 and 51, respectively. The evaluation of sperm staging, showed no abnormalities in the testes, epididymides and accessory sex glands that could be related to treatment. Single lesions were in the range of control lesions that may be recorded also in control animals.
Beside the lesions noted for decedent animals, there were no findings that distinguished controls from test item-treated animals.
Under the conditions of this study, the test item did not induce lesions. A number of animals died during the study (2 animals in MD and 4 animals is HD). The cause of death was deemed to be related to test item aspiration.
Therefore, based on the pathology evaluation, the NOAEL may be established at 675.50 mg/kg/day bw.

Thyroid Hormone (T4) Analysis
In adult males, the serum level of T4 hormone was higher in the LD (105.94 nmol/L) and the HD (102.46 nmol/L) groups compared to the control group (85.82 nmol/L). As there was no dose-response relationship and mean values in test item groups were not statistically significantly different compared to the control group, these findings were not considered to be adverse.

Estrous Cyclicity
The estrous cyclicity was not affected in test item groups when compared to the control group.

Precoital Interval and Duration of Gestation
The precoital interval and the duration of gestation were not affected in test item groups compared to the control.

Pre- and Postnatal Data
The pre-natal and post-natal parameters including the number of corpora lutea, the number of implantations and the number of live pups were not statistically significantly different in test item groups compared to the control.
The percentage of pre- and post- implantation losses were higher in HD group (10.20% and 24.36, respectively) compared to corresponding control (3.77% and 19.33, respectively). This was due to single female no. 79 whose number of implantation site was lower and the animal did not litter. In absence of associated findings this is assumed to be without relation to the test item.

Reproductive Indices
The copulation, fertility and viability indices were not affected in test item groups compared to the corresponding control group.
The delivery index was lower in HD group i.e., 83% compared to 100% in the control group. This excludes the gravid females (71, 77 and 80) those died during the gestation. The lower delivery index in HD group could be related to test item treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

675.5 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: No clinical substance related effects seen in any of the dose groups

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results F1: Pup External Findings

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

see Details on results F1

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results F1

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

see Details on results F1

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results F1

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results F1: Thyroid Hormone (T4) Analysis, Thyroid Weight on PND 13

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Litter Data
The litter parameters including the total number of pups born on PND 0, the number of live pups, number of male and female pups and sex ratio (m/f) on PND 0, 4 and 13 were not statistically significantly different in test item groups compared to the control group. However, the total number of pups (live and dead) on PND 0 and total live pups, total number of male pups and sex ratio (male/female) on PND 0, 4 and 13 were slightly lower in the HD group. As the means were not statistically significantly different compared to the control group the findings were not considered to be adverse.

Litter Weight Data
The pup mean weight on PND 0, 4 and 13 were not affected in test item groups compared to the control group. The mean values were not statistically significantly different in test item groups compared to the corresponding control group.
The total litter weight, male litter weight, female litter weight were not affected in test item groups compared to corresponding control group. However, there was a statistically significantly higher total litter weight on PND 0 in the MD group. Because there was no dose-response relationship, the statistical significance was not of toxicological relevance.

Pup Survival Data
The survival of the pups from PND 0 to PND 4 and from PND 4 to PND 13 was not affected in test item groups when compared to the corresponding control group.
Anogenital Distance and Nipple Retention
The relative anogenital distance in male and female pups measured on PND 0 was not affected in test item groups when compared to the corresponding control group.
There was no test item treatment-related increase in the number of pups with nipple retention in test item groups compared to the control group.

Thyroid Hormone (T4) Analysis and Pup Thyroid Weight on PND 13
In pups, the serum level of T4 hormone in the HD group was comparable to the corresponding control group. The serum level of T4 hormone in the LD (97.46 nmol/L) and the MD (100.83 nmol/L) groups was higher compared to the control group (80.20 nmol/L), however, not statistically significantly different and without dose-response relationship. Thus, the finding was not of test item treatment relevance.
The thyroid weight of male pups measured on PND 13 was not affected in test item groups compared to control group. The thyroid weight was higher in female pups of the MD and HD group, however not statistically significantly different compared to the control group. Thus, the finding was not considered to be adverse.

Pup External Findings
There were no adverse gross pup external findings in test item groups compared to the corresponding control group.
In the control group, all pups of female no. 45 showed no indication of suckling and were cold and weak. Hematoma on the snout and right forelimb were seen in pup no. 7 of dam no. 45 and pup no. 5 of dam no. 50 was small. In the LD group, absent hair coat was seen in pups 1-12 of female no. 60. In the MD group, crust on thorax and snout was observed in pup no. 1 and 7, respectively of female no. 65. Pup no. 6 of female no. 66 was small. In the HD group, a hematoma in the neck region was seen in pup no. 6 of female no. 74.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

675.5 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: No clinical substance related effects seen in any of the dose groups

Reproductive effects observed:

no

Conclusions:

On the basis of this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with Coco iminodiglycinate (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1) in male and female Wistar rats with dose levels of 67.55, 202.65 and 675.50 mg/kg body weight/day the following conclusions can be made:
The body weight and food consumption were lower in male HD group compared to the control. But in the absence of other adverse findings in male rats, these findings were of little toxicological relevance.
The percentage of pre- and post- implantation losses were higher in HD group (10.20 % and 24.36, respectively) compared to corresponding control (3.77 % and 19.33, respectively). This is based on an incidental finding, single female no. 79 whose number of implantation sites was lower and who did not litter. In absence of associated findings is assumed to be without relation to the test item.
The delivery index was lower in HD group i.e., 83 % compared to 100 % of control. The lower delivery index in HD group is based on single female no. 79 who did not litter. In absence of associated findings is assumed to be without relation to the test item.
Based on the generated data the high dose (HD) level 675.50 mg/kg body weight/day is considered to be the NOAEL for the systemic toxicity for male and female rats. The high dose (HD) level 675.50 mg/kg body weight/day is considered to be the NOAEL for the reproduction/ developmental toxicity.

Executive summary:

The aim of this study was to assess the possible effects of Coco iminodiglycinate (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1) on male and female fertility and development after repeated dose administration in Wistarrats.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of a maximum of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed. Animals of an additional control group were handled identically as the dose groups but received aqua ad iniectabilia (sterile water), the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistarrats.

Before initiation of dosing, all females were screened for two weeks for regular estrous cyclicity and animals (10 females / group) with regular estrous cycle (4-5 day cycle) were used in the study.

The following doses were evaluated:

Control (C):                0           mg/kg body weight/day

Low Dose (LD):          67.55    mg/kg body weight/day

Medium Dose (MD):   202.65  mg/kg body weight/day

High Dose (HD):         675.50  mg/kg body weight/day

 

The dose levels referred to active ingredient Coco iminodiglycinate (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1). In order to correct for the purity of 40.8 % of active ingredient, the test material was weighed under consideration of a correction factor of 2.451.

The test item formulation was prepared once in 10 days and stored at room temperature. The test item was dissolved inaqua ad iniectabilia and further vortexing it for 2-3 minutes. The vehicle was also used as control item. Dose volumes were adjusted individually based on the body weight most recently measured. The administration volume was 5 mL/kg body weight.

During the period of administration, the animals were observed each day for signs of toxicity. Animals that died or were euthanised for animal welfare reasons were examined macroscopically and at the conclusion of the test all surviving animals were sacrificed and observed macroscopically.

Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behavior observations were performed before the treatment period and in the last week of treatment in five randomly selected males and females of each group.

Body weight and food consumption were measured weekly, except for food consumption measurements which were not made during the mating period in males and females and the post-mating period in males. During pregnancy, females were weighed on gestation days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4 and PND 13 along with the pups.

After 14 days of treatment, male and female animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.

Live pups were counted, sexed and litters weighed within 24 hours of parturition, on day 4 and day 13 post-partum.

The anogenital distance of each pup was measured on PND 0. The number of nipples/areolae in male pups was counted on PND 12. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4).

Males were sacrificed after completion of the mating period on day 29 and 30 and females along with their pups were sacrificed on post-natal day 13. Non-pregnant females were sacrificed on day 26 from the day of sperm positive vaginal smear or form the last day of mating period. The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Pups sacrificed on post-natal day 4 for interim sacrifice for blood sampling and those found dead were carefully examined for gross external abnormalities.

At the conclusion of the test, all surviving adult animals were sacrificed and observed macroscopically. The wet weight of a subset of tissues was taken and a set of organs/tissues was preserved.

Haematological and clinical biochemistry evaluations and coagulation measurements were performed on blood samples collected at terminal sacrifice from five randomly selected males and females from each group. Urinalysis was performed on samples collected at terminal sacrifice from five randomly selected males and females from each group.

A full histopathological evaluation of preserved organs/tissues was performed in five randomly selected males and females of the control and HD group and in all animals and in non pregnant female animals of the LD and MD animals. Gross lesion macroscopically identified were examined microscopically in all animals.

 

 

Summary Results

There was mortality in males and females during the treatment. In MD group, male no. 21 was found dead on day 17 and female no. 68 was found dead on GD 6. In HD group females 71, 75, 77 and 80 were found dead on GD 20, premating day 6, GD 16 and GD 10, respectively. Microscopically, the cause of death was deemed to be related to test item aspiration. 

In surviving and decedent males and females, there were no adverse clinical symptoms in test item treated groups compared to the control. However, moving the bedding and salivation were observed immediately after the dose administration in mostly all male and female rats of MD and HD groups. These finding were considered to be related to local effect of test item treatment by gavage. Additionally, decedent female no. 68 showed abnormal breathing and reduced spontaneous activity and these symptoms were secondary to test substance aspiration.

The functional neurological assessment revealed no significant differences between the control and test item group animals. There were no ophthalmoscopic findings in any of the animals of this study.

The body weight and body weight gain of males and females were not adversely affected in test item groups compared to control. However, in males the mean body weight was statistically significantly lower in HD (-7 % to -6 %) group from day 14 onwards. The body weight gain was lower during the entire treatment period (premating day 1 to terminal sacrifice) in MD (24.33 g) and HD (12.60 g) groups compared to control (33.10 g) attaining statistical significance in HD group. The terminal body weight of males in HD group was statistically significantly lower compared to the control group. This finding in the absence of other adverse changes and also considering the absolute body weight being slightly lower (<7 %) compared to control, the statistically significantly lower weight gain was of little toxicological relevance.

The food consumption was not statistically significantly different in females of test item groups compared to the corresponding control. However, the food consumption was slightly lower in male MD group and moderate to markedly lower in HD group compared to the control. The lower food consumption in males corroborated lower weight gain.

The estrous cyclicity was not affected by test item treatment. The precoital interval and the duration of gestation were not affetcted in test item groups compared to the control.

The pre-natal and post-natal parameters including number of corpora lutea, number of implantations, number of live pups were not statistically significantly different in test item groups compared to the control group. The percentage of pre- and post- implantation losses were higher in HD group (10.20 % and 24.36, respectively) compared to corresponding control (3.77 % and 19.33, respectively). This was due to a single female no. 79 whose number of implantation sites was remarkably low and the animal did not litter. In the absence of adverse findings in other reproduction parameters in this animal (with the exception of the related parameter of delivery index) and as all other animals of this group were not affected, this is assumed to be an incidental finding without relation to the test item. Due to this animal delivery index was lower in HD group i.e., 83 % compared to 100 % of control. The copulation, fertility and the viability indices were not adversely affected in test item groups compared to the corresponding control group.

The litter parameters including the total pups born on PND 0, number of live pups, number of male and female pups and sex ratio (m/f) on PND 0, 4 and 13 were not adversely affected in test item groups compared to the control. There was no considerable number of stillbirths and no runts in the control and the test item groups.

The pup mean weight and litter weight including total litter, male litter and female litter weight on PND 0, 4 and 13 were not affected in test item groups compared to control.

The survival of the pups from PND 0 to PND 4 and from PND 4 to PND 13 was not affected in test item groups compared to the corresponding control.

The relative anogenital distance in male and female pups measured on PND 0 was not affected in test item groups compared to the corresponding control. There was no test item treatment-related increase in the number of pups with nipple rention in test item groups compared to the control group.

In adult males and pups, the serum level of T4 hormone was not adversely affected in test item groups compared to the corresponding control.

There were no adverse gross pup external findings in test item groups compared to the corresponding control.

In males and females, the measured hematological, blood coagulation and clinical biochemistry parameters were not adversely affected in test item groups compared to the corresponding control group.

In males and females, the urinalysis indicated no significant differences between the test item and the corresponding control group.

There were no macroscopic findings that could be attributed to treatment with the test item.

The organ weights of spleen (MD group), pituitary (HD group) and epididymides (HD group) of males and thymus (HD group) and liver (HD group) of females statistically significantly deviated from corresponding control. These findings in the absence of macroscopic and/or microscopic changes were not considered to be adverse.

The thyroid weight of pups measured on PND 13 was not adversely affected in test item groups compared to the control group.

Under the conditions of this study, the test item did not induce lesions. A number of animals died during the study (2 animals in MD and 4 animals is HD). The cause of death was deemed to be related to test item aspiration. 

Concentration analysis of formulation samples was determined at three concentrations, 13.5 mg/mL, 40.5 mg/mL and 135 mg/mL in study weeks 1, 3, 5 and last. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 98.1 %, 96.0 % and 92.1 % of the nominal concentration, respectively. Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10 %.

 

Conclusion

On the basis of this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1) in male and female Wistar rats with dose levels of 67.55, 202.65 and 675.50 mg/kg body weight/day the following conclusions can be made:

The body weight and food consumption were lower in male HD group compared to the control. But in the absence of other adverse findings in male rats, these findings were of little toxicological relevance.

The percentage of pre- and post- implantation losses were higher in HD group (10.20 % and 24.36, respectively) compared to corresponding control (3.77 % and 19.33, respectively). This is based on an incidental finding, single female no. 79 whose number of implantation sites was lower and who did not litter. In absence of associated findings is assumed to be without relation to the test item. The delivery index was lower in HD group i.e., 83 % compared to 100 % of control. The lower delivery index in HD group is based on single female no. 79 who did not litter. In absence of associated findings is assumed to be without relation to the test item.

Based on the generated data the high dose (HD) level 675.50 mg/kg body weight/day is considered to be the NOAEL for the systemic toxicity for male and female rats. The high dose (HD) level 675.50 mg/kg body weight/day is considered to be the NOAEL for the reproduction/ developmental toxicity.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

675.5 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Good. The study is performed according to OECD 422 guideline and under GLP and has reliability rating 1. It is sufficient to cover the information requirement for this tonnage band.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

There were no indications of any substance related toxicity to reproduction in any of the dose groups of Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1). Therefore the substance does not have to be classified and has no obligatory labelling requirement according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011), nor the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.